- Email: [email protected]
More thoughts on plaques and tangles
432
Y }~N
78. Tomlinson, B. E., G. Blessed with M. Roth. Observations on the brain of demented old people..I Neurol Sci 11: 205-243. 1970. 79. Tomlinson, B. E. and G. Henderson. Some quantitative cerebral findings in normal and demented old people. In: Neurobiology of Aging, edited by R. D. Terry and S. Gershon. New York: Raven Press, 1976, pp. 183-204. 80. Torack, R. M. and R. G. Lynch. Cytochemistry of brain amyloid in adult dementia. Acta Neuropathol (Berl) 53: 189196, 1981. 81. Vanley, C. T., M. J. Aguilar, R. J. Kleinhenz and M. D. Lagios. Cerebral amyloid angiopathy. Hum Pathol 12: 609-616, 1981. 82. Whitehouse, P. J., D. L. Price, R. G. Struble, A. W. Clark, J. T. Coyle and M. R. DeLong. Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain. Science 215: 1237-1239, 1982. 83. Wilcock, G. K. and M. M. Esiri. Plaques, tangles and dementia: A quantitative study. J Neurol Sci 56: 343-356, 1982. 84. Wilcock, G. K., M. M. Esiri, D. M. Bowen and C. C. T Smith. Alzheimer's disease: Correlation of cortical choline acetyltransferase activity with the severity of dementia and histological abnormalities. J Neurol Sci 57: 407-417, 1982. 85. Wischik, C. M., R. A. Crowther, M. Stewart and M. Roth. Subunit structure of paired helical filaments in Alzheimer's disease. J Cell Biol 100: 1905-1912, 1985. 86. Wisniewski, H. M., M. E. Bruce and H. Fraser. Infectious etiology of neuritic (senile) plaques in mice. Science 190:1108-11t0, 1975. 87. Wisniewski, H. M., H. K. Narang and R. D. Terry. Neurofibrillary tangles of paired helical filaments. J Neurol Sci 27: 173-181, 1976.
88. Wisniewski, H. M. and R. D. Terry. Reexamination of the pathogenesis of the senile plaque. Prog Ne,ropathol 2: 1-26. 1973. 89. Wisniewski, H. M. and G. Y. Wen. A comparative study on the substructures of neurofilaments and paired helical filaments from Alzheimer neurofibrillary tangles. In: Intermediate f:71,m~,,ts, edited by E. Wang, D. Fischman, R. K. H. Liem and T. T. Sun. Ann N Y A c a d Sci 455: 814-815, 1985. 90. Wong, C. W., V. Quaranta and G. G. Glenner. Neuritic plaques and cerebrovascular amyloid in Alzheimer disease are antigenically related. Proc Natl Acad Sci USA 82: 872%8732. 1985. 91. Wood, J. G., S. S. Mirra, N. J. Pollock and L. 1. Binder. Neurofibrillary tangles of Alzheimer's disease share antigenic determinants with the axonal microtubule-associated protein tau. Proc Natl Acad Sci USA 83: 4040-4043, 1986. 92. Yagashita, S., T. Itoh, W. Nan and N. Amano. Reappraisal of the fine structure of Alzheimer's neurofibrillary tangles. Acta Ncuropathol (Berl) 54: 239-246, 1981. 93. Yen, S-H., A. Crowe and D. W. Dickson. Monoclonal antibodies to Alzheimer's neurofibrillary tangles. I. Identification of polypeptides. Am J Pathol 120: 282-291, 1985. 94. Yen. S-H., Gaskin and R. D. Terry. Immunocytochemical studies of neurofibrillary tangles. Am J Pathol 104: 77-89, 1981. 95. Yen, S.-H. and Y. Kress, The effect of chemical reagents or proteases on the ultrastructure of paired helical filaments. In: Banbury Report 15: Biological Aspects of Alzheimer's Disease. edited by R. Katzman. New York: Cold Spring Harbor Laboratory, 1983, pp. 155-165. 96. Yoshimura, N. Evidence that paired helical filaments originate from neurofilaments. Clin Ncuropathol 3: 22-27. 1984.
COMMENTARIES More Thoughts on Plaques and Tangles SHU-HUI
YEN
Department of Pathology A l b e r t E i n s t e i n Co l l eg e o f M e d i c i n e , B r o n x , N Y 10461 Alzheimer neurofibrillary tanlges (NFT) contain unique antigenic determinants and determinants in common with several normal cytoskeletal proteins which include the high molecular weight neurof'flament proteins, microtubule associated proteins and vimentin. The unique determinants are found in nearly all NFT whereas the determinants in common with normal cytoskeletal proteins are generally detected only in a fraction of NFT. It remains to be determined whether a particular type of cytoskeletal protein is more involved than others in the formation of NFT and if posttranslational modification of certain normal proteins is responsible for the expression of unique antigenic determinants.
N E U R O F I B R I L L A R Y tangles ( N F T ) and senile plaques h a v e long b e e n r e c o g n i z e d as two of the m o s t p r o m i n e n t histopathological hallmarks o f A l z h e i m e r disease and senile d e m e n t i a of the A l z h e i m e r type. The pathogenesis o f these extraordinary structures remains unknown. H o w e v e r , considerable progress has been m a d e during the last d e c a d e in understanding the composition of both neurofibrillary tangles and in particular the amyloid fibrils o f cerebral vascular
amyloid and senile plaques. During this period, n u m e r o u s investigations have b e e n c o n d u c t e d with i m m u n o c y t o c h e m i cal techniques and biochemical methods. The r e v i e w written by Dr. Selkoe is timely; it summarizes rather nicely the adv a n c e s in this field and indicates a n u m b e r of problems that remain to be solved. He describes the n e w approaches that are currently being taken by several laboratories in studying the structural protein abnormalities in A l z h e i m e r ' s disease.
COMMENTARY
433
Several issues that have not been addressed or only briefly mentioned are discussed in this commentary. A definitive answer to the composition of Alzheimer neurofibrillary tangles undoubtedly depends heavily on biochemical characterization of a pure paired helical filament (PHF) preparation. As stated in the review, all the methods available so far have generated only enriched fractions. Significant contamination of the preparations with lipofuscin and in some cases amyloid fibrils remains a major obstacle. The difficulty of solubilizing partially purified P H F has been emphasized in the past. A recent study has reported the presence of two types of PHF (one with left handed twist and the other with right handed twist) in vivo and in vitro [12]. The two ultrastructural forms of PHF have different physiochemical properties. The susceptibility of one type of PHF to solubilization is different from that of the other. In view of this information, it may be important in future studies to focus on the isolation of the relatively soluble PHF since they can be more easily studied by conventional biochemical analysis. Alzheimer neurofibrillary tangles cross react with antibodies to various normal cytoskeletal proteins that have no apparent morphological and biochemical similarity [14]. These observations have generated confusion about the characteristics of neurofibrillary tangles and the significance of immunocytochemical studies. Selkoe indicates in the review that the problem may soon be resolved since he and coworkers have found in a preliminary study that all tangles reactive with anti-neurofilament (anti-NF) antibodies recognize the phosphorylated, heat stable microtubule associated protein tau. Other investigators in several recent studies have demonstrated that other cytoskeletal proteins share epitopes most of which are phosphorylated [7, 8, 13]. The cross-reactivity between NFT and normal cytoskeletal proteins, therefore, may be due to the sharing of phosphorylated epitopes. Evidence suggesting that NFT may contain phosphoproteins derives primarily from the observation that antibodies that bind to NFT recognize phosphorylated epitopes. Pretreatment of brain sections with phosphatase blocks the staining of axon as well as that of NFT by certain antibodies raised against NF [10]. However, not all of the normal cytoskeleton epitopes detected in NFT respond to phosphatase-induced dephosphorylation in a manner similar to their counter part in normal proteins [11,14]. This differential property may be due to conformational changes of proteins which are incorporated into NFT. Alternatively, it may suggest that NFT are composed of unique phosphoproteins which by coincidence share epitopes with normal cytoskeletal phosphoproteins. Regardless of whether PHF contain unique proteins or proteins similar to those in normal
neurons, a direct analysis of phosphate content will demonstrate the involvement of phosphate in PHF. The immunocytochemical staining of N F T by all anti-tau antibodies tested to date and the Western blotting of tau proteins with certain anti-NFT antibodies have drawn considerable interest on these heat stable microtubule associated proteins. An important question is whether tau is a major component of N F T as it has been suggested recently [5,6] or whether other cytoskeletal proteins are also present in the NFT. The sharing of epitopes between seemingly unrelated proteins makes an over zealous interpretation of the immunochemical results hazardous. Furthermore, in several studies it has been noted that none of the antibodies to normal cytoskeletal proteins react with all of the NFT in a given tissue section or isolated preparation. In our hands, anti-tau antibodies often stain only about half of the N F T in sections double-labelled with thioflavine S (a fluorochrome that binds to amyloid and amyloid-like material such as NFT), whereas antibodies that recognize epitopes unique to N F T react with nearly all N F T [15]. One can always raise the possibility that the lack of binding of antibodies to all N F T is due to biochemical modification of normal protein in some NFT. But this argument will not be resolved unless other parameters beside the antigen-antibody reaction are measured. A major breakthrough in the characterization of cerebral amyloid was the purification and amino acid sequencing of a 4kD component of vascular amyloid by Glenner and Wong [4]. It now appears that the time has finally come for finding out if any particular cell type or tissue is responsible for the production of cerebral amyloid. Since amyloid in cerebral vessel is soluble in guanidine hydrochloride but that in plaques is not, it will be important to determine the relationship between vascular and plaque amyloid. Inorganic matter such as aluminum silicate has been found in senile plaques core [2], however this issue remains controversial [9]. If inorganic substances are present in plaque core amyloid then it will be necessary to find out if the accumulation of aluminum or other trace metals precedes or follows the deposition of amyloid in order to understand the pathogenesis of senile plaque. Antibodies raised against amyloid synthetic peptides have been reported to stain amyloid cores and material in the periphery of classical senile plaques [1]. We found that the antibodies also stain senile plaque with no amyloid core [3]. Whether some of the staining is due to reaction of antibodies with amyloidogenic precursor proteins remains to be determined. The relationship between aluminum deposition and the amyloid proteins can most easily be understood by studying human tissues with early stage of the disease since an animal model for Alzheimer disease is not available.
REFERENCES
I. AIIsop, D., M. Landon, M. Kidd, J. S. Lowe, G. P. Reynolds and A. Gardner. Monoclonal antibodies raised against a subsequence of senile plaque core protein react with plaque cores, plaque periphery and cerebrovascular amyloid in Alzheimer disease. Neurosci Lett 68: 252-256, 1986. 2. Candy, J. M., A. E. Oakley, J. Klinowski, T. A. Carpenter, P. H. Perry, J. K. Atack, E. K. Perry, G. Blessed, A. Fairbairn and J. A. Edwardson. Aluminosilicates and senile plaque formation in Alzheimer's disease. Lancet 1: 354--358, 1985. 3. Dickson. D. W. and S. H. Yen. Manuscript in preparation.
4. Glenner. G. G. and C. W. Wong. Alzheimer disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res (~otnmun 120: 885-890, 1984. 5. Iqbal, K., I. Grunke-Iqbal and H. M. Wisniewski. Microtubule associated protein Tau is a major component of Alzheimer paired helical filaments. X International Congress of Neuropathology, 296, 1986.
434
!I)BA[
6. Kosik, K. S., C. L. Joachim and D. J. Selkoe. Micrombule associated protein tau is a major antigenic component of paired helical filaments in Alzheimer disease. Proc Natl Acad Sci U~SA 83: 4044-4048, 1986. 7. Ksiezak-Reding, H. and S. H. Yen. Two monoclonal antibodies recognize Alzheimer's neurofibrillary tangles, neurofilaments, and microtubule-associated proteins. ,I Neuro('hem, in press. 1987. 8. Luca, F. C., G. S. Bloom and R. B. Vallee. A monoclonal antibody that cross reacts with phosphorylated epitopes on two microtubule associated proteins and two neurofilament polypeptides. Proc Natl Acad Sci USA 83: 1006-1010, 1986. 9. Stem, A. J., D. P. Perl, D. Munoz-Garcia, P. F. Good, C. Abraham and D. Selkoe. Investigation of silicon and aluminum content in isolated senile plaque cores by laser microprobe mass analysis. J Neuropathol Exp Neurol 45: 361, 1986. 10. Sternberger, N. H., L. A. Sternberger and J. Ulrich. Aberrent neurofilament phosphorylation in Alzheimer disease. Proc Natl Acad Sci USA 82: 4274-4276. 1985.
11. Ulrich, J., M. Haugh, B. H. Anderton and A. Probst. Phosphorylated epitopes on Pick bodies and Alzheimer neurofibrillary tangles. J Neuropathol l:'xp Neurol 44: 367, 1985. 12. Wisniewski, H. M. and G. Y. Wen. Ultrastructure of paired helical filaments in Alzheimer's disease. X International Congress of Neuropathology, 295, 1986. 13. Wood, J. N., N. B. Lathangue, D. R. McLachlan, B. J. Smith, B. H. Anderton and A. J. Dowding. Chromatin proteins share antigenic determinants with neurofilaments..I Ncurochem 44: 149-154, 1985. 14. Yen, S. H., D. W. Dickson, C. Peterson and J. 1-2. Goldman. Cytoskeletal abnormalities in neuropathology. In: Progress in N~,uropathoh)gy. Vol 6. edited by H. M. Zimmerman. New York: Raven Press, 1986, pp. 63-90. 15. Yen, S. H., A. Crowe and D. W. Dickson. Monoclonal antibodies to Alzheimer neurofibriltary tangles. I. identification of polypeptides. Am ,1 Pathol 120: 282-291, 1985.
Biology of Alzheimer's Disease Through Biochemistry of Tangles and Plaques KHALID
IQBAL
N e w York S t a t e Ojfice o f M e n t a l R e t a r d a t i o n a n d D e v e l o p m e n t a l Disabilities I n s t i t u t e f o r B a s i c R e s e a r c h in D e v e l o p m e n t a l Disabilities, S t a t e n Island, N Y 10314
Recent advances on identification of the polypeptides making the paired helical filaments of Alzheimer neurofibrillary tangles and the neuritic (senile) plaque amyloid have provided a new insight into the pathogenesis of Alzheimer's disease. Major breakthroughs towards establishing the underlying primary defect in Alzheimer's disease are now in sight. The strength of various data towards identification of the polypeptides of tangles and plaques and the role of these proteins in the biology of Alzheimer's disease are discussed.
T H E central point of this review, as put in the title, is what we k n o w about altered structural proteins in plaques and tangles. Our k n o w l e d g e in this area o f r e s e a r c h is expanding rapidly and the gaps and disagreements b e t w e e n various laboratories, in what p r o v e d to be a very difficult area o f research, are b e c o m i n g smaller e v e r y day. The v e r y first studies on the polypeptide composition of A l z h e i m e r paired helical filaments ( P H F ) which were published in 1974 [20] have led, several years later, to the identification of the microtubule associated protein tau in P H F [4, 10, 11,24, 29, 37, 39]. It is most gratifying to see that several different laboratories have independently c o m e to the same conclusions. Identification of a c e r e b r o v a s c u l a r amyioid peptide and its amino acid s e q u e n c e by G l e n n e r ' s group [7,8] have started v e r y m u c h interest in this area o f r e s e a r c h and m o r e and more data from different laboratories is by and large confirming these findings (see c o m m e n t a r y by Wisniewski). STRUCTURAL PROTEINS OF TANGLES AND PLAQUES First of all it is important to point out that A t z h e i m e r neurofibrillary tangles (ANT) are h e t e r o g e n e o u s both in their
solubility properties and in morphology Most of the A N T are m a d e up of paired helical filaments IPHF). Only a few A N T are a mixture of P H F and straight 10-15 nm filaments. S o m e A N T mostly in rare atypical cases o f A i z h e i m e r disease/senile d e m e n t i a of the A l z h e i m e r type I A D / S D A T ) are made up of 15 nm straight filaments: the neurofibrillary tangles of 15 nm filaments are generally seen in cases o f progressive supranuclear palsy [33]. T w o general populations of A N T . I and 11. have been identified based on the solubility and insolubility, respectively in 2% SDS at r o o m temperature for 3-5 minutes [211. The A N T II are, however, solubilized on repeated extractions in SDS and /3-mercaptoethanol at 90-100°C o r more effectively by ultrasonication followed by heating in Laemlli's sample buffer (which c o n t a i n s 1% e a c h of S D S and /3-mercaptoethanoi) as carried out routinely for SDSpolyacrylamide gel electrophoresis [18]. In a g r e e m e n t with these biochemical studies, ultrastructural studies of P H F have r e v e a l e d that, indeed, there are two general populations o f P H F , i.e., P H F with right-handed helices and P H F with left-handed helices [36]. The right-handed P H F are larger than the left-handed P H F both in d i a m e t e r and in periodicity
Y }~N
78. Tomlinson, B. E., G. Blessed with M. Roth. Observations on the brain of demented old people..I Neurol Sci 11: 205-243. 1970. 79. Tomlinson, B. E. and G. Henderson. Some quantitative cerebral findings in normal and demented old people. In: Neurobiology of Aging, edited by R. D. Terry and S. Gershon. New York: Raven Press, 1976, pp. 183-204. 80. Torack, R. M. and R. G. Lynch. Cytochemistry of brain amyloid in adult dementia. Acta Neuropathol (Berl) 53: 189196, 1981. 81. Vanley, C. T., M. J. Aguilar, R. J. Kleinhenz and M. D. Lagios. Cerebral amyloid angiopathy. Hum Pathol 12: 609-616, 1981. 82. Whitehouse, P. J., D. L. Price, R. G. Struble, A. W. Clark, J. T. Coyle and M. R. DeLong. Alzheimer's disease and senile dementia: loss of neurons in the basal forebrain. Science 215: 1237-1239, 1982. 83. Wilcock, G. K. and M. M. Esiri. Plaques, tangles and dementia: A quantitative study. J Neurol Sci 56: 343-356, 1982. 84. Wilcock, G. K., M. M. Esiri, D. M. Bowen and C. C. T Smith. Alzheimer's disease: Correlation of cortical choline acetyltransferase activity with the severity of dementia and histological abnormalities. J Neurol Sci 57: 407-417, 1982. 85. Wischik, C. M., R. A. Crowther, M. Stewart and M. Roth. Subunit structure of paired helical filaments in Alzheimer's disease. J Cell Biol 100: 1905-1912, 1985. 86. Wisniewski, H. M., M. E. Bruce and H. Fraser. Infectious etiology of neuritic (senile) plaques in mice. Science 190:1108-11t0, 1975. 87. Wisniewski, H. M., H. K. Narang and R. D. Terry. Neurofibrillary tangles of paired helical filaments. J Neurol Sci 27: 173-181, 1976.
88. Wisniewski, H. M. and R. D. Terry. Reexamination of the pathogenesis of the senile plaque. Prog Ne,ropathol 2: 1-26. 1973. 89. Wisniewski, H. M. and G. Y. Wen. A comparative study on the substructures of neurofilaments and paired helical filaments from Alzheimer neurofibrillary tangles. In: Intermediate f:71,m~,,ts, edited by E. Wang, D. Fischman, R. K. H. Liem and T. T. Sun. Ann N Y A c a d Sci 455: 814-815, 1985. 90. Wong, C. W., V. Quaranta and G. G. Glenner. Neuritic plaques and cerebrovascular amyloid in Alzheimer disease are antigenically related. Proc Natl Acad Sci USA 82: 872%8732. 1985. 91. Wood, J. G., S. S. Mirra, N. J. Pollock and L. 1. Binder. Neurofibrillary tangles of Alzheimer's disease share antigenic determinants with the axonal microtubule-associated protein tau. Proc Natl Acad Sci USA 83: 4040-4043, 1986. 92. Yagashita, S., T. Itoh, W. Nan and N. Amano. Reappraisal of the fine structure of Alzheimer's neurofibrillary tangles. Acta Ncuropathol (Berl) 54: 239-246, 1981. 93. Yen, S-H., A. Crowe and D. W. Dickson. Monoclonal antibodies to Alzheimer's neurofibrillary tangles. I. Identification of polypeptides. Am J Pathol 120: 282-291, 1985. 94. Yen. S-H., Gaskin and R. D. Terry. Immunocytochemical studies of neurofibrillary tangles. Am J Pathol 104: 77-89, 1981. 95. Yen, S.-H. and Y. Kress, The effect of chemical reagents or proteases on the ultrastructure of paired helical filaments. In: Banbury Report 15: Biological Aspects of Alzheimer's Disease. edited by R. Katzman. New York: Cold Spring Harbor Laboratory, 1983, pp. 155-165. 96. Yoshimura, N. Evidence that paired helical filaments originate from neurofilaments. Clin Ncuropathol 3: 22-27. 1984.
COMMENTARIES More Thoughts on Plaques and Tangles SHU-HUI
YEN
Department of Pathology A l b e r t E i n s t e i n Co l l eg e o f M e d i c i n e , B r o n x , N Y 10461 Alzheimer neurofibrillary tanlges (NFT) contain unique antigenic determinants and determinants in common with several normal cytoskeletal proteins which include the high molecular weight neurof'flament proteins, microtubule associated proteins and vimentin. The unique determinants are found in nearly all NFT whereas the determinants in common with normal cytoskeletal proteins are generally detected only in a fraction of NFT. It remains to be determined whether a particular type of cytoskeletal protein is more involved than others in the formation of NFT and if posttranslational modification of certain normal proteins is responsible for the expression of unique antigenic determinants.
N E U R O F I B R I L L A R Y tangles ( N F T ) and senile plaques h a v e long b e e n r e c o g n i z e d as two of the m o s t p r o m i n e n t histopathological hallmarks o f A l z h e i m e r disease and senile d e m e n t i a of the A l z h e i m e r type. The pathogenesis o f these extraordinary structures remains unknown. H o w e v e r , considerable progress has been m a d e during the last d e c a d e in understanding the composition of both neurofibrillary tangles and in particular the amyloid fibrils o f cerebral vascular
amyloid and senile plaques. During this period, n u m e r o u s investigations have b e e n c o n d u c t e d with i m m u n o c y t o c h e m i cal techniques and biochemical methods. The r e v i e w written by Dr. Selkoe is timely; it summarizes rather nicely the adv a n c e s in this field and indicates a n u m b e r of problems that remain to be solved. He describes the n e w approaches that are currently being taken by several laboratories in studying the structural protein abnormalities in A l z h e i m e r ' s disease.
COMMENTARY
433
Several issues that have not been addressed or only briefly mentioned are discussed in this commentary. A definitive answer to the composition of Alzheimer neurofibrillary tangles undoubtedly depends heavily on biochemical characterization of a pure paired helical filament (PHF) preparation. As stated in the review, all the methods available so far have generated only enriched fractions. Significant contamination of the preparations with lipofuscin and in some cases amyloid fibrils remains a major obstacle. The difficulty of solubilizing partially purified P H F has been emphasized in the past. A recent study has reported the presence of two types of PHF (one with left handed twist and the other with right handed twist) in vivo and in vitro [12]. The two ultrastructural forms of PHF have different physiochemical properties. The susceptibility of one type of PHF to solubilization is different from that of the other. In view of this information, it may be important in future studies to focus on the isolation of the relatively soluble PHF since they can be more easily studied by conventional biochemical analysis. Alzheimer neurofibrillary tangles cross react with antibodies to various normal cytoskeletal proteins that have no apparent morphological and biochemical similarity [14]. These observations have generated confusion about the characteristics of neurofibrillary tangles and the significance of immunocytochemical studies. Selkoe indicates in the review that the problem may soon be resolved since he and coworkers have found in a preliminary study that all tangles reactive with anti-neurofilament (anti-NF) antibodies recognize the phosphorylated, heat stable microtubule associated protein tau. Other investigators in several recent studies have demonstrated that other cytoskeletal proteins share epitopes most of which are phosphorylated [7, 8, 13]. The cross-reactivity between NFT and normal cytoskeletal proteins, therefore, may be due to the sharing of phosphorylated epitopes. Evidence suggesting that NFT may contain phosphoproteins derives primarily from the observation that antibodies that bind to NFT recognize phosphorylated epitopes. Pretreatment of brain sections with phosphatase blocks the staining of axon as well as that of NFT by certain antibodies raised against NF [10]. However, not all of the normal cytoskeleton epitopes detected in NFT respond to phosphatase-induced dephosphorylation in a manner similar to their counter part in normal proteins [11,14]. This differential property may be due to conformational changes of proteins which are incorporated into NFT. Alternatively, it may suggest that NFT are composed of unique phosphoproteins which by coincidence share epitopes with normal cytoskeletal phosphoproteins. Regardless of whether PHF contain unique proteins or proteins similar to those in normal
neurons, a direct analysis of phosphate content will demonstrate the involvement of phosphate in PHF. The immunocytochemical staining of N F T by all anti-tau antibodies tested to date and the Western blotting of tau proteins with certain anti-NFT antibodies have drawn considerable interest on these heat stable microtubule associated proteins. An important question is whether tau is a major component of N F T as it has been suggested recently [5,6] or whether other cytoskeletal proteins are also present in the NFT. The sharing of epitopes between seemingly unrelated proteins makes an over zealous interpretation of the immunochemical results hazardous. Furthermore, in several studies it has been noted that none of the antibodies to normal cytoskeletal proteins react with all of the NFT in a given tissue section or isolated preparation. In our hands, anti-tau antibodies often stain only about half of the N F T in sections double-labelled with thioflavine S (a fluorochrome that binds to amyloid and amyloid-like material such as NFT), whereas antibodies that recognize epitopes unique to N F T react with nearly all N F T [15]. One can always raise the possibility that the lack of binding of antibodies to all N F T is due to biochemical modification of normal protein in some NFT. But this argument will not be resolved unless other parameters beside the antigen-antibody reaction are measured. A major breakthrough in the characterization of cerebral amyloid was the purification and amino acid sequencing of a 4kD component of vascular amyloid by Glenner and Wong [4]. It now appears that the time has finally come for finding out if any particular cell type or tissue is responsible for the production of cerebral amyloid. Since amyloid in cerebral vessel is soluble in guanidine hydrochloride but that in plaques is not, it will be important to determine the relationship between vascular and plaque amyloid. Inorganic matter such as aluminum silicate has been found in senile plaques core [2], however this issue remains controversial [9]. If inorganic substances are present in plaque core amyloid then it will be necessary to find out if the accumulation of aluminum or other trace metals precedes or follows the deposition of amyloid in order to understand the pathogenesis of senile plaque. Antibodies raised against amyloid synthetic peptides have been reported to stain amyloid cores and material in the periphery of classical senile plaques [1]. We found that the antibodies also stain senile plaque with no amyloid core [3]. Whether some of the staining is due to reaction of antibodies with amyloidogenic precursor proteins remains to be determined. The relationship between aluminum deposition and the amyloid proteins can most easily be understood by studying human tissues with early stage of the disease since an animal model for Alzheimer disease is not available.
REFERENCES
I. AIIsop, D., M. Landon, M. Kidd, J. S. Lowe, G. P. Reynolds and A. Gardner. Monoclonal antibodies raised against a subsequence of senile plaque core protein react with plaque cores, plaque periphery and cerebrovascular amyloid in Alzheimer disease. Neurosci Lett 68: 252-256, 1986. 2. Candy, J. M., A. E. Oakley, J. Klinowski, T. A. Carpenter, P. H. Perry, J. K. Atack, E. K. Perry, G. Blessed, A. Fairbairn and J. A. Edwardson. Aluminosilicates and senile plaque formation in Alzheimer's disease. Lancet 1: 354--358, 1985. 3. Dickson. D. W. and S. H. Yen. Manuscript in preparation.
4. Glenner. G. G. and C. W. Wong. Alzheimer disease: initial report of the purification and characterization of a novel cerebrovascular amyloid protein. Biochem Biophys Res (~otnmun 120: 885-890, 1984. 5. Iqbal, K., I. Grunke-Iqbal and H. M. Wisniewski. Microtubule associated protein Tau is a major component of Alzheimer paired helical filaments. X International Congress of Neuropathology, 296, 1986.
434
!I)BA[
6. Kosik, K. S., C. L. Joachim and D. J. Selkoe. Micrombule associated protein tau is a major antigenic component of paired helical filaments in Alzheimer disease. Proc Natl Acad Sci U~SA 83: 4044-4048, 1986. 7. Ksiezak-Reding, H. and S. H. Yen. Two monoclonal antibodies recognize Alzheimer's neurofibrillary tangles, neurofilaments, and microtubule-associated proteins. ,I Neuro('hem, in press. 1987. 8. Luca, F. C., G. S. Bloom and R. B. Vallee. A monoclonal antibody that cross reacts with phosphorylated epitopes on two microtubule associated proteins and two neurofilament polypeptides. Proc Natl Acad Sci USA 83: 1006-1010, 1986. 9. Stem, A. J., D. P. Perl, D. Munoz-Garcia, P. F. Good, C. Abraham and D. Selkoe. Investigation of silicon and aluminum content in isolated senile plaque cores by laser microprobe mass analysis. J Neuropathol Exp Neurol 45: 361, 1986. 10. Sternberger, N. H., L. A. Sternberger and J. Ulrich. Aberrent neurofilament phosphorylation in Alzheimer disease. Proc Natl Acad Sci USA 82: 4274-4276. 1985.
11. Ulrich, J., M. Haugh, B. H. Anderton and A. Probst. Phosphorylated epitopes on Pick bodies and Alzheimer neurofibrillary tangles. J Neuropathol l:'xp Neurol 44: 367, 1985. 12. Wisniewski, H. M. and G. Y. Wen. Ultrastructure of paired helical filaments in Alzheimer's disease. X International Congress of Neuropathology, 295, 1986. 13. Wood, J. N., N. B. Lathangue, D. R. McLachlan, B. J. Smith, B. H. Anderton and A. J. Dowding. Chromatin proteins share antigenic determinants with neurofilaments..I Ncurochem 44: 149-154, 1985. 14. Yen, S. H., D. W. Dickson, C. Peterson and J. 1-2. Goldman. Cytoskeletal abnormalities in neuropathology. In: Progress in N~,uropathoh)gy. Vol 6. edited by H. M. Zimmerman. New York: Raven Press, 1986, pp. 63-90. 15. Yen, S. H., A. Crowe and D. W. Dickson. Monoclonal antibodies to Alzheimer neurofibriltary tangles. I. identification of polypeptides. Am ,1 Pathol 120: 282-291, 1985.
Biology of Alzheimer's Disease Through Biochemistry of Tangles and Plaques KHALID
IQBAL
N e w York S t a t e Ojfice o f M e n t a l R e t a r d a t i o n a n d D e v e l o p m e n t a l Disabilities I n s t i t u t e f o r B a s i c R e s e a r c h in D e v e l o p m e n t a l Disabilities, S t a t e n Island, N Y 10314
Recent advances on identification of the polypeptides making the paired helical filaments of Alzheimer neurofibrillary tangles and the neuritic (senile) plaque amyloid have provided a new insight into the pathogenesis of Alzheimer's disease. Major breakthroughs towards establishing the underlying primary defect in Alzheimer's disease are now in sight. The strength of various data towards identification of the polypeptides of tangles and plaques and the role of these proteins in the biology of Alzheimer's disease are discussed.
T H E central point of this review, as put in the title, is what we k n o w about altered structural proteins in plaques and tangles. Our k n o w l e d g e in this area o f r e s e a r c h is expanding rapidly and the gaps and disagreements b e t w e e n various laboratories, in what p r o v e d to be a very difficult area o f research, are b e c o m i n g smaller e v e r y day. The v e r y first studies on the polypeptide composition of A l z h e i m e r paired helical filaments ( P H F ) which were published in 1974 [20] have led, several years later, to the identification of the microtubule associated protein tau in P H F [4, 10, 11,24, 29, 37, 39]. It is most gratifying to see that several different laboratories have independently c o m e to the same conclusions. Identification of a c e r e b r o v a s c u l a r amyioid peptide and its amino acid s e q u e n c e by G l e n n e r ' s group [7,8] have started v e r y m u c h interest in this area o f r e s e a r c h and m o r e and more data from different laboratories is by and large confirming these findings (see c o m m e n t a r y by Wisniewski). STRUCTURAL PROTEINS OF TANGLES AND PLAQUES First of all it is important to point out that A t z h e i m e r neurofibrillary tangles (ANT) are h e t e r o g e n e o u s both in their
solubility properties and in morphology Most of the A N T are m a d e up of paired helical filaments IPHF). Only a few A N T are a mixture of P H F and straight 10-15 nm filaments. S o m e A N T mostly in rare atypical cases o f A i z h e i m e r disease/senile d e m e n t i a of the A l z h e i m e r type I A D / S D A T ) are made up of 15 nm straight filaments: the neurofibrillary tangles of 15 nm filaments are generally seen in cases o f progressive supranuclear palsy [33]. T w o general populations of A N T . I and 11. have been identified based on the solubility and insolubility, respectively in 2% SDS at r o o m temperature for 3-5 minutes [211. The A N T II are, however, solubilized on repeated extractions in SDS and /3-mercaptoethanol at 90-100°C o r more effectively by ultrasonication followed by heating in Laemlli's sample buffer (which c o n t a i n s 1% e a c h of S D S and /3-mercaptoethanoi) as carried out routinely for SDSpolyacrylamide gel electrophoresis [18]. In a g r e e m e n t with these biochemical studies, ultrastructural studies of P H F have r e v e a l e d that, indeed, there are two general populations o f P H F , i.e., P H F with right-handed helices and P H F with left-handed helices [36]. The right-handed P H F are larger than the left-handed P H F both in d i a m e t e r and in periodicity