Heparan Sulfate Proteoglycan in Diffuse Plaques of ... - Europe PMC

American Journal of Pathology, Vol. 144, No. 2, February 1994 Copyright © American Society for Invcstigative Pathology

Heparan Sulfate Proteoglycan in Diffuse Plaques of Hippocampus but Not of Cerebellum in Alzheimer's Disease Brain

Alan D. Snow,* Raymond T. Sekiguchi,* David Nochlin,* Rajesh N. Kalaria,t and Koji Kimatat From the Department of Pathology,* Neuropathology

Laboratories, University of Washington, Seattle, Washington; Department of Neurology,t Case Western University School of Medicine, Cleveland, Ohio; and the Institute for Molecular Science of Medicine,* Aichi Medical University, Yazako Nagakute, Aichi, Japan

Previous studies have shown the basement membrane form of heparan sulfate proteoglycan (HSPG) known as perlecan, co-localized to 3-amyloid protein (A (3)-containing amyloid deposits in brains ofpatients with Alzbeimer's disease (AD) and Down's syndrome. Although HSPG was localized to diffuseA( 6plaques in hippocampus, amygdala, and neocortex, it is not known whether they are present in diffuseA A3 plaques in cerebellum. In the present study, Alcian blue staining and immunocytochemical techniques were used to determine whether highly sulfated glycosaminoglycans (GAGs) and/or HSPG (perlecan) were also present in diffuse A 13 plaques of cerebelum. Tissues from cases ofAD were examined for the co-localization of highly sulfated GAGs, HSPGs, and A 3 in diffuse plaques in cerebellum in comparison with hippocampus. Consecutive serial sections of AD brain tissue were stained or immunostained with 1) the modified Bielschowsky stain,i 2) a polyclonal antibody directed against synthetic A 36(1-40); 3) Congo red, 4) Alcian blue (pH 5.7) with varying concentrations of magnesium chloride for identification of sulfated and highly sulfated GAGs; and 5) polyclonal and monoclonal antibodies recognizing either the core protein or a specific GAG epitope on perlecan. AUlcases (7of 7) ofAD contained diffuse AjBplaques in the cerebelum as identified bypositive Bielschowsky staining and A 1 immunoreactivity. None of the cases demonstratedpositiveAl-

cian blue staining (at 0.3 and 0. 7 mol/L MgCl2), HSPG, or HS GAG immunoreactivity in the same diffuse cerebellarplaques on adjacent serial sections. However, Alcian blue staining, HSPG, and/or HS GAG immunoreactivity were observed in blood vessel walls, choroid plexus, and within Purkinje cells, suggesting that the techniques used were reliable and specific. In cerebelum, aUl plaques containing amyloid cores that were Congo red-positive were also positive for highly sulfated GAGs (byAlcian blue staining at 0. 7mol/L MgCI) and HSPG (both core protein and GAG chain) immunoreactivity. Even though HSPG immunoreactivity was not present in cerebeUar diffuse plaques, aUl cases (4 of 4) examined demonstrated HSPG (both core protein and GAG chain) immunoreactivity in diffuse A (3 plaques in hippocampus. Therefore, by Alcian blue staining and immunocytochemical methods, highly sulfated GAGs and HSPGs are not present in Af3 diffuse plaques in cerebellum. Since previous studies indicate that the cerebelum contains relatively few amyloid-containing plaques in comparison with diffuse plaques, these studies suggest that HSPG may be an essential component neededfor amyloidformation and/orpersistence in brain as observed in cortical areas. The presence of HSPG in diffuse plaques in hippocampus but not in cerebellum may explain why amyloid-containing plaques in hippocampus may be selectively formed orpersist in comparison with cerebelum. Furthermore, the lack of a neuritic response in cerebellar diffuse plaques may also be due to the lack ofHSPGs, which have been shown to play a role in potentiating neurite outgrowth. (Am J Pathol 1994, 144:33 7-34 7) Supported by the Alzheimer's Disease Research Program of the American Health Assistance Foundation, NIH grants AG05136, AG10030, and AG08012, and the Seikagaku Corporation. Accepted for publication October 18, 1993. Address reprint requests to Dr. Alan D. Snow, University of Washington, Department of Pathology, Neuropathology Labs RJ05, Seattle, WA 98195.

337

338

Snow et al

AJP Febnuary 1994, Vol. 144, No. 2

The primary protein component present within the amyloid deposits in the brains of individuals with Alzheimer's disease (AD) is a 39- to 43-amino-acid peptide termed the ,3-amyloid protein,1 2 A4,3 /A4,4 or AP.5 This small peptide is a major component within the amyloid deposits of neuritic plaques and in the walls of blood vessels (ie, congophilic angiopathy) in AD brain, and is derived from larger precursor molecules (termed amyloid precursor proteins or fPPs) by an unknown pathogenic mechanism. f3PPs have been demonstrated to be normally processed in constitutive secretory69 and endosomallysosomal10-12 pathways. In the secretory pathway, previous studies indicated that during normal PP catabolism, the intact amyloidogenic fragment containing A,B was not generated. This was thought to be due to proteolytic cleavage at or near position 612 of I3PP (Lys-16 of AP3), resulting in the release of a soluble secreted protein of undefined function. 13,14 In the endosomal-lysosomal pathway, a complex set of carboxyl-terminal derivatives were identified, including potential amyloidogenic forms.12'15 These latter studies also initially suggested that altered PP processing was required to release the AP3 domain. However, more recent studies suggest that AP is produced by cultured cells during normal metabolism,16'17 implying that A,B accumulation in AD brain may be more dependent on post-translational modifications of the peptide and factors or co-associated macromolecules that may be important in affecting its removal, solubility, and/or production. The studies described above therefore implied that other amyloid components may be necessary and involved in post-translational modifications of PP, ultimately leading to the accumulation of the AP3 fragment. One such component may be the basement membrane form of the heparan sulfate proteoglycan (HSPG) known as perlecan. Previous studies have shown that this specific PG accumulates and colocalizes to the amyloid deposits containing Af in neuritic plaques and amyloidotic blood vessels in AD18-22 and Down's syndrome20 brain. Immunocytochemical18-20 and cationic dye studies23'24 suggest an intimate ultrastructural association between this class of PGs (ie, HSPG) and amyloid fibrils containing AP. In Down's syndrome, HSPG accumulation was specifically associated with the first appearance of diffuse AP cortical deposits in young patients (aged 18-24 years) before the appearance of congophilic amyloid (found at later ages), suggesting an early role for HSPG in amyloid fibril formation, accumulation, and/or persistence.20 Recent studies also demonstrated a high binding affinity between perlecan and

Af,` as well as to the different isoforms of ppR26 The high affinity binding of HSPG to A,B is postulated to play an important role in the accumulation of AP and/or stabilization of amyloid deposits once formed. These studies therefore suggest that HSPG accumulation in conjunction with AP3 may be a critical event in the pathogenesis of AP3 amyloidosis. Although both amyloid core-containing neuritic plaques and diffuse plaques both exist in AD brain in regions such as hippocampus, amygdala, and neocortex, the majority of Af3 deposits in the cerebellum are primarily in a diffuse nonfibrillar form.27-31 The cerebellar diffuse plaques are primarily located in the molecular layer of cerebellar cortex and apparently contain no dystrophic neurites,28 32 in contrast to the presence of neurites surrounding amyloid-containing plaques in other areas of AD brain. It is not known why diffuse plaques primarily exist in cerebellum, but other factors may be necessary to cause the ultimate formation, accumulation, and/or persistence of AP in vivo into a fibrillar structure. One such factor as discussed above may be the presence and/or accumulation of HSPG in conjunction with A,B and/or its precursors. Therefore, in the present study, we used Alcian blue staining and immunocytochemical techniques to test the hypothesis that the relative lack of amyloid-containing plaques in cerebellum correlates with an absence of highly sulfated glycosaminoglycans (GAGs) and/or HSPG (ie, perlecan) in diffuse AP3 plaques in this region.

Materials and Methods

Autopsy Material Cerebellum obtained within 6 hours after death from seven selected cases of AD (confirmed at autopsy by neuropathological diagnosis) was used in the present study. These cases were selected by demonstrating numerous diffuse cerebellar plaques using Bielschowsky stain. Four of these seven cases also included the entire hippocampus and parahippocampal gyrus to allow comparison of the distribution of AP, sulfated GAGs, and HSPGs in diffuse plaques of hippocampus versus cerebellum. The brain tissue obtained included patients in the range of 60 to 95 years of age. These tissues were obtained from cases of rapid autopsy (usually 2-6 hours postmortem) at the University of Washington Alzheimer's Disease Research Center and the Case Western University School of Medicine.

Selective Distribution of HSPG in Diffuse Plaques 339 AJP February 1994, Vol. 144, No. 2

Fixation and Processing of Tissue Four of the seven AD brain tissues obtained at rapid autopsy were fixed in either 10% formalin for 24 hours or Carnoy's solution for 4 hours33 before routine paraffin embedding. From each block of paraffin-embedded material, 6-,u serial sections were cut and placed on gelatin-coated slides. Three of the seven cases were snap-frozen unfixed for immunocytochemical studies. Sections measuring 10 p were cut on a cryostat and immunostained after 10-minute acetone fixation.

Alcian Blue Staining Histochemical staining of sulfated and highly sulfated GAGs was accomplished using the Alcian blue method at pH 5.7 with 0.3 mol/L or 0.7 mol/L magnesium chloride (MgCI2).3435 At a concentration of 0.3 mol/L MgCI2, all sulfated GAGs stain (ie, non-sulfated hyaluronic acid does not stain), whereas at a concentration of 0.7 mol/L, only heparan sulfate, heparin, and keratan sulfate stain.

Staining and Immunocytochemistry In addition to Alcian blue staining as described above, in each case (either cerebellum or hippocampus), consecutive serial sections were stained or immunostained as follows: 1) modified Bielschowsky stain36 to demonstrate both diffuse and neuritic plaques; 2) a polyclonal antibody to synthetic A,B(1-40) (generous gift of Dr. C. Masters) (1:400 dilution) to demonstrate localization of A,B; 3) Congo red staining as viewed under polarized light37 to demonstrate fibrillar amyloid; 4) a polyclonal antibody (1:50 dilution) to the core protein of HSPG38; 5) a monoclonal antibody (HK-102) (undiluted hybridoma supernatant) to the core protein of HSPG39; and 6) a monoclonal antibody (HK-249) (undiluted hybridoma supernatant) to a specific GAG chain epitope (glucosamine sulfate a 1 >4 glucuronic acid determinant) in HSPG isolated from the Engelbreth-Holm-Swarm tumor.20'40 Immunostaining of tissue sections was accomplished using the avidin-biotin-immunoperoxidase method, using the appropriate biotin-labeled secondary antibodies, followed by incubation with avidin-conjugated horseradish peroxidase complex (Vector Laboratories, Burlingame, CA). Peroxidase activity was produced by treatment with 3,3diaminobenzidine as previously described. 18 For immunocytochemical staining, the primary antibody

was used initially through a series of dilutions to obtain the best specificity with the least background staining. Only the optimal dilutions of primary antibody are reported. In all cases using the HSPG core protein or GAG chain antibodies, sections were also pretreated for 5 minutes with 88% formic acid before immunostaining.41 In most instances, tissue sections after immunostaining were counterstained with hematoxylin.

Results Lack of Sulfated GAGs in Cerebellar Diffuse Plaques of Alzheimer's Disease Brain Seven cases of confirmed AD were analyzed for the presence of AP, sulfated GAGs (using Alcian blue staining in the presence of MgCI2), and HSPGs in diffuse plaques of the cerebellum. Both paraffinembedded (four cases) and frozen tissue (three cases) were used to ensure that lack of Alcian blue staining and/or HSPG immunostaining was not due to an artifact of fixation. As shown in Figures 1A and 2A, modified Bielschowsky silver staining clearly demonstrated diffuse plaques in the molecular layer of the cerebellum. On adjacent serial sections, these diffuse plaques were positive with A,B(1-40) antibodies (Figures 1B and 2B). These diffuse plaques were negative for Congo red (not shown) on adjacent serial sections, as previously demonstrated.27 31 In all seven cases, diffuse plaques demonstrated by Bielschowsky staining and A,B immunoreactivity were negative for sulfated GAGs as shown by lack of Alcian blue staining with 0.3 mol/L MgCI2 (Figure 1C). However, positive Alcian blue staining (with 0.3 mol/L MgCI2) was observed in the same tissue sections in the walls of blood vessels (primarily medium and large vessels lying outside the cerebellum) (Figure 1 D), in choroid plexus (Figure 1E), and within Purkinje cells (Figure 1F), indicating that this staining technique was reliable and effective in specific localization of highly sulfated GAGs.

Lack of Highly Sulfated GAGs and HSPG Immunoreactivity in Cerebellar Diffuse Plaques of Alzheimer's Disease Brain Diffuse cerebellar AP deposits identified by positive Bielschowsky staining (Figure 2A) and AP3 immunoreactivity (Figure 2B) were found to be also negative

340

Snow et al

A/P Febnuary 1994, Vol. 144, No. 2

Figure 1. Lack ofsulfated GAGs in cerebellar diffuse plaques. Tissue obtainedfrom a 95-year-old womani with AD. A: Modified Bielschousky sib er in the molecular layer. B: Conscucltive serial section from A demonstrating Ap(1-40) immunostaining (arrouheads) in difftise cerebellar plaques. These diffuise plaque-s were Conzgo red negative on the adjacent serial section (not shown). Avidin-biotin-immunoperoxidase method. C: Serial section from B demonstrating Alcian bluie staining with 0.3 mol/L MgCGI2. No positive Alcian blue stainiing is observed in diffuse cerebellar plaques, suiggesting the lack of sulfated GAGs in these deposits. D: High magnification of' positive Alcian bluie staining uith 0.3 mol/L MgCI2 in blood tessel u'all (arrows) from the same tissue (as in A-C), inidicatintg specific localizationi oj' sulfated GAGs in blood vessel u'all. Most medium and large-sized blood vessels lying ouitside the cerebellum nvere positive. No positii'e Alcianl blue staining with 0.3 mol/L MgCGU was observed in small vessels of the cerebellar parenchyma. E: Positive Alciant blue staining u'ith 0.3 mol/L MgCG, in the choroid plexus basement membranie (arrou'heads) from the same tissue (as in A-D), suggesting the presenice of sulfated GAGs. F: Positii'e Alcian blue staininig wvith O.3 mnollL MgCGU of Purkinje cells (arrou,heads) from the same tissue (as in A-E), indicating the presence of stulfated GAGs.

stainiing of cerebelluim demonstrating diffuse plaques (arrouheads)

Scale bars

=

.O. 100

serial sections for highly sulfated GAGs as demonstrated by Alcian blue staining (at 0.7 mol/L MgCl2) (Figure 2C). These cerebellar diffuse deposits were also negative for HSPG core protein immu-

on

noreactivity when using either a monoclonal antibody (HK-102) (Figure 2D) or a polyclonal antibody (not shown) directed against the core protein of the HSPG. Similar lack of HS GAG chain (HK-249,

9;ti:lXS,!.EUF'_[m1I85

Selective Distribution of HSPG in Diffuse Plaques 341 AJP February 1994, Vol. 144, No. 2

V

I

Figure 2. Lack of highly sulfated GAGs and HSPG core protein immunostaining in cerebellar diffuse plaques. Tissue obtained from of a 75-yearold man with AD. A: Modified Bielchowsky silver staining of cerebellum demonstrating diffuse plaques (arrowheads) in the molecular layer. B: Consecutive serial section from A demonstrating A/3(1-40) immunostaining (arrowheads) in diffuse cerebellar plaques. Avidin-biotinimmunoperoxidase method. C: Serial section demonstrating lack of Alcian blue staining with 0. 7 mol/L MgCl2 in diffuse cerebellar plaques (compare with Figuire 2, A and B). Positive staining of blood vessels (arrows) and Purkinje cells (arrowheads) indicates specific staining for highly sulfated GAGs in these locales. D: Serial section demonstrating lack of HSPG core protein (HK-102, monoclonal) immunostaining in diffuse plaqtes in the molecular layer of the cerebellum (compare uvith Figure 2, A and B). Similar lack of HSPG core protein immunostaining in cerebellar diffuse plaques was observed using the HSPG polyclonal antibody. Formic acid pretreatment; avidin-biotin-immunoperoxidase method. E: Positive HSPG core protein (HK-102, monoclonal) immunostaining in the wall of a large blood vessel (arrows) lying outside the cerebellum indicates that the antibody is active. Formic acid pretreatment; avidin-biotin-immunoperoxidase method. F: Positive HSPG core protein (HK-102, monoclonal) immunostaining of choroid plexus basement membrane (arrows) further indicates that the antibody is active. Scale bars = 100 p.

monoclonal) immunoreactivity was observed in diffuse cerebellar plaques (Figure 3E). However, HSPG core protein and HS GAG chain immunoreactivity were similarly observed in the walls of largeand medium-sized blood vessels, lying primarily

outside the cerebellum (Figure 2E) as previously described42 and in the choroid plexus basement membrane (Figure 2F), indicating that the antibodies were active. Essentially identical results showing a lack of HS GAG or HSPG core protein were

342

Snow et al

AJP February 1994, Vol. 144, No. 2

obtained in the absence or presence of formic acid '4 | | i 1 1 1 = pretreatment.41

Presence of HSPG Immunoreactivity in

Amyloid Plaque Cores in Cerebellum in Alzheimer's Disease Brain Both Alcian blue staining and HSPG immunoreactivity indicated the lack of highly sulfated GAGs and HSPGs in cerebellar diffuse plaques. However, ;,.g, the same cerebellar tissues, occasional .,;,,,within plaques were observed containing an amyloid core. These amyloid core-containing plaques were identified by positive Bielschowsky staining (Figure 3, A _ and D), AP immunoreactivity (not shown), and Congo red birefringence (as observed under polarized light) (not shown). Positive staining of the amyloid cores in these plaques with Alcian blue at 0.7 mol/L MgCl2 (Figure 3B) indicated the presence of sulfated GAGs. This was confirmed by posi~~~~~~~~~~~highly tive HSPG immunoreactivity using antibodies dii ; ir..l rected against either the HSPG core protein (Figure 30) or HS GAG chains (Figure 3E). Both Alcian blue staining and HSPG immunostaining clearly indi~~~ ~~~~ ~cated that the diffuse deposits identified by Bielschowsky staining in the vicinity of these amyloid cores were again negative, indicating that HSPGs _ were primarily localized to amyloid deposits in a fit : X : ~ . ,brillar form in the cerebellum.

.....

B

_ '' e '.

V

r ..

Heparan Sulfate GAGs and Core Protein ..in

Hippocampal Diffuse Plaques of

Alzheimer's Disease Brain

Z. "I.,

Four of the seven cases were also analyzed for the presence of HSPG immunostaining in diffuse and amyloid-containing plaques in hippocampus. HS GAG (HK-249 antibody) (Figure 4, A-C) and HSPG core protein (Figure 4, D-F) immunoreactivity were

~~~~~~

lm demonstrating diffuse deposits (arrows) and an amyloid plaque ,...~~~~~coe (arrowvhead) in the molecuilar layer. B: Serial section demon. ~~~strating positive Alcian blue staining (with 0. 7 mol MgCU) of the amyvloid core (arrowhead) indicating the presence of highly sulfated Note lack of Alcian blue staining of diffuse deposits as shown ~~~~~~~~~~GAGs.

4 ....

_a

,'.m'.; ',"''!!

jm fl-

'8 '@ gj t

in A. C: Serial section demonstrating positive HSPG core protein (HK-

102, monoclonal) immunostaining in amyloid plaque core (arrow-

X 40. D: Bielschowsky staining in cerebellum (molecular h~~~~~~~~~~ead). layer) in a 95- yerold woman with AD. Note the presence of two amyvloid plaque cores (arrowheads) and dfftise silver-stainied depos

..

y.-jj

~~~~~Figure 3. Alciani blue .staining and HSPG immtinoreactivity in am~y loid plaquie cores in cerebellum. Tissue obtained from of a 75-yearold man with AD. A: Modified Bielchousky silver staining of cerebel-

.

-.^;

- ts. E: Serial section from D demonstrating positive HS GAG chain immunostaining (HK-249, monoclonaf) of the tuo atmyloid plaqte cores (arrowheads). Note lack of HS GAG immunoreactiviUy in the diffiase deposits shouwn in D. Scale bar = 100 I.

Selective Distribution of HSPG in Diffuse Plaques

343


Figure 4. Heparan sulfate GAG chain and proteog yvcan core protein immunostaining in diffuse and amyloid plaques in hippocampus of Alzbeimer's disease braini. A: HS GAG chain (HK-249, monoclonal) immunostaining of amyloid core containing plaques (arrows) in hippocampus of a 67-year-old man uwith Al). Formic acid pretreatment; aiidin-biotim -immunoperoxidase method. B: HS GAG chain (HK-249, monoclonal) immunostainzing oJ nleuitic components (arrouwheads) in hippocampuis of a 64-year-old man with AD. Formic acid pretreatment; avidini-biotinimmuiinoperoxiclase method. C: HS GAG chaini (HK-249, monioclonal) immutnostaining of amyloid and diffitse plaques (arrowheads) in hippocamplus fromn the same 95-year-old woman with AD shouwn in Figutre 1. Formic acid pretreatment; avidin-biotin-immunoperoxidase method. D-F: Hippocamnpus oJ the samtie 75-year-old man with AD shown in Figutre 2. D: HSPG core protein (HK-102, monoclonal) immunostaining of amyloid core containing plaques (arrous) in hippocamnputs. Formiic acid pretreatment; avidin-biotin-immunoperoxidase method. E: HSPG core protein (HK-102, monoclonal) immnitiostaininig of an amnyloid (arrow) anid neuittic (arrowheads) containing plaque. Formic acid pretreatment; acidinbiotin-immunoperoxidase miethod. F: HSPG core proteini (HK-102, nmonioclonial) immuinostaining of dciffuse (arrowheads) plaques in hippocampuis.

Formnic acid pretreatment; avidin-biotin-immunoperoxidase miiethod. Scale bars

observed in the hippocampus in all cases analyzed. HS GAG chain (HK-249) immunoreactivity was localized to the amyloid cores (Figure 4A) and neuritic components of plaques (Figure 4B). Additionally, diffuse plaques that were A,P immunoreactive and Congo red negative (not shown) demonstrated HS GAG (Figure 4C) immunoreactivity. Similar results were observed using the HSPG core protein

=

100 y.

antibodies with positive immunostaining of amyloid cores (Figure 4D), neuritic components (Figure 4E), and diffuse plaques (Figure 4F). All four cases that demonstrated lack of HSPG immunoreactivity in diffuse plaques of cerebellum showed positive immunostaining of diffuse plaques in hippocampus (for example, compare Figures 3E and 4C, which were obtained from the same patients).

344

Snow et al


Discussion The results from the present study suggest the absence of highly sulfated GAGs and HSPG in AP cerebellar diffuse plaques in AD brain. It is possible that the lack of Alcian blue staining for highly sulfated GAGs or HSPG and HS GAG chain immunoreactivity in diffuse cerebellar plaques may be due to masking of HSPG or HS GAG antigenicity due to other protein components. However, both formic acid pretreatment (used to unmask blocked antigenic sites)41'43 and use of frozen sections (to preserve antigenicity) did not seem to alter the results. Furthermore, positive Alcian blue staining, HSPG, and HS GAG immunoreactivity were observed in specific areas of the same tissues known to contain highly sulfated GAGs and HSPG (ie, blood vessel walls, choroid plexus, Purkinje cells), suggesting that the staining and immunocytochemical techniques employed in this study were reliable and effective. This study also suggests that the selective distribution of AP3 in a fibrillar amyloid form in brain may ultimately be due to the presence or absence of HSPGs. Although highly sulfated GAGs, HSPGs, and HS GAGs were not found in diffuse plaques of cerebellum, they were present in diffuse plaques of hippocampus in the same AD patients. Furthermore, the few plaques in cerebellum that contained amyloid cores were positive for highly sulfated GAGs (Alcian blue staining at 0.7 mol/L MgCl2) and HSPG immunoreactivity. Since few amyloid containing plaques are observed in AD cerebellum in comparison with hippocampus, we postulate that this may be due to the relative lack of HSPG in these AP3 deposits. A previous study has demonstrated that heparan sulfate (and not heparin or chondroitin-6-sulfate) influences the specific AA amyloid precursor known as SAA2 to form a predominant ,8-pleated sheet structure.44 This study implicates heparan sulfate in conformational alterations of amyloidogenic precursors which may be important in the ultimate formation of the 3-pleated sheet structure characteristic of all amyloids. Recently, Fraser et al.45 found that the sulfate moieties present on GAGs (the carbohydrate component of PGs) caused various Af3 peptides derived from the AP3 amyloid protein of AD to undergo extensive lateral aggregation and axial growth. These latter studies implicate that the presence of HSPG in AP deposits may provide the nucleation events needed for in vivo facilitation of protein conformations that induce fibril formation,

stabilize preexisting fibrils, and/or decrease amyloid susceptibility to proteolysis. Additionally, Snow et al.46'47 demonstrated that coinfusions of AP3 and HSPG (perlecan) into rat brain or endogenous accumulation of HSPG at A,B sites when infused alone causes consistent deposition and persistence of AP3 in a fibrillar form. These latter studies suggest that HSPG may not only play an important role in amyloidogenesis but may also play a role in stabilization of the fibrillar A,B amyloid deposits once formed. Although the present investigation suggests that highly sulfated GAGs and HSPG are lacking in cerebellar diffuse plaques, it is possible that other plaque components may also be absent. However, two primary components of AP3 amyloid deposits, a1-antichymotrypsin46 and P component49-51 appear to be present in cerebellar diffuse plaques. Although an initial study by Rozemuuler et al.52 implied that P component is not present in diffuse cerebellar plaques, recent studies by Kalaria et al.53 indicate that amyloid P component is present in diffuse cerebellar deposits. Additionally, Shoji et al.54 clearly demonstrated a1-antichymotrypsin inmunoreactivity in diffuse plaques in cerebral cortex and cerebellum of AD patients. These studies imply that P component and a1-antichymotrypsin may not have a direct role in AP fibrillogenesis in vivo and/or the persistence of AP3 in a fibrillar form in brain. It is also known that cerebellar diffuse plaques contain no dystrophic neurites and are not recognized by antibodies to neurofilaments, tau, and paired helical filaments,28 all which detect dystrophic neurites in cerebral cortical neuritic plaques. If one assumes that the diffuse AP3 plaques eventually form neuritic plaques containing a central amyloid core as observed in brains of Down's syndrome patients of increasing ages, 20 then what prevents further progression of diffuse plaques in cerebellum to neuritic plaques? Based on the present study, we postulate that the apparent absence of neuritic plaques and a neuritic response in cerebellar molecular cortex is due to the absence of HSPG in these deposits. Previous studies have clearly demonstrated that HSPG and its carbohydrate components are important inducers of neuritic outgrowth.55-0 The association of HS GAGs with neurites in AD hippocampus implicates these macromolecules as potential key components in neuritic response in vivo. Additionally, HSPGs known ability to bind to certain growth factors such as basic fibroblast growth factor61 62 may be important for the accumulation of growth factors at sites of A,B-HSPG complexes such as observed in the neuritic plaque.


345

AJP Febnuary 1994, Vol. 144, No. 2

References 1. Glenner GG, Wong CW: Alzheimer's disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res Commun 1984, 120:885-890 2. Wong CW, Quaranta V, Glenner GG: Neuritic plaques and cerebrovascular amyloid in Alzheimer's disease are antigenically related. Proc Natl Acad Sci USA 1985, 82:8729-8732 3. Masters CL, Simms G, Weinman NA, Multhaup G, McDonald BL, Beyreuther K: Amyloid plaque core protein in Alzheimer's disease and Down's syndrome. Proc Natl Acad Sci USA 1985, 82:4245-4249 4. Beyreuther K, Masters CL: Nomenclature of amyloid A4 proteins and their precursors in Alzheimer's disease and Down's syndrome. Neurobiol Aging 1990, 11:66-68 5. Husby G, Araki S, Benditt EP, Benson MD, Cohen AS, Frangione B, Glenner GG, Natvig JB, Westermark P: Nomenclature of amyloid and amyloidosis. Bull WHO 1993, 71:105-108 6. Palmert MR, Podlisny MB, Witker DS, Oltersdorf T, Younkin LH, Selkoe DJ, Younkin SG: The 3-amyloid protein precursor of Alzheimer disease has soluble derivatives found in human brain and cerebrospinal fluid. Proc Natl Acad Sci USA 1989, 86:6338-6342 7. Palmert MR, Siedlak SL, Podlisny MB, Greenberg B, Shelton ER, Chan HW, Usiak M, Selkoe DJ, Perry G, Younkin SG: Soluble derivatives of the 1 amyloid protein precursor of Alzheimer's disease are labelled by antisera to the 1 amyloid protein. Biochem Biophys Res Commun 1989, 165:182-188 8. Schubert D, LaCorbiere M, Saitoh T, Cole G: Characterization of an amyloid 1 precursor protein that binds heparin and contains tyrosine sulfate. Proc Natl Acad Sci USA 1989, 86:2066-2069 9. Weidemann A, Konig G, Bunke P, Fisher P, Salbaum JM, Masters CL, Beyreuther K: Identification, biogenesis and localization of precursors of Alzheimer's disease A4 amyloid protein. Cell 1989, 57:115-126 10. Cole GM, Huynh TV, Saitoh T: Evidence for lysosomal processing of amyloid 13-protein precursor in cultured cells. Neurochem Res 1989, 14:933-939 11. Caporaso GL, Gandy SE, Buxbaum JD, Greengard P: Chloroquine inhibits intracellular degradation but not secretion of Alzheimer P/A4 amyloid precursor protein. Proc Natl Acad Sci USA 1992, 89:2252-2256 12. Estus S, Golde TE, Kunishita T, Blades D, Lowery D, Eisen M, Usiak M, Qu X, Tabira T, Greenberg BD, Younkin SG: Potentially amyloidogenic carboxylterminal derivatives of the amyloid protein precursor. Science 1992, 255:726-728 13. Sisodia SS, Koo EH, Beyreuther K, Unterbeck A, Price DL: Evidence that f3-amyloid protein in Alzheimer's disease is not derived from normal processing. Science 1990, 248:492-495

14. Esch FS, Keim PS, Beattie EC, Blancher RW, Culwell AR, Oltersdorf T, McClure D, Ward PJ: Cleavage of amyloid , peptide during constitutive processing of its precursor. Science 1990, 248:1122-1124 15. Golde TE, Estus S, Younkin LH, Selkoe DJ, Younkin SG: Processing of the amyloid protein precursor to potentially amyloidogenic derivatives. Science 1992, 255:728-730 16. Haass C, Schlossmacher MG, Hung AY, Vigo-Pelfrey C, Mellon A, Ostaszewski BL, Lieberburg I, Koo EH, Schenk D, Teplow DB, Selkoe DJ: Amyloid ,B-peptide is produced by cultured cells during normal metabolism. Nature 1992, 359:322-325 17. Shoji M, Golde TE, Ghiso J, Cheung TT, Estus S, Shaffer LM, Cai X, McKay DM, Tintner R, Frangione B, Younkin SG: Production of the Alzheimer f3 protein by normal proteolytic processing. Science 1992, 248: 492-495 18. Snow AD, Mar H, Nochlin D, Kimata K, Kato M, Suzuki S, Hassell H, Wight TN: The presence of heparan sulfate proteoglycans in the neuritic plaques and congophilic angiopathy in Alzheimer's disease. Am J Pathol 1988, 133:456-463 19. Snow AD, Wight TN: Proteoglycans in the pathogenesis of Alzheimer's disease and other amyloidoses. Neurobiol Aging 1989, 10:481-497 20. Snow AD, Mar H, Nochlin D, Sekiguchi RT, Kimata K, Koike Y, Wight TN: Early accumulation of heparan sulfate in neurons and in the f-amyloid protein containing lesions of Alzheimer's disease and Down's syndrome. Am J Pathol 1990, 137:1253-1270 21. Perlmutter LS, Chui HC, Saperia D, Athanikar J: Microangiopathy and the colocalization of heparan sulfate proteoglycan with amyloid in senile plaques of Alzheimer's disease. Brain Res 1990, 508:13-19 22. Su JH, Cummings BJ, Cotman CW: Localization of heparan sulfate glycosaminoglycan and proteoglycan core protein in aged brain and Alzheimer's disease. Neuroscience 1992, 51:801-813 23. Snow AD, Nochlin D, Lara S, Wight TN: Cationic dyes reveal proteoglycans structurally integrated within the characteristic lesions of Alzheimer's disease. Acta Neuropathol 1989, 78:113-123 24. Young ID, Willmer JP, Kisilevsky R: The ultrastructural localization of sulfated proteoglycans is identical in the amyloids of Alzheimer's disease and AA, AL, senile cardiac and medullary carcinoma associated amyloidosis. Acta Neuropathol 1989, 78:202-209 25. Snow AD, Kinsella MG, Prather PB, Nochlin D, Podlisny MB, Selkoe DJ, Kisilevsky R, Hassell JR, Wight TN: A characteristic binding affinity between heparan sulfate proteoglycans and the A4 amyloid protein of Alzheimer's disease. J Neuropathol Exp Neurol 1989, 48:352 (abstract) 26. Narindrasorasak S, Lowery D, Gonzalez-DeWhitt P, Poorman RA, Greenberg B, Kisilevsky R: High affinity

346

Snow et al


27.

28.

29.

30.

31.

32.

33. 34.

35. 36.

37.

38.

39.

40.

41.

interactions between the Alzheimer's ,B-amyloid precursor proteins and the basement membrane form of heparan sulfate proteoglycan. J Biol Chem 1991, 266: 12878-12883 Braak H, Braak E, Bohl J, Lang W: Alzheimer's disease: amyloid plaques in the cerebellum. J Neurol Sci 1989, 93:277-287 Joachim CL, Morris JH, Selkoe DJ: Diffuse senile plaques occur commonly in the cerebellum in Alzheimer's disease. Am J Pathol 1989, 135:309-319 Yamaguchi H, Hirai S, Morimatsu M, Shoji M, Nakazato Y: Diffuse type of senile plaques in the cerebellum of Alzheimer-type dementia demonstrated by ,B protein immunostain. Acta Neuropathol 1989, 77:314319 Yamaguchi H, Nakazato Y, Hirai S, Shoji M: Immunoelectron microscopic localization of amyloid ( protein in the diffuse plaques of Alzheimer type dementia. Brain Res 1990, 508:320-324 Yamaguchi H, Nakazato Y, Shoji M, Takatama M, Hirai S: Ultrastructure of diffuse plaques in senile dementia of the Alzheimer type: comparison with primitive plaques. Acta Neuropathol 1991, 82:13-20 Rozemuller JM, Stam FC, Eikelenboom P: Acute phase proteins are present in amorphous plaques in the cerebral but not in the cerebellar cortex of patients with Alzheimer's disease. Neurosci Lett 1990, 119: 75-78 Gatenby J, Cowdry EV: Microtomist's Vade-mecum. Philadelphia, P. Blakiston's Son & Co, 1929, p 53 Scott JE, Dorling J: Differential staining of acid glycosaminoglycans (mucopolysaccharides) by Alcian blue in salt solutions. Histochemie 1965, 5:221-233 Dorling J: Critical electrolyte concentration method in histochemistry. J Med Lab Technol 1969, 26:124-130 Yamamoto T, Hirano A: A comparative study of modified Bielchowsky, Bodian and thioflavin S stains on Alzheimer's neurofibrillary tangles. Neuropathol Appl Neurobiol 1986, 12:3-9 Puchtler H, Sweat F, Levine M: On the binding of Congo red by amyloid. J Histochem Cytochem 1962, 10:355-364 Hassell JR, Robey PG, Barrach H, Wilczek J, Rennard SI, Martin GR: Isolation of a heparan sulfatecontaining proteoglycan from basement membrane. Proc Natl Acad Sci USA 1980, 77:4494-4498 Kato M, Koike Y, Suzuki S, Kimata K: Basement membrane proteoglycan in various tissues: characterization using monoclonal antibodies to the EngelbrethHolm-Swarm mouse tumor low density heparan sulfate proteoglycan. J Cell Biol 1988, 106:2203-2210 Koike Y, Kato M, Suzuki S, Kimata K: A monoclonal antibody against the heparan sulfate of EHS-tumor proteoglycan. IX International Symposium on Glycoconjugates, 1987, p B8 (abstract) Kitamoto T, Ogomori K, Tateishi K, Prusiner SB: Formic acid pretreatment enhances immunostaining of cere-

42.

43.

44.

45.

46.

47.

48.

49.

50.

51.

52.

53.

54.

bral and systemic amyloids. Lab Invest 1987, 57:230236 Snow AD, Wight TN, Nochlin D, Koike Y, Kimata K, DeArmond SJ, Prusiner SB: Immunolocalization of heparan sulfate proteoglycans to the prion protein amyloid plaques of Gerstmann-Straussler syndrome, Creutzfeldt-Jakob disease and scrapie. Lab Invest 1990, 63:601-611 Cammarata S, Mancardi G, Tabaton M: Formic acid treatment exposes hidden neurofilament and tau epitopes in abnormal cytoskeletal filaments from patients with progressive nuclear palsy and Alzheimer's disease. Neurosci Lett 1990, 115:351-355 McCubbin WD, Kay CM, Nadrindrasorasak S, Kisilevsky R: Circular dichroism and fluorescence studies on two murine serum amyloid A proteins. Biochem J 1988, 256:775-783 Fraser PE, Nguyen JT, Chin DT, Kirschner DA: Effects of sulfate ions on Alzheimer f/A4 peptide assemblies: implications for amyloid fibril-proteoglycan interactions. J Neurochem 1992, 59:1531-1540 Snow AD, Sekiguchi RT, Nochlin D, Kimata K, Schreier WA, Morgan DG: Brain amyloid accumulation in rats within 1 week of continuous infusion of 3-amyloid protein (1-40) and an amyloid plaque co-component. Neurobiol Aging 1992, 13:S101 (abstract) Snow AD, Sekiguchi RT, Nochlin D, Kimata K, Schreier WA, Morgan DG: A rat model to study the effects of BAP-containing amyloid in brain. Soc Neurosci Abstr 1992, 18:1465 Abraham R, Selkoe DJ, Potter H: Immunochemical identification of the serine protease inhibitor a1antichymotrypsin in the brain amyloid deposits of Alzheimer's disease. Cell 1988, 52:487-492 Coria F, Castano E, Prelli F, Larrondo-Lillo M, Van Duinen S, Shelanski ML, Frangione B: Isolation and characterization of amyloid P component from Alzheimer's disease and other types of cerebral amyloidosis. Lab Invest 1988, 58:454-458 Holck M, Husby G, Sletten K, Natvig JB: The amyloid P component (protein AP): an integral part of the amyloid substance? Scand J Immunol 1979, 10:55-60 Kalaria RN, Galloway PG, Perry G: Widespread serum amyloid P immunoreactivity in cortical amyloid deposits and the neurofibrillary pathology of Alzheimer's disease and other degenerative disorders. Neuropathol Appl Neurobiol 1991, 17:189-201 Rozemuller JM, Stam FC, Eikelenboom P: Acute phase proteins are present in amorphous plaques in the cerebral but not in the cerebellar cortex of patients with Alzheimer's disease. Neurosci Lett 1990, 119: 75-78 Kalaria RN, Perry G: Amyloid P component and other acute-phase proteins associated with cerebellar Af3 deposits in Alzheimer's disease. Br Res 1993, 631: 151-155 Shoji M, Hirai S, Yamaguchi H, Harigaya Y, Ishiguro K,


347


55.

56.

57.

58.

Matsubara E: Alpha 1-antichymotrypsin is present in diffuse senile plaques. A comparative study of f-protein and al-antichymotrypsin immunostaining in the Alzheimer brain. Am J Pathol 1991, 138:247-257 Margolis RK, Salton SRJ, Margolis RU: Effects of nerve growth factor-induced differentiation on the heparan sulfate of PC 12 pheochromocytoma cells and comparison with developing brain. Biochemistry 1987, 257:107-114 Ard MD, Bunge RP: Heparan sulfate proteoglycan and laminin immunoreactivity on cultured astrocytes: relationship to differentiation and neuritic growth. J Neurosci 1988, 8:2844-2858 Damon DH, D'Amore PA, Wagner JA: Sulfated glycosaminoglycans modify growth factor-induced neurite outgrowth in PC12 cells. J Cell Physiol 1988, 135: 293-300 Katoh-Semba R, Oohira A, Kashiwamata S: Changes

59. 60.

61.

62.

in glycosaminoglycans during the neuritogenesis in PC12 pheochromocytoma cells induced by nerve growth factor. J Neurochem 1990, 55:1749-1757 Riopelle RJ, Dow KE: Functional interactions of neuronal heparan sulfate proteoglycans with laminin. Brain Res 1990, 525:92-100 Schwarz MA, Mitchell M, Emerson DL: Reconstituted basement membrane enhances neurite outgrowth in PC 12 cells induced by nerve growth factor. Cell Diff 1990, 1:313-318 Raprager AC, Krufka A, Owlin BB: Requirement of heparan sulfate for bFGF-mediated fibroblast growth and myoblast differentiation. Science 1991, 252:17051708 Turnbull JE, Fernig DG, Ke Y, Wilkinson MC, Gallagher JT: Identification of the basic fibroblast growth factor binding sequence in fibroblast heparan sulfate. J Biol Chem 1992, 267:10337-10341