Neurohiolog3 qfAging. Vol. 10, pp. 510-512. e~ Pergamon Press pie. 1989. Printed m the U,S.A {ll i~-4580/89 Sq011 C~i!

themselves, plays an important role. Probably, interaction be- tween amyloid protein and such component (e.g., formation of a compound) would trigger fibril formation, or high affinity between amyloid protein and such component creates high concentration (significantly higher than the systemic level and enough for fibril formation) of the amyloid protein locally wherever the component located. PGs and GAGs are the highly potential candidate for such component, and indeed the results of recent studies support the possibility (15). Likely possibilities of the role that PGs and GAGs

play in amyloid fibrillogenesis are 1) to act as a catalyzer for polymerization of amyloid proteins, 2) to influence the localiza- tion of amyloid deposis by attracting a certain type of amyloid proteins to the locations where a certain specific type of PGs are present, and 3) to solidify formed amyloid fibrils. If so, PGs and GAGs hold a key for the development of the second phase of amyloidogenesis, and in turn for entire amyloidogenesis since the second phase constitutes one of its two essential and independent phases. Further studies in this field are anxiously awaited.

REFERENCES

t. Cohen, A. S.; Shirahama, T.; Sipe, J. D.; Skinner, M. Amyloid proteins, precursors, mediator, and enhancer. Lab. Invest. 48:1--4; 1983.

2. Connors, L. H.; Shirahama, T.; Skinner, M.; Fenves, A.; Cohen, A. S. In vitro formation of amyloid fibrils from intact [32-microglobulin. Biochem. Biophys. Res. Commun. 131:1063-1068; 1985.

3. Gejyo, F.; Homma, N.; Suzuki, Y.; Arakawa, M. Serum levels of 13z-microglobulin as a new form of amyloid protein in patients undergoing long-term hemodialysis. N. Engl. J. Med. 314:585-586; 1986.

4. Glenner, G. G.; Amyloid deposits and amyloidosis. The 13-fibrillosis. N. Engl. J. Med. 302:1283-1292; 1333-1343; 1980.

5. Glenner, G. G.; Ein, D.; Eanes, E. D.; Bladen, H. A.; Terry, W.; Page, D. L. Creation of "amyloid" fibrils from Bence-Jones proteins in vitro. Science 174:712-714; 1971.

6. Glenner, G. G.; Osserman, E. F.; Benditt, E. P.; Calkins, E.; Cohen, A. S.; Zucker-Franklin, eds. Amyloidosis. New York: Plenum Press; t986.

7. Hardt, F.; Ranlov, P. Transfer amyloidosis. Int. Rev. Exp. Pathol. 16:273-334; 1976.

8. Isobe, T.; Araki, S.; Uchino, F.; Kito, S.; Tsubura, E., eds. Amyloid and amyloidosis. New York: Plenum Press; 1988.

9. Kisilevsky, R. Biology of disease. Amyloidosis: a familiar problem in the light of current pathogenetic developments. Lab. Invest. 49: 381-390; 1983.

10. Marrink, J.; van Rijswijk, M. H., eds. Amyloidosis. Dordrecht, The Netherlands: Martinus Nijhoff; 1986.

II. Shirahama, T. Amyloidogenesis and light chains. In: Minetti, L.; D'Amico, G.; Ponticelli, C., eds. The kidney in plasma cell dyscra- sias. Dordrecht, The Netherlands: Kluwer Academic Publishers; 1988:57-66.

12. Shirahama, T.; Benson, M. D.; Cohen, A. S.; Tanaka, A. Fibrillar assemblage of variable segments of immunoglobulin light chains. An electron microscopic study. J. Immunol. 110:21-30; 1973.

13. Shirahama,T.; Cohen, A. S. High-resolution electron microscopic analysis of the amyloid fibril. J. Cell Biol. 33:679-708; 1967.

14. Shirahama, T.; Cohen, A. S.; Skinner, M. Immunohistochemistry of amyloid. In: DeLellis, R. H., ed. Advances in immunohistochemisry. New York: Masson Publishing; 1984:277-302.

15. Snow, A. D.; Wight, T. N. Proteoglycans in the pathogenesis of Alzheimer's disesase and other amyloidoses. Neurobiol. Aging 10: 481-497; 1989.

16. Teilum, G. Pathogenesis of amyloidosis. The two-phase cellular theory of local secretion. Acta Pathol. Microbiol. Scand. 6t:21-45; 1964.

17. Varga, J.; Flinn, M. S. M.; Shirahama, T.; Rodgers, O. G.; Cohen. A. S. The induction of accelerated murine amyloidosis with human spleen extract. Probable role of amyloid enhancing factor. Virchows Arch. [B] 51:177-185; 1986.

18. Virchow, R. Zur Cellulose-frage. Virchows Arch. 6:416-426; 1954.

Authors' Response to Commentaries

A L A N D. S N O W A N D T H O M A S N. W I G H T

Department of Pathology SM-30, University of Washington, Seattle, WA 98195

EXCELLENT commentaries were provided by our colleagues which included Drs. Caputo, Linker, Kisilevsky, Schubert, Shira- hama, Benditt and Margolis. Each author has expertise in several different areas of amyloid and/or proteoglycan (PG) research, and through their commentaries have added further insight and inquir- ies as to the possible roles that PGs play in the pathogenesis of amyloidosis in general, and in Alzheimer's disease in particular. In this summation, we will address some of the important points conveyed by these authors.

SPECIFIC INVOLVEMENT OF HEPARAN SULFATE PROTEOGLYCANS IN THE PATHOGENESIS OF AMYLOIDOSIS

As effectively discussed by Schubert, a number of different

PGs are most likely synthesized by individual cell types within the central nervous system (CNS). Although there are potentially many different types of PGs and/or GAGs (i.e., heparin, heparan sulfate, chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sul- fate, keratan sulfate and the GAG hyaluronic acid/ that can be synthesized by cells, both within and outside the CNS, evidence suggests that only the heparan sulfate proteoglycan (HSPG) class is localized to a variety of different amyloids (25-28). The specific involvement of HSPGs in a variety of different amyloids suggests that the HSPG-amyloid association may be more than simply a charge effect, since similar charged PGs such as keratan sulfate do not appear to be present in association with these different amyloid proteins (Snow and Wight, unpublished data). Immunocytochem- ical techniques involving antibodies against basement membrane

RESPONSE 511

HSPGs have suggested that this specific subclass of PGs are localized to a variety of different amyloids (25-28). However, we have not eliminated the possibility that other HSPGs may be present. Only when we isolate and structurally characterize these HSPGs can we determine whether unique subclasses (i.e., differ- ences in MW, core protein sequence, and/or number and size of GAG chains) exist in different amyloids. If structural differences exist, it may affect the interaction of PGs with different amy- loidogenic precursor proteins, proteases and their inhibitors or other unknown components, which may in turn affect different stages of the pathogenetic sequence of events leading to amyloid deposition. On the other hand, if a structurally similar HSPG is common to a variety of different amyloids, it suggests that a common mechanism involving similar HSPGs may be important in the pathogenesis of amyloid deposition.

Both Kisilevsky and Shirahama have raised the issue concern- ing the in vitro formation of amyloid fibrils, presumably in the absence of GAGs or PGs. As we originally discussed, the in vitro studies involving the formation of amyloid fibrils (2, 3, 6, 7, 12), using diverse conditions, do not recapitulate the process which takes place in vivo. Both commentators make the important point that not only do these studies not use physiological salt concen- trations, but the concentrations of precursors and products do not reflect the amounts found in vivo at the sites of amyloid de- position.

The potential importance of P component in the pathogenesis of amyloidosis is addressed by both Linker and Caputo. Previous studies have demonstrated that P component, like HSPGs, is a common constituent of a variety of different amyloids (4, 11, 17, 20). Its role in the pathogenesis of amyloidosis is largely un- known. However, as mentioned by Linker, recent studies suggest that its presence in a variety of different amyloids may be due to its binding affinity with heparan sulfate, localized at the site of amyloid deposition (9,10). Further studies are needed to determine the role of amyloid P component in amyloidosis and its association with HSPGs at the sites of amyloid accumulation.

Both Linker and Benditt raise the question of whether the increased accumulation of heparan sulfate or other GAGs in amyloid-containing organs is due to a direct response of the GAG metabolic system to the amyloidogenic stimulus (most likely inflammation), or secondary to the primary deposition of unusual proteins. This question was specifically addressed in our early studies using the AA experimental mouse model (22), when GAG accumulation in organs (i.e., spleen, liver, kidney) occurred only during amyloid deposition and was not part of an effect which the inflammatory stimulus had on the organ which eventually con- tained amyloid.

HEPARAN SULFATE PROTEOGLYCANS IN ALZHEIMER'S DISEASE

In his commentary, Linker cautions the use of heparan sulfate antibodies for their identification and localization in amyloid deposits due to their potential cross-reactivity with laminin. It is true that new evidence based on DNA sequencing indicates that the basement membrane HSPG core protein shows homology to portions of laminin (16). However, several lines of evidence suggest that the identification of HSPGs in AD amyloid deposits is real and not due to the presence of other matrix proteins such as laminin. Antibodies to laminin (27) and fibronectin (14) do not immunostain amyloid deposits in Alzheimer's brain. Additionally, a monoclonal antibody that recognizes the GAG chains of HSPGs (15) also demonstrate that HS GAG chains are present in amyloid deposits in AD (Snow, Kimata and Wight, unpublished data).

We agree with Linker that one should be cautious in using strictly histological data based on Alcian blue (21,23) or Cupro- linic blue (24,29) staining to identify classes of PGs and/or GAGs

that may be present in amyloid deposits, since considerable overlap may exist between different classes of GAGs. However, these techniques were originally employed essentially as screening methods to determine whether PGs were present in different types of amyloids and later were used as supplements to methods involving biochemical analysis (22) and/or immunocytochemistry (25,27).

We agree with Caputo that it will be important to not only precisely identify the particular PGs associated with AD amyloid, but also to identify those present in association with intracellular or extracellular paired helical filaments. New evidence from our lab (Snow, Mar, Kimata and Wight, unpublished data) suggests that heparan sulfate GAG chains are present intracellularly in neurons in the hipocampus in Alzheimer's and Down's syndrome brain. We are now assessing to what extent heparan sulfate is associated with intraneuronal and extraneuronal tangles. As suggested by Caputo, we are also trying to determine whether the accumulation of HS in neurons is a function of aging and to what extent this occurs in Down's syndrome. Preliminary evidence (Snow, Kimata and Wight, unpublished data) suggests that HSPGs are present in association with "preamyloid" deposits (those immunopositive with antibodies to the beta-amyloid protein) in young Down's syndrome brain prior to the appearance of a fibrillar amyloid substance detected by Congo red or Thioflavin S. This would be contradictory to the possibility suggested by Benditt that PGs accumulate in amyloid simply as a common response to the presence of amyloid fibrillar deposits. This latter finding would agree with Kisilevsky's suggestion that PGs/GAGs may be inter- acting with amyloidogenic precursors prior to any proteolytic attack and that the precursor-PG/GAG interaction may be respon- sible for fibril formation prior to proteolytic cleavage. Intriguing new evidence for this latter effect is the observation that SAA, the precursor protein to AA amyloid, once bound to GAGs is much more resistant to proteolytic attack than free SAA (l 3).

In reference to the paper by Fillit et al. (5) concerning HSPG autoantibodies in the sera of Alzheimer's patients, Benditt sug- gests that such autoantibodies are common in situations of brain injury and stroke. However, in the original article by Fillit et a l . , patients with multi-infarct dementia, multiple sclerosis, stroke, Parkinson's disease, progressive supranuclear palsy and myasthe- nia gravis did not appear to have autoantibodies to HSPGs. This suggests that the presence of such autoantibodies in Alzheimer's patients may not be a nonspecific response to injury, but may play a significant role in altering blood-brain barrier function.

As discussed by Schubert, since HSPGs are a component of synaptic vesicles and may play a role in synaptic transmission, alterations in HSPG metabolism at synaptic sites may have important consequences on the transport and/or function of neu- rotransmitters and other proteins and should be evaluated as a potential contributor to synaptic alterations that may be occurring in AD.

In reference to the commentaries by Benditt and Margolis concerning the possibility of the beta-amyloid precursor protein (BAPP) being a HSPG (18), we think this question is at present unresolved. Strong arguments agains this hypothesis are given by Margolis in his commentary, and recently by Gowda et al. (8). However, as suggested by Schubet (8), we also agree that it will be necessary to completely sequence the PC 12 PG core protein or at least a portion of the NHz-terminal sequence for the 65 kDa HSPG core protein (as suggested by Margolis) to thoroughly demonstrate that it is not the BAPP.

As recommended by Caputo and Kisilevsky, it will be impor- tant to determine the mode of binding between HSPGs and the BAPP of AD. Schubert has demonstrated that residues 176-186 of the BAPP (referred to as the GID antigen) bind to heparin sepharose columns (19). Although heparin is structurally similar to

512 SNOW AND WIGHT

heparan sulfate, one must be cautious in making the extrapolation that heparan sulfate will also bind this portion of the BAPP. Likewise, the arguments by Kisilevsky when discussing the paper by Cardin and Weintraub (1) which suggest possible protein binding sites for GAGs, must be taken with caution, as in many instances protein-GAG models do not always conform to what is actually found in vivo. For example, the binding of residues 176-186 of the BAPP to heparin as demonstrated by Schubert (19) does not conform to one of the potential GAG binding regions suggested by Kisilevsky based on Cardin's paper (11. However, our preliminary evidence suggests that the beta-amyloid protein (residues 597-614 and 597-635) does bind HSPGs in situ (30). Additionally, when beta-amyloid protein (residues 597-624) is put on an affinity column (using Affi-gel 10), and a mixture of S-35 labeled PGs (mostly heparan sulfate and chondroitin sulfate)

isolated from the cell layer of bovine aortic endothelial cells are put through the column, preliminary evidence suggests that a high molecular weight HSPG from this mixture of PGs bind to this region of the beta-amyloid protein. This implies that the presence of HSPGs in amytoid deposits in AD may be due to its specific binding affinity with the beta-amyloid protein itself. It will be important to determine whether other domains in the BAPP demonstrate binding to HSPGs and the mode of binding that exists (i.e., protein-protein or protein-GAG interaction).

Although some insight into the possible role of PGs/GAGs in Alzheimer's disease and other amyloidoses is implicated in these early studies, several possibilities still exist. Within the next few years we hope to determine to what extent these macromolecules can be further implicated in the pathogenesis of these diseases.

R E F E R E N C E S

1. Cardin, A.D.; Weintraub, H. J. R. Molecular modeling of protein- glycosaminoglycan interactions. Arteriosclerosis 9:21-32; 1989.

2. Castano, E. M.; Ghiso, J.; Prelli, F.; Gorevic, P. D.; Migheli, A.; Frangione, B. In vitro formation of amyloid fibrils from two synthetic peptides of different lengths homologous to Alzheimer's disease beta-protein. Biochem. Biophys. Res. Commun. 141:782-789; 1986.

3. Conners, L. H.; Shirahama, T.; Skinner, M.; Fenves, A.; Cohen, A. S. In vitro formation of amyloid fibrils from intact beta~-microglob- ulin. Biochem. Biophys. Res. Commun. 131:1063-1068; 1985.

4. Coria, F.; Castano, E.; Prelli, F.; Larrondo-Lillo, M.; Van Duinen, S.; Shelanski, M. L.; Frangione, B. Isolation and characterization of amyloid P component from Alzheimer's disease and other types of cerebral amyloidosis. Lab. Invest. 58:454-458; 1988.

5. Fillit, H. M.; Kemeny, E.; Luine, V.; Weksler, M. E.; Zabriskie, J. B. Antivascular antibodies in the sera of patients with senile dementia of the Alzheimer's type. J. Gerontol. 42:180-184; 1987.

6. Glenner, G. G.; Ein, D.; Eanes, E. D.; Bladen, H. A.; Terry, W.; Page, D. L. Creation of "amyloid" fibrils form Bence Jones proteins in vitro. Science 174:712-714; 1971.

7. Gorevic, P. D.; Castano, E. M.; Sarma, R.; Frangione, B. Ten to fourteen residue peptides of Alzheimer's disease protein are sufficient for amyloid fibril formation and its characteristic x-ray diffraction pattern. Biochem. Biophys. Res. Commun. 147:854-862; 1987.

8. Gowda, D. C.; Margolis, R. K.; Frangione, B.; Ghiso, J.; Larrondo- Lillo, M.; Margolis, R. U. Relation of the amyloid beta-protein precursor to heparan sulfate proteoglycans. Science 244:826--828; 1989.

9. Hamazaki, H. Ca2+-mediated association of human serum amyloid P component with heparan sulfate and dermatan sulfate. J. Biol. Chem. 262:1456-1460; 1987.

10. Hamazaki, H. Calcium-mediated hemagglutination by seurm amyloid P component and the inhibition by specific glycosaminogtycans. Biochem. Biophys. Res. Commun. 150:212-218; 1988.

11. Holck, M.; Husby, G.; Sletten, K.; Natvig, J. B. The amyloid P component (protein AP): an integral part of the amyloid substance? Scand. J. Immunol. 10:55--60; 1979.

12. Kirschner, D. A.; Inouye, H.; Duffy, L. K.; Sinclair, A.; Lind, M.; Selkoe, D. J. Synthetic peptide homologous to beta protein from Alzheimer's disease forms amyloid-like fibrils in vitro. Proc. Natl. Acad. Sci. USA 84:6953-6957; 1987.

13. Kisilevsky, R. Glycosaminoglycans and amyloid proteins. In: Gins- berg, M. D.; Dietrich, W. B., eds. Cerebrovascular Diseases, 16th Research (Princeton) Conference, Miami, FL, 1988. New York: Raven Press; 1989:223-229.

14. Koike, F.; Kunishita, T.; Nakayama, H.; Tabira, T. Immunohis- tochemicat study of Alzheimer's disease using antibodies to synthetic amyloid and fibronectin. J. Neurol. 85:9-15; 1988.

15. Koike, Y.; Kato, M.; Suzuki, S.; Kimata, K. A monoclonal antibody against the heparan sulfate of EHS-tumor proteoglycan. IX Interna- tional Symposium on Glycoconjugates, 1987 (abstract).

16. Noonan, D. M.; Horigan, E. A.; Ledbetter, S. R.; Vogeli, G.; Sasaki, M.; Yamada, Y.; Hassell, J. R. Identification of cDNA clones

encoding different domains of the basement membrane heparan sulfate proteoglycan. J. Biol. Chem. 263:16379-16387; 1988.

17. Saperia, D.; Perlmutter, L. S.; Athanikar, J.; Chui, H. C. Amyloid P component in dementia. Soc. Neurosci. Abstr. 12:636; 1988.

18. Schubert, D.; Schroeder, R,; LaCorbiere, M.; Saitoh, T.; Cole, G. The Alzheimer's beta-protein precursor is possibly a heparan sulfate proteoglycan core protein. Science 241:223-226; 1988.

19. Schubert, D.; LaCorbiere, M.; Saitoh, T.; Cole, G. Characterization of an amyloid beta-protein precursor that binds heparin and contains tyrosine sulfate. Proc. Natl. Acad. Sci. USA 86:2066-2069; 1989.

20. Skinner, M.; Cohen, A. S. Aspects of the amyloid P component. In: Wegelius, O.; Pasternack, A., eds. Amyloidosis. New York: Aca- demic Press; 1976:339-352.

21. Snow, A. D.; Kisilevsky, R. Temporal relationship between glyco- saminoglycan accumulation and amyloid deposition during experimen- tal amyloidosis. A histochemicai study. Lab. Invest. 53:37-44; 1985.

22. Snow, A. D.; Kisilevsky, R.; Stephens, C.; Anastassiades, T. Characterization of tissue and plasma glycosaminoglycans during experimental AA amyloidosis and acute inflammation. Qualitative and quantitative analysis. Lab. Invest. 56:665-675; 1987.

23. Snow, A. D.; Willmer, J.; Kisilevsky, R. Sulfated glycosaminogly- cans in Alzheimer's disease. Hum. Pathol. 18:506-510; 1987.

24. Snow, A. D.; Willmer, J.; Kisilevsky, R. A close ultrastructural relationship between sulfated proteoglycans and AA amyloid fibrils. Lab. Invest. 57:687-698; 1987.

25. Snow, A. D.; Kisilevsky, R.: Wight, T. N Immunolocaiization of heparan sulfate proteoglycans to AA amyloid deposition sites m spleen and liver during experimental amyloidosis. In: Isobe. T.: Araki, S.; Uchino, F.; Kito. S.: Tsubura. E.. eds. Arnyloid and amyloidosis. New York: Plenum Press: 1988:87-93.

26. Snow, A. D.; Nochlin, D.: Sumi. S.: Bird. T. D.: Wight, T. N. Immunolocalization of heparan sulfate proteoglycans to "primitive plaques" and multi-core prion positive plaques in familial dementia Alzheimer Dis. Assoc. Disord. 2:182; 1988 (abstract P.

27. Snow, A. D.; Mar, H.; Nochlin. D.; Kimata. K.: Sato. M.; Suzuki, S.; Hassell, J.; Wight, T. N. The presence of heparan sulfate proteoglycans in he neuritic plaques and congophilic angiopathy in Alzheimer's disease. Am. J. Pathol. 133:456-453: 1988.

28. Snow, A. D.; Nochlin, D.; DeArmond. S. L; Pursiner, S. B.: Bird. T. D.; Sumi, S. M.; Wight, T. N. Identification and localization of heparan sulfate proteoglycans in PrP amyloid plaques in Gerstmann- Straussler syndrome, Creutzfeldt-Jakob disease and scrapie Alzhei- mer Dis. Assoc. Disord. 3(Suppl. 11:40:1989 (abstract).

29. Snow, A. D.; Nochlin, D.; Lara, S.: Wight, T. N. Cationic dyes reveal pmteoglycans structurally integrated within the characteristic lesions of Alzheimer's disease. Acta Neuropathol. (Berl.) 78:113- 123; 1989.

30. Snow, A. D.; Kinsella, M. G,; Prather. P. B.; Nochtin, D.: Podlisny, M. B.; Selkoe, D. J.; Kisilevsky, R.: Wight, T. N. A characteristic binding affinity between heparan sulfate proteoglycans and the A4 amyloid protein of Alzheimer's disease. J. Neuropathol. Exp. Neurol 48:352; 1989 (abstract).