NEURODEGENERATION, Vol. 4, pp 291-297 (1995)

Distribution of Beta Amyloid Associated Proteins in Plaques in Alzheimer's Disease and in the

Non-demented Elderly Shan-Shan Zhan, 1 Robert Veerhuis, 1,2 Wouter Kamphors t 2

and Piet Eikelenboom 1

Graduate School of Neurosciences Amsterdam, Research Institute Neurosciences Vrije Universiteit, Depts of Psychia tr f and Neuropathology 2 of the Medical Faculty, Amsterdam

Recent studies have shown that cerebral beta amyloid (A[5) protein deposition is a necessary, but not sufficient, factor to develop the pathology of Alzheimer's disease (AD). In the present immunohistochemical study, we have investigated in AD the distribution of AI5 associated pro- teins in the cerebral neocortex, in the cerebellar cortex where A~ plaques are mainly of the diffuse type, and also in the cerebral neocortex of non-demented patients with A[5 plaques. Results show that immunolabeling for Clq, C4c, C3d, 0~I-ACT and Apolipoprotein E (ApoE) occurs in the great majority of A~ plaques in all groups. ApoJ is present in A~ plaques of the cerebral neocortex in AD and in non-demented elderly, but is almost absent from those of the AD cerebellar cortex. C4Bp and P-component, in contrast to AD, rarely occurs in A[5 plaques of the cerebral neocortex in the non-demented elderly. Heparan sulphate proteoglycan (HSPG) core protein and inter- cellular adhesion molecule-1 (ICAM-1) are absent in the diffuse A[5 plaques in the AD cerebellum. These differences in distribution and expression of A~ associated proteins may be determined by brain region specific factors (cerebral cortex versus cerebellar cortex) and clinical state (demented versus non-demented cases). We suggest that, besides A~ peptide, certain A[5 associated proteins are required for both amyloid plaque formation and for the induction of neurofibrillary changes. © 1995 Academic press Limited

K e y w o r d s : Alzheimer's disease, A[5 associated proteins

THE AMYLOID PLAQUE is one of the pathological hall- marks of Alzheimer 's disease (AD). Beta amyloid pro- tein (AIS), a 39- to 43-amino-acid peptide derived from a precursor membrane glycoprotein (amyloid precur- sor protein, APP), is the principal protein component. Recently, it has become apparent that a number of other proteins are associated with A[5 deposits---so called 'pathological chaperones ' - -but which are not themselves components of the A[5 fibrils. It has been suggested that some of these proteins are involved in a balancing between maintaining the solubility of A~ peptide (Ghiso et al., 1993) and its deposition, and /o r

Correspondence to: P. Eikelenboom, Dept of Psychiatry, PCA Valeriuskliniek, Valeriusplein 9,1075 BG Amsterdam, The Netherlands

Received 9 March 1995; revised and accepted for publication 3 May 1995

© 1995 Academic Press Limited 1055-8330/95/030291 +7 $12.00/0

amyloid fibril formation (Snow et a l., 1994a). However, their precise functions and roles in the brain in AD are still unclear.

A[3 amyloid plaques can be principally divided into two types: neuritic or senile plaques containing amy- loid filaments and dystrophic neurites with glial reac- tion; and diffuse or amorphous plaques without neuritic and glial changes (Rozemuller et al., 1989a). A[~ deposits in the cerebellum in AD consist mainly of diffuse plaques and they do not usually progress to form amyloid fibrils, neuritic alterations and glial changes (Joachim et al., 1989). Recently, we and others (Arai et al., 1990; Cras et al., 1991; Schmidt et al., 1991; Yasuhara et al., 1994; Zhan et al., 1995) have shown that the altered neurites of neuritic plaques in the cerebral cortex differ in AD from those in the non-demented elderly, since these can be immunostained by anti- bodies to tau, in contrast to those in the non-demented elderly which cannot. These data imply that although

291

292 S-S. Zhan et al.

A~ is necessary it m a y also require other components to accelerate fibril formation and to induce neuro- fibnllary pathology.

Accordingly, in the present immunohistochemical study, we have investigated the distribution of A[3 associated proteins including complement proteins (Eikelenboom et al., 1982, 1989), C4-binding protein (C4Bp) (Kalaria & Kroon, 1992), (zl-antichymotrypsin (o~I-ACT) (Abraham et al., 1988; Rozemuller et al., 1991), intercellular adhesion molecule-1 (ICAM-1) (Rozemuller et al., 1989b; Verbeek et al., 1994), apolipoprotein J (ApoJ) (McGeer et al., 1992; Ghiso et al., 1993; Zhan et al., 1994), apolipoprotein E (ApoE) (Wisnlewski & Frangione, 1992), amyloid P-compo- nent protein (Kalaria & Perry, 1993), ¢z2-macroglobulin (Bauer et al., 1991; Strauss et al., 1992; Wood et al., 1993), lactoferrin (Osmand & Switzer, 1991; Kawamata et al., 1993), and heparan sulfate proteoglycan (Snow et al., 1994a, 1994b) in both the cerebral neocortex and the cerebellar cortex in AD and in the cerebral neocortex (with A~ plaques) in the nondemented elderly.

Ma te r i a l s a n d Methods

Frozen specimens of neocortex and cerebellar cortex from cases of AD (aged 47-85 years) and six controls (aged 62-80 years) were obtained from the Netherlands Brain Bank, Amsterdam (Coordinator Dr R. Ravid). Postmortem time was in all instances less than 6 h. A diagnosis of AD was clin- ically and pathologically confirmed. Control cases were free from neurological disease and without record of dementia.

A n t i b o d i e s

The antibodies used are listed in Table 1.

Immunostaining Serial 4 ~ n cryostat sections were cut and mounted onto poly-L-lysine coated glass slides, air-dried, and fixed in ace- tone for 10 min. Sections were preincubated with normal swine serum for 20 min followed by incubation overnight at 4°C with primary antibodies diluted in phosphate-buffered saline (pH 7.4) containing 1% bovine serum albumin. Immunolabelling for polyclonal antibodies and monoclonal antibodies was detected with PAP kit (DAKO) and ABC kit 0decta-stain), respectively, and visualized with the DAB method (5 mg 3,3-diaminobenzidine in 10 ml phosphate- buffered saline, PH 7.4, containing 0.02% H202 for 2 to 5 min). Controls included incubation of the sections with nonim- munized rabbit serum or with PBS only instead of primary antibodies. Secondary antibodies and reagents were appropriately tested for absence of cross-reactivity and for non-specific staining. All sections were counterstained with haematoxylin and Congo red, dehydrated and mounted.

Table 1. Antibodies used

Antibody Source Type Dilution

A~ (1-28) Dr Van Nostrand Rabbit IgG, 1:300 (Univ. California) polyclonal

Clq Zymed Rabbit IgG, 1:50 polyclonal

C3d CLB Monoclonal 1:50 C4c DAKO Rabbit, 1:50

polyclonal C4Bp Calbiochem Rabbit, 1:50

polyclonal SP40,40 Quidel Monoclonal 1:100 (xl-ACT DAKO Rabbit, 1:800

polyclonal ApoE Chemicon Inc. Goat, 1:500

polyclonal P-component DAKO Rabbit, 1:100

polyclonal Lactoferrin DAKO Rabbit, 1:200

polyclonal a2-Macro- DAKO Rabbit, 1:200

globulin polyclonal HSPG (JM13) Nijmegen Monoclonal 1:50

HS-GAGs specific

HSPG (JM403) Nijmegen Monoclonal 1:800 HS-GAGs specific

HSPG (JM72) Nijmegen Monoclonal 1:200 (core of HSPG)

CLB: Central Laboratory Blood Transfusion Service, Amsterdam. HSPG: Heparan sulfate proteoglycan (Van den Born J et al., 1994). Anti-HSPG-core monoclonal is specific for the core protein of HSPG and does not react with the core protein of other proteoglycans.

Results

The cerebral neocortex in all AD and non-demented elderly cases showed plaques identifiable by A~ immunostaining. Based on A~ immunostaining, the morphological plaque type was classified according to Delaere et al. (1991). In AD, both diffuse and neuritic plaques were seen. However, in the non-demented elderly mainly diffuse plaques and only few neuritic plaques were present. In the cerebellar cortex in AD, diffuse A~ plaques were found in the molecular layer, and a few congophilic cored plaques were occasion- ally seen at the borders between the molecular layer and the Purkinje cell layer, or between the Purkinje cell layer and the granular cell layer.

In the present immunohistochemical study, on serial sections, immunolabeUing for C1 q, C4c, C3d, 0~1 - ACT and ApoE was present in nearly all A~ immuno- stained plaques in the neocortex in AD and in those in the non-demented elderly, and also in plaques in the

Distribution of beta amyloid associated proteins 293

Table 2. Distribution of associated proteins in A~ plaques

AD Non-demented elderly Neocortex cerebellum neocortex

Diffuse Neuritic Diffuse Amyloid cored Diffuse Neuritic

Clq ++ ++ ++ ++ ++ ++ C3d + + + + + + + + + + + + + + + + + + C4c ++ ++ ++ ++ ++ ++ cd-ACT + + + + + + + + + + + + + + + + + + ApoE ++ ++ ++ ++ ++ ++ ApoJ ++ ++ + + ++ ++ Pcomp ++ ++ ++ ++ - + C4Bp + + + + + + ++ ++ + + Lactoferrin + + + - + - ___ o~2-macro-globulin ± + + - + - ± HSPG (core) + + + + - +_ + + + + HSPG (HS) + + + + - + + + + + + ICAM ±/+ +-/+ - - +/+ +/+

- No immunoreactivity in A~ plaques. --- Positive immunoreactivity occasionally occurring in A~ plaques. + Positive immunoreactivity in some A~ plaques.

+ + Positive immunoreactivity in a majority of A~ plaques. + + + Positive immunoreactivity in nearly all A~ plaques.

cerebellar cortex in AD (Table 2, Fig. la,b). Anti-ApoJ (SP40, 40) immunoreact iv i ty was present in the major- i ty of the A[3 plaques in the cerebral neocortex in AD and in the non-demented elderly cases, but was vir- tually absent from cerebellar cortical plaques in AD (Fig. lc). The forementioned antibodies stained both the diffuse and the neuritic plaques. ICAM-1 anti- body stained more than half the A[~ amylo id plaques in the cerebral neocortex in some of the AD and non- demented elderly cases, but d id not stain plaques in the cerebellums in AD ( not shown). P component immunoreact iv i ty was present in the A[~ plaques in the cerebral neocortex (Fig. 2c) and the cerebellar cortex in AD, but was only occasionally present in the diffuse and the neuritic plaques in the cerebral neocortex in the non-demented elderly (Fig. 2a,b). Immuno- labelling for C4Bp was present in nearly all A~ plaques in the cerebral neo-cortex and in the cerebellar cortex in AD, but was seen to a much lesser extent in A[} p l a q u e s in the n o n - d e m e n t e d e l d e r l y (not shown). Lactoferr in and (z2-macroglobulin immunoreact ivi ty was seen mainly in the neuritic plaques in the neocortex in AD, and occasionally in neuritic plaques in the cerebral neocortex of one of the non-demented elderly (but not in the other five non- demented cases) (Fig. 3a-c). Neither of these latter proteins were seen in the diffuse plaques, but were present in the congophilic cored plaques, in the cere- bellar cortex in AD (not shown). HSPG core protein

immunoreact ivi ty was present in the majority of the A~ plaques in the cerebral neocortex in AD and in the non-demented elderly. However, the diffuse plaques in the molecular layer of the cerebeUar cortex in AD were not immunolabeUed for HSPG core protein (Fig. 4a,b), though the few cored plaques in the cerebellar cortex d id show a clear immunosta in ing for heparan sulfate but not, or only weakly so, for the core protein of HSPG (Fig. 4c). All f indings are summar ized as semiquanti tat ive data in Table 2.

D i s c u s s i o n

This present s tudy has shown that Clq , C4c, C3d, col- ACT and ApoE are present in all types of A[~ plaques in AD and in those of the neocortex in non-demented elderly subjects, and also in those of the cerebellar cor- tex in AD. It has been suggested that a l - A C T ( a pro- tease inhibitor) o c ~ g in diffuse plaques, a n early stage of plaque development , m a y be involved in the generation of amyloidogenic fragments (Rozemuller et al., 1989a) and promote the formation of A~ filaments (Ma et al., 1994). It has also been. repor ted that A[}I~ and especially A~1~ pept ides when aggre- gated b ind the collagen-like domain of the C l q A c h a i n to activate the classical complement pa thway (Rogers et al., 1992; Jiang et al., 1994). Recently we have shown that C5, C7, C9 and the C5b-9 membrane attack corn-

294 S-S. Zhan et al.

~ . ~ , , ~ . ~ ~;4'.'~.~. . ~ . ~ : ~ ; . . . . . . : " •~.~ ~ ,~ :

... • , ',...-..2~

,, ,.:. : . . . . .:,..,:~ ,~. .: ... ~- -.- .~. ,r(K~2"~ ,

-.: - _ ~..~ - ..,: ,: -... .'.,:.. .~.'"

"" ~ %:',_' "-'.~'.'. : -: "'4" "

,_

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. - } . ~i~ d, ' k " . . y ~. - - . ' . 2 ~

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Figure 1. Adjacent sections of cerebellar cortex in AD. (a) ApoE is present in the majority of A~ plaques, (b) Figure 2. Serial sections of the cerebral neocortex from a Immunostaining for AI]g, (c) ApoJ (SP40,40) is virtually non-demented elderly patient (a & b) showing that plaques absent from the A~ plaques (x40). immunostained by AIt antibody (a) are not stained for P-

component (b); (c) cerebral n~ocortex in AD case, which in contrast to (a) and (b), shows P-component to be present in

plex (MAC) are not present in plaques in AD, which the A[] plaques (x40). means that complement activation in AD does not pro- ceed further than C3 (Zhan et al., 1994; Veerhuis et al., 1995a). The role of complement proteins in plaque ApoJ (SP40, 40) has been suggested to be involved formation is not clean It has been proposed that ~-amy- in maintaining the solubility of A~ in biological fluids loid-mediated complement activation could lead to (Ghiso et al., 1993). In this study, ApoJ was present in recruitment of microglia and increased synthesis of the neocortical plaques in both AD and non-demented inflammatory mediators (Veerhuis etal. , 1995b). ApoE elderly, but, surprisingly, it was present to a much is involved in lipid transport and metabolism and lesser extent in the diffuse plaques in the cerebellum. found in amyloid deposits. Possession of the ApoE4 We found that much less P component and C4Bp allele is associated with increased risk of late-onset AD was present in AI] plaques of the cerebral cortices in and is correlated with a higher deposition of A~ the non-demented elderly cerebral cortex in compar- (Schmechel et al., 1993). The presence of ApoE in all ison to AD. Degradation of P component can occur morphological types of plaques of all patient groups under two conditions. It is remarkably resistant to pro- in this study indicates that ApoE may play a critical teolytic degradation in the presence of calcium, but, in role in A~ plaque formation, contrast, in the absence of calcium it is cleaved by

Distribution of beta amyloid associated proteins 295

"'- *.;. ' " " - " ~ ~;. . . . . " -" "'- " , ":"!: . ' % " ' " ' i t""" - " ~

Figure 3. Adjacent sections of cerebral neocortex from a non-demented elderly patient. No immunostaining for o.2- macroglobulin (a) or lactoferrin (c) is seen in A~ immuno- labelled (b) (x40).

°- ; • . - . , .4v?~'~ " ~ ,

: ,... ~,~ t .- ~ . . ~ ~ ¢ ~ ' ~ I R W ~ g , / . ~

• 1' • "" ".'~Y.-~"

Figure 4. Sections of cerebellar cortex in AD (a & b); dif- fuse A~ plaques seen in (a) do not contain HSPG core pro- tein (b) though the side chain of HSPG immunostained with JM 403 antibody is present in cored plaques (c) (x40).

enzymes such as 0~-chymotrypsin and pronase. Furthermore, P component binding to amyloid fibrils is absolutely calciumdependent at physiological pH and ionic strength. Therefore, it has been suggested that P component is a significant factor maintaining the persistence of amyloid deposits, due to its pro- teinase resistance (Emsley et al., 1994). C4Bp is not only an inhibitor of the activation pathway of the comple- ment system (at C3 stage) but is also one autologous ligand for P component (Kalaria & Kroon, 1992). The question is whether P component participates more in A[3 plaques in AD than in the non-demented elderly, or whether P component is more degraded in the non- demented elderly than in AD brain.

0~2-macroglobulin and lactoferrin showed a very

similar localization. Both proteins are present in neuritic plaques in the cerebral cortex and in the con- gophilic cored plaques in the cerebellar cortex in AD, but are rarely present in the plaques of the cerebral cor- tex in the non-demented elderly. In agreement with this result, it has been reported that rx2-macroglobulin was absent in the amyloid plaques of aged non-AD control brains (Strauss et al., 1992). 0~2-macroglobulin is an acute phase protein, also involved in lipoprotein metabolism, promoting neurite growth (Wood et al., 1993). Lactoferrin is considered to be an iron scavenger and may also be involved in the production of reac- tive oxygen metabolites (Kawamata et al., 1993). Their widespread presence in neuritic plaques in the neocortex in AD but only rare presence in such plaques

296 S-S. Zhan et al.

in the non-demented elderly suggest these two mole- cules may be related to A[~ fibril formation and neurofibriUary pathology.

ICAM-1 is present in amyloid plaques in the cere- bral cortex but not in the cerebellar cortex, suggesting that inflammatory factors might also play a role in amyloid fibril formation.

HSPG core protein is present in A~ plaques in the cerebral cortex, but is absent in the cerebeUar cortex. The observations that HSPG is not present in diffuse plaques of cerebellum but present in neocortex and that HSPG is present in the amyloid cored plaques in the cerebellum agree with previous studies (Snow et al., 1994a). This supports the hypothesis that HSPG is an essential component required for amyloid fibril formation and persistence in the brain (Snow et al., 1994a,b).

It has been suggested that these A[3 associated pro- reins are involved in the evolution of amyloid plaques (Rozemuller et al., 1989a; Eikelenboom et al., 1991; Selkoe et al., 1991). Recent studies have shown that Clq (Webster et al., 1994), %-antichymotrypsin (Ma et al., 1994), apolipoprotein E (Ma et al., 1994), and HSPG (Snow et al., 1994a,b) stimulate A~ fibrillogen- esis, while it is suggested that ApoJ (clusterin, SP40, 40) may prevent A~ peptide from deposition. In this study, we have shown that Clq, 0~I-ACT, ApoE are all present in diffuse plaques in the cerebral neocortex in AD. Such findings indicate that there is no single A[3 associated protein responsible for A[3 fibrillogenesis. A[3 associated proteins may therefore act as a complex network, which, in AD, have a net effect that may lead to amyloid formation.

Although little information is available about poss- ible interactions between the A~ associated proteins, interactions between HSPG and ApoE or P component have recently been reported (Ji et al., 1994). The net effect of the A~ associated proteins may depend on the particular balance that might exist between some com- ponents which maintain solubility of the A~ peptide, and other components which promote amyloid fibril formation. Differences in distribution of the A~ asso- ciated proteins may determine the region-specific occurence of A[~ fibril formation and neurofibrillary pathology.

Acknowledgements

We thank Ms Thea Tadema, Ms Ineke de Moor and Ms Ingrid Janssen for technical assistance. Brain t~ssue was obtained from the Nether lands Brain Bank, Amsterdam (coordinator Dr R. David). We thank Drs J.H.M. Berden and J. van den

Born for generously providing the HSPG antibodies and their helpful comments dur ing the preparation of this manuscript.

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