Bwchimica et Biophysica Acta, 1096 (1991) 1 - 9
1
Elsevier
BBADIS 61002
Review
Structural aspects of pathology in Alzheimer's disease R.A. Crowther Medical Research Council, Laboratory of Molecular Biology, Cambridge (U.K.) (Received 24 May 1990)
Key words: Alzheimer's disease; Neurofibrillary tangle; Paired helical filament; Neuritic plaque; /~-Amyloid
Contents I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
II.
Structure of paired helical filaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
III.
Tau protein is a component of the P H F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
IV.
Structure of the tangle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
V.
/~-Amyloid and its precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
VI.
Homology of ]~APP with other proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
VII.
Topographical relationships between/~-amyloid and tangles . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
The characteristic lesions of Alzheimer's disease, neurofibrillary tangles and neuritic plaques, are the sites of accumulation of abnormal fibrillar material. The structure of the paired helical filament from tangles has been analysed by electron microscopy and biochemical studies have shown that it contains microtubule associated protein tau as a component. Fibrils of jS-amyloid in the neuritic plaque arise by polymerization of a small proteolytic fragment of a much larger precursor protein. It is not yet clear what triggers the events that lead to assembly of the abnormal structures nor why the structures once formed are so resistant to turnover.
I. Introduction Structural changes in brain tissue were reported in certain cases of senile dementia by Alzheimer in 1907
Abbreviations: PHFs, paired helical filaments; SF, straight filament; GAGs, glycosaminoglycans; flAPP, fl-amyloid precursor proteins. Correspondence: R.A. Crowther, Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K.
[1]. The neuritic plaques and neurofibrillary tangles he described are now recognized as characteristic of the disease that bears his name. The severity of the dementia in life correlates with the numbers of pathological lesions found in particular areas of the brain postmortem [2]. Although small numbers of the lesions can be found in otherwise apparently normal aged individuals, the incidence of the lesions in cases of Alzheimer's disease is typically a hundred- to a thousand-fold increased. In severely affected parts of the brain, particularly the hippocampal formation and certain areas of
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the cortex, the devastation of tissue is so extensive that it is hard to imagine any normal neurons remaining amongst the lesions. Since the areas most profoundly affected are believed to be involved in memory and higher associative functions, it is not surprising that affected individuals become demented. The extensive disruption of tissue correlates at the molecular level with the assembly of various abnormal fibrous polymers, whose origin is only now beginning to be understood, though the nature of the primary molecular cause of the disorder remains unknown (for recent reviews see Refs. 3 and 4). The finding of rare kindreds in which a particularly virulent form of the disease is inherited in an autosomal dominant fashion, testifies to the existence, at least in these cases, of a single molecular cause. Whether the vast majority of so-called sporadic cases arises in an identical manner is not known, though the limited evidence available suggests that a genetic factor or factors may be involved here, too [5]. The gene predisposing to the familial form of Alzheimer's disease has been mapped to chromosome 21 [6], so it is noteworthy that long-lived individuals with Down's syndrome, caused by trisomy of chromosome 21, also develop Alzheimer pathology in their brains [7]. The plaques and tangles (Fig. 1) Alzheimer saw by light microscopy were revealed by contrasting the tissue with a silver stain and subsequently fluorescent dyes were used to fight up the pathology. Both stains pick
out, against a background of more normal neuropil, dense deposits of what proved on detailed electron microscopic examination to be fibrous aggregates. Thus the neurofibrillary tangles formed inside pyramidal cells were shown to consist principally of paired helical filaments (PHFs), whilst the extra-cellular neuritic plaques were found to be the sites of deposition of fibrils of fl-amyloid. Most recently, antibodies to the various abnormal deposits have been developed and these more sensitive probes have shown that the pathology is even more extensive than suggested by silver or fluorescent stains. In particular, the neurophil often appears disrupted by abnormal fine neuritic elements and extensive diffuse deposits of fl-amyloid may also be seen. Furthermore, the topographical relationships revealed by the antibodies suggest that the formation of tangles and the deposition of fl-amyloid might be linked. Some structural aspects of the different pathologies will now be described.
II. Structure of paired helical filaments PHFs form the principal fibrous component of neurofibrillary tangles. They were initially seen in electron micrographs of sectioned material [8], in which detailed ultrastructural analysis is difficult, though some tilting experiments were performed [9]. The PHFs appeared as double stranded structures, the images of which showed periodic variations in width between about 8 nm and 20
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;
.
Fig. 1. General view of neurofibrillary tangles and neuritic plaques. (a) Tangles form inside pyramidal cells, filling the cell body and apical dendrite. (b) Plaques represent extracellular deposits of fl-amyloid and often also have a neuritic element formed by dystrophic nerve terminals. Scale bar 100 um.
Fig. 2. Paired hefical filaments in various states of untwisting. (a) Native PHFs, showing characteristic width variation with a spacing between cross-overs of about 80 nm. (b-e) Successively more untwisted states, showing PHFs treated with 2% formic acid that have unwound into flat ribbons. In (d) and (e) each ribbon has a region (arrowed) where one of the two strands of subunits that constitute the ribbon is missing. Scale bar 100 nm.
nm with a spacing between cross-overs of about 80 nm. Subsequent studies of isolated negatively stained P H F s (Fig. 2a) showed that the P H F does indeed consist of two strands of structural subunits and that the whole P H F can be considered as a twisted ribbon [10]. Treatment of P H F s with acid leads to untwisting and production of long stretches of flat ribbon about 20 n m wide (Fig. 2 b - e ) . N o ribbons are seen in native untreated P H F preparations, though a similar untwisting effect can also be produced with alkali [10]. All stages of untwisting between standard P H F images with slightly lengthened cross-overs to completely untwisted flat ribbons can be found, indicating that that the ribbons really do arise from PHFs. The substructure of the ribbons shows four parallel stain-excluding lines of density, best seen by viewing the prints obliquely along the axis of the filament, when a double tram-track
appearance is clear. Such an appearance can also often be seen in the wide parts of P H F images. In rare instances one finds a region of a ribbon where one pair of lines is missing (Fig. 2d and e), leaving a single tram-track. However, regions exhibiting one or three of the usual four stain excluding white lines have never been observed. These half-ribbons presumably represent the untwisted form of the half-PHFs reported earlier [10]. The existence of such half structures, but not of structures containing one or three lines of density argues for an organization of the P H F based on two equivalent strands of subunits. Furthermore, the observation that breakage of P H F s leads to well-defined blunt ends and not to frayed ends suggests that the structural subunit, whatever it is, has relatively short axial extent, rather than itself being a long fibrous molecule. Mass measurements using scanning transmission electron microscopy of unstained specimens showed that pronase treatment of P H F s during their preparation leads to a loss of material, the mass per unit length being 79 k D a / n m without pronase treatment but 65 k D a / n m after pronase treatment [11]. The material removed appeared to be in the form of a morphogically ill-defined fuzzy coat around a core structure consisting of the double tram-track just described. Using computer image reconstruction techniques the cross-section through the P H F was shown to consist of two C-shaped features arranged base-to-base [12]. This elongated form of cross-section for the helical structure gives rise to the alternation in width between 8 and 20 nm, characteristic of the images of native PHFs. In the untwisted flat ribbons, each C-shaped unit gives rise to one of the tram-tracks, the penetration of stain into the cleft of the C-shaped unit and between the C-shaped units giving rise to the double tram-track appearance. In some images of native P H F s an axial spacing of 3 nm can be detected [12], though whether this really represents the axial extent of the subunit is not clear. Another form of abnormal filament, the so-called straight filament (SF), is also found in Alzheimer tangles [13] and similar looking filaments are found in other neurological disorders. The filaments are termed 'straight' because they do not exhibit the characteristic width variations shown by P H F images but appear rather to have a more or less uniform width of about 15 nm. SFs and P H F s share epitopes [14] and other observations indicate that they m a y well represent alternative forms of assembly of the same structural subunit (Crowther, R.A., unpublished results). III. Tau protein is a component of the P H F A monoclonal antibody (6/423) raised against a P H F preparation decorated the pronase-stripped P H F s more strongly than the fuzzy unstripped P H F s [11,15]. It proved to recognize protein fragments of about 10
k D a extracted by formic acid treatment of core PHFs. Protein sequence from the fragments corresponded to the repeat region of microtubule associated protein tan [15,16]. Thus the repeat region of tau, which is known to represent the microtubule binding domain of tau, forms part of the P H F core. Other parts of the tan molecule contribute to the fuzzy coat of the PHF, since some antibodies against tau decorate fuzzy P H F s but not stripped ones [11]. It is unlikely that P H F s are formed solely of tau protein. Although formic acid treatment of P H F cores released fragments of tau, the bulk of the material left after such treatment appears to be of very high molecular weight and does not enter the gel when subjected to polyacrylamide gel eletrophoresis. It is difficult to estimate what proportion of the core mass of the P H F is contributed by the repeat region of tan. If one assumes that the 3 nm axial spacing observed in some P H F images does indeed represent the axial extent of the C-shaped subunit, then that subunit would have a mass of 120 kDa before pronase treatment and 100 k D a after
pronase treatment. If the stoichiometry of tau is such that there is one tau molecule per 3 nm structural subunit, then the repeat region of tau would contribute only 10% of the mass of the core of the PHF. However, the repeating domain of each tau molecule could span more than one core subunit and the pronase treatment could be removing other material in addition to parts of the tan molecule. The identity of the remainder of the core is presently unknown. There are at least six different isoforms of human tau protein, which differ in having three or four repeats in the microtubule binding domain and by the presence or absence of additional inserts near the N-terminus of the protein [17]. The different isoforms arise from a single gene by alternative m R N A splicing. Antibodies raised against synthetic peptides corresponding to different parts of the longest tau isoform all detect neurofibrillary tangles by immunocytochemistry [17]. Protein sequencing also indicates the presence of three and four repeat regions in the P H F core [15]. It thus appears likely that all isoforms of tan protein are incorporated into PHFs.
Fig. 3. General view of a tangle in a specimen prepared by the quick-freeze deep-etch technique (reproduced with permission from Ref. 24). The nucleus (Nu) of the pyramidal cell is seen and dense bundles of PHFs push cellular organelles against the cytoplasmic membrane (arrows). Scale bar 1 urn.
N o differences in the levels of expression of mRNAs coresponding to the different isoforms could be detected in Alzheimer's disease compared with agedmatched normal controls [17,18]. Thus, if an abnormality of tau protein contributes to the assembly of PHFs in Alzheimer's disease, it is likely to be some form of post-translational modification, rather than the level of expression per se, which is operative. It does appear that abnormal forms of tau protein can be detected in Alzheimer's disease and the modification may reside in the phosphorylation state of the protein [19-22]. In this connection it is interesting to note that microtubule associated protein MAP2, which shows a high degree of homology to tau protein in its repeat region and which might therefore be expected to participate in the assembly of PHFs, appears not to do so [23], though the evidence on this point is not yet compelling.
IV. Structure of the tangle The structural studies described so far have concentrated on isolated PHFs. However, a recent study by quick-freezing and deep-etching of tissue has provided graphic images of tangles in situ [24]. An overall view of a tangle inside a pyramidal cell (Fig. 3) gives a vivid impression of dense bundles of PHFs forming in the cell body and pushing the cellular organelles up against the cytoplasmic membrane of the cell. At higher magnification (Fig. 4) the PHFs are visualized as twostranded ropes twisted in a left-handed sense, consistent with images of shadowed preparations of extracted filaments [10]. More interestingly, slender bridges about 6 nm thick can be seen linking neighbouring PHFs at intervals of about 26 nm. The chemical nature of these cross-bridges is unknown. However, it is likely that they
Fig. 4. Detailed views of a tangle in a specimen prepared by the quick-freezedeep-etch technique (reproduced with permission from Ref. 24). Many PHFs show anti-clockwise helical structure (arrows) and thin cross bridges (arrow heads) can be seen linking neighbouring PHFs. Scale bars 100 nm.
represent part of the fuzzy coat seen on extracted PHFs that have not been treated with proteinase. It is thus possible that they could be formed by tau protein, since this has been shown to form rather similar looking bridges between microtubules [25]. Neurofibrillary tangles also contain glycosaminoglycans (GAGs) [26,27], which are visualized in sectioned material, when stained with cationic dyes. Ruthenium red gives electron-dense granules associated with PHFs with a rough periodicity of 40-70 nm. Cuprolinic blue produces electron-dense tapered filaments, again associated with PHFs. The latter staining was abolished by high concentrations of magnesium chloride, suggesting that the GAGs in tangles are not highly sulphated [26]. How much the GAGs contribute to the three-dimensional organization of the tangle is unclear. Nor is it clear whether the GAGs act in some primary way during formation of the tangle or only secondarily become associated with the tangle after its formation. Possibly, as may also be the case with ubiquitination of the tangle [28,29], the decoration with GAGs represents an attempt by the body to turn over or mask the abnormal assembly. Tangles evolve from intracellular forms, such as that shown in Fig. 1, to extracellular fibrous masses. It appears that, as a tangle-bearing cell dies, the tangle becomes invaded by astrocytic processes [30]. At the light microscope level the morphology of the tangle changes from a compact flame-shaped structure to a more open, fibrous-looking expanded structure of variable shape. The immunocytochemical properties also change, in a way consistent with proteolysis of the outer fuzzy coat of the P H F [31], suggesting attempted removal of the abnormal material.
V. fl-Amyloid and its precursors The B-amyloid precursor proteins (BAPP) are a family of giycoproteins of presently unknown function. The first to be cloned corresponded to a protein of 695 amino acids, which contained putative N-terminal extracellular and C-terminal intracellular domains linked by a membrane spanning helix [32]. Subsequently variants containing an additional domain homologous to the Kunitz family of serine proteinase inhibitors and another smaller insert were found [33-35], respectively 751 and 770 amino acids long. In cultured cells BAPPs are tyrosine-sulphated, N- and O-glycosylated proteins, which appear transiently on the cell-surface [36]. A shorter, secreted, form lacking cytoplasmic and membrane-spanning regions has also been described [37]. The B-amyloid, which accumulates in neuritic plaques and in blood vessels in Alzheimer's disease, represents a small 4 kDa fragment of BAPP corresponding to the region of the extracellular domain adjacent to the membrane and part of the membrane spanning region (Fig.
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638
DAEFRItD SGYL'VItHQKLVFFAEDVGSN KC'~I IGLMVGGW'r A 1 42 Fig. 5. Relationship between /9-amyloid and its precursor protein. The sequence of the 42 amino-acid A4 protein derives from a region of the precursor just outside and within the putative membranespanning region.
5). It is presumed that the fragment, also known as A4 protein, arises by abnormal proteolytic processing but it is not known whether abnormal cleavage of normal BAPP is the primary event or whether malfunction of the normal mechanism for/3APP turnover leads to an abnormal configuration of /3APP which then gets cleaved. Whatever its mode of origin, the A4 fragment polymerizes to produce fibrils of B-amyloid which then seem to be resistant to further breakdown. In order to understand the nature of the polymerization of B-amyloid, various groups have studied synthetic peptides corresponding to different parts of the A4 sequence. What emerges very clearly is that quite different, non-overlapping, parts of the sequence can give rise to amyloid-like fibrils. Thus peptides corresponding to regions 1-28, 12-28 and 14-28 of A4, believed to lie outside the membrane in BAPP, all formed fibrils with a diameter of 8-10 nm, whereas peptides 16-28 and 18-28 formed sheets or ribbons [38,39]. X-ray diffraction patterns showed a sharp ring at a spacing of 0.47 nm and a more diffuse ring in the 1 nm region, which were taken as support for a cross-B structure, in which B-sheet strands run normal to the axis of the fibril. It was suggested that this region of the molecule in some way conferred amyloidogenic potential. However, regions 26-33 and 34-42 lying in the hydrophobic supposed membrane-spanning region of BAPP have also recently been shown to form fibrils [40] and the authors suggest that this part of the sequence is a major contributor to the formation of the fibrils. It is thus difficult to be sure that the assembly process of the complete A4 peptide and the factors that govern it in vivo are being realistically modelled by the studies of peptide fragments of A4. Equally, although small peptides may well adopt a cross-B conformation, especially when dried, it is not clear that this is the dominant secondary structure in the A4 fibrils. The critical molecular properties that drive assembly and make the resulting B-amyloid fibrils so stable and resistant to turnover still have to be identified.
VI. Homology of BAPP with other proteins The different kinds of /3APP arise by alternative m R N A splicing from a single large gene with many
exons [41]. However, it appears that the flAPP proteins are themselves members of a larger family of cell surface proteins. A protein from Drosophila that may be required for the development of the embryonic nervous system shows three regions of homology to flAPP, two regions being in the extracellular domain and the third corresponding to the whole of the cytoplasmic domain [42]. More recently a human sperm membrane protein has been shown to have a region homologous to the membrane segment and cytoplasmic domain of flAPP
[431. A further region of homology between flAPP and the fl-integrins is shown in Fig. 6. The homology in this region between flAPP and the fl-integrins is about the same as between the fl-integrins themselves. The fl-integrins provide one of the two polypeptide chains in a family of heterodimeric cell surface receptors involved in various aspects of cell adhesion (reviewed in Ref. 44). The cytoplasmic domain of the fl-integrins interacts with cytoskeletal components. However, although the region of homology corresponds to that part of the
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~I
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~3
KL
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Fig. 6. Region of homology between fl-amyloid precursor and fl-integrins, flAPP - sequence starting from residue 302 of the 695 amino-acid flAPP [32]. fll - human fibronectin receptor from residue 752 [45]. f12 - human leukocyte adhesion protein from residue 716 [46]. f13 - human platelet glycoprotein l l l a from residue 742 [47]. Amino-acids underlined in the flAPP sequence occur in at least one of the fl-integrin sequences.
cytoplasmic domain closest to the membrane in the fl-integrins, in flAPP it lies in the extracellular domain. Thus the biological significance, if any, of the homology is unclear but it may possibly represent a common binding site or effector region for a presently unknown protein. Whatever the normal function of flAPP proves to be, the protein clearly contains regions homologous
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Fig. 7. Staining with antibodies against different parts of the A4 protein (See text). (a) Amyloid plaques stained by 4G8. (b) Plaque cores stained strongly but the remainder of the plaque less strongly by BR89. (c) Diffuse amyloid stained weakly by BR88. (d) Tangle-bearing cells stained by BR88. Scale bar 80 t~m.
to several other different types of protein and these homologies may give clues to that unknown function.
VII. Topographical relationships between fl-amyloid and tangles The dense deposits of fl-amyloid in plaques and blood vessels are intensely stained by Congo-red dye and the staining shows the apple-green birefringence, taken as the defining characteristic of all amyloids. Such staining does not show the more subtle and complex distribution of fl-amyloid revealed by antibodies raised against synthetic peptides corresponding to different parts of the A4 protein. A surprising feature of the antibody staining is that antibodies to different parts of something as small as the A4 protein can give radically different staining patterns. In particular, different antibodies stain different regions of plaques and some of the antibodies strongly stain neurofibrillary tanglebearing cells. The results [31,48,49] from three such antibodies will be briefly described: BR88 was raised against A4 1-12; 4G8 against A4 17-24 [50]; BR89 against A4 28-40 (see Fig. 5 for A4 numbering). Thus, 4G8 stains amyloid plaques and diffuse amyloid deposits (Fig. 7a). BR89 stains the cores of plaques intensely, the halo surrounding the core more weakly and diffuse deposits not at all (Fig. 7b). BR88 does not stain plaques, stains diffuse amyloid deposits weakly (Fig. 7c), but stains numerous tangle-beating cells strongly (Fig. 7d). From double labelling experiments, using an antibody against amyloid and an antibody against tau protein that labels tangles, it appears that the region of amyloid staining is always outside the region of tau protein staining [49]. Thus, although at first sight the appearance of tangle-bearing cells labelled with anti-amyloid is identical to those labelled with anti-tau, a more critical examination shows that different regions of the cell are being stained and that the amyloid staining is always peripheral to the tau staining. It is most likely that the anti-tau antibodies are labelling the PHFs, since they are known to do so in isolated preparations, but that the anti-amyloid antibodies are labelling regions apposed to the membrane in ceils with intracellular tangles and material on the surface of extracellular tangles. These observations argue for a complex processing and assembly pathway for fl-amyloid on its way from being part of a large precursor molecule to its final state as a dense mass of refractory fibrils. They suggest a model [48] in which the A4 sequence exists in different configurations and in different states of aggregation. Thus the BR88 staining of tangle bearing cells indicates that the flAPP has been cleaved to expose the Nterminus of what will become the A4 protein. Further proteolysis might then release the A4 protein in an uncondensed state in which it can be labelled by 4G8.
Finally, the A4 becomes a dense mass which can be stained by BR89 but in which the epitopes for BR88 and 4G8 are masked, either by altered folding of individual monomers or by assembly into fibrils or both. It does appear that some cells containing tangles have the amino-terminal region of B-amyloid exposed on their surface and this raises the possibility that the pathology in plaques and tangles, though biochemically distinct, might be related in its development.
VIII. Conclusions It is thus clear that there are many challenging structural problems still to be solved in connection with the pathology of Alzheimer's disease. These range in scale from the level of cells and tissue to the level of atomic detail. We need to know much more about the sites of appearance of abnormal molecules and how they get transported to their sites of final deposition. We need to establish the identity of all the molecules involved in the pathology, to determine the reasons for their abnormal assembly and to discover why the assembled structures are so stable. But most crucially we need to discover the primary cause that triggers the cascade of molecular pathology associated with Alzheimer's disease.
Acknowledgements I am grateful to Dr. K. Ohtsubo for providing Figs. 3 and 4 and to M.G. Spillantini for providing Figs. 1 and 7.
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7 8 9 10 11
12 13
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33 Ponte, P., Gonzales-DeWitt, P., Schilling, J., Lieberburg, I., Fuller, F. and Cordell, B. (1988) Nature 331, 525-527. 34 Tanzi, R.E., McClatchey, A.I., Lamperti, E.D., Villa-Komaroff, L., Gusella, J.F. and Neve, R.L. (1988) Nature 331,528-530. 35 Kitaguchi, N., Takahashi, Y., Tokushima, Y., Shiojiri, S. and Ito, H. (1988) Nature 331, 530-532. 36 Weidemann, A., KOnig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters C.L. and Beyreuther, K. (1989) Cell 57, 115-126. 37 De Sauvage, F. and Octave, J-N. (1989) Science 245, 651-653. 38 Gorevic, P.D., Castano, E.M., Sarma, R. and Frangione, B. (1987) Biochem. Biophys. Res. Commun. 147, 854-862. 39 Kirschner, D.A., Inouye, H., Duffy, L.K., Sinclair, A., Lind, M. and Selkoe, D.J. (1987) Proc. Natl. Acad. Sci. USA 84, 6953-6957. 40 Halverson, K., Fraser, P.E., Kirschner, D.A. and Lansbury, P.T. (1990) Biochemistry 29, 2639-2644. 41 Lemaire, H.G., Salbaum, J.M., Multhaup, G., Kang, J., Bayney, R.M., Unterbeck, A., Beyreuther, K. and Mailer-Hill, B. (1989) Nucleic Acids Res. 17, 517-522. 42 Rosen, D.R., Martin-Morris, L., Luo, L. and White, K. (1989) Proc. Natl. Acad. Sci. USA 86, 2478-2482. 43 Yan, Y.C., Bai, Y., Wang, L., Miao, S. and Koide, S.S. (1990) Proc. Natl. Acad. Sci. USA 87, 2405-2408. 44 Geiger, B. (1989) Curr. Opin. Cell Biol. 1, 103-109. 45 Argraves, W.S., Suzuki, S., Arai, H., Thompson, K. and Piersch° bacher, M.D. (1987) J. Cell Biol. 105, 1183-1190. 46 Law, S.K.A., Gagnon, J., Hildreth, J.E., Wells, C.E., Willis, A.C. and Wong, A.J. (1987) EMBO J. 6, 915-919. 47 Fitzgerald, L.A., Steiner, B., Rail, S.C., Lo, S. and Phillips, D.R. (1987) J. Biol. Chem. 262, 3936-3939. 48 Spillantini, M.G., Goedert, M., Jakes, R. and Klug, A. (1990) Proc. Natl. Acad. Sci. USA 87, 3947-3951. 49 Spillantini, M.G., Goedert, M., Jakes, R. and Klug, A. (1990) Proc. Natl. Acad. Sci. USA 87, 3952-3956. 50 Kim, K.W., Miller, D.L., Sapienza, V.J., Chen, C.M.J., Bai, C., Grundke-lqbal, I., Currie, J.R. and Wisniewski, H.M. (1988) Neurosci. Res. Commun. 2, 121-130.
1
Elsevier
BBADIS 61002
Review
Structural aspects of pathology in Alzheimer's disease R.A. Crowther Medical Research Council, Laboratory of Molecular Biology, Cambridge (U.K.) (Received 24 May 1990)
Key words: Alzheimer's disease; Neurofibrillary tangle; Paired helical filament; Neuritic plaque; /~-Amyloid
Contents I.
Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1
II.
Structure of paired helical filaments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2
III.
Tau protein is a component of the P H F . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3
IV.
Structure of the tangle . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
5
V.
/~-Amyloid and its precursors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
VI.
Homology of ]~APP with other proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6
VII.
Topographical relationships between/~-amyloid and tangles . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8
The characteristic lesions of Alzheimer's disease, neurofibrillary tangles and neuritic plaques, are the sites of accumulation of abnormal fibrillar material. The structure of the paired helical filament from tangles has been analysed by electron microscopy and biochemical studies have shown that it contains microtubule associated protein tau as a component. Fibrils of jS-amyloid in the neuritic plaque arise by polymerization of a small proteolytic fragment of a much larger precursor protein. It is not yet clear what triggers the events that lead to assembly of the abnormal structures nor why the structures once formed are so resistant to turnover.
I. Introduction Structural changes in brain tissue were reported in certain cases of senile dementia by Alzheimer in 1907
Abbreviations: PHFs, paired helical filaments; SF, straight filament; GAGs, glycosaminoglycans; flAPP, fl-amyloid precursor proteins. Correspondence: R.A. Crowther, Medical Research Council, Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K.
[1]. The neuritic plaques and neurofibrillary tangles he described are now recognized as characteristic of the disease that bears his name. The severity of the dementia in life correlates with the numbers of pathological lesions found in particular areas of the brain postmortem [2]. Although small numbers of the lesions can be found in otherwise apparently normal aged individuals, the incidence of the lesions in cases of Alzheimer's disease is typically a hundred- to a thousand-fold increased. In severely affected parts of the brain, particularly the hippocampal formation and certain areas of
0925-4439/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
the cortex, the devastation of tissue is so extensive that it is hard to imagine any normal neurons remaining amongst the lesions. Since the areas most profoundly affected are believed to be involved in memory and higher associative functions, it is not surprising that affected individuals become demented. The extensive disruption of tissue correlates at the molecular level with the assembly of various abnormal fibrous polymers, whose origin is only now beginning to be understood, though the nature of the primary molecular cause of the disorder remains unknown (for recent reviews see Refs. 3 and 4). The finding of rare kindreds in which a particularly virulent form of the disease is inherited in an autosomal dominant fashion, testifies to the existence, at least in these cases, of a single molecular cause. Whether the vast majority of so-called sporadic cases arises in an identical manner is not known, though the limited evidence available suggests that a genetic factor or factors may be involved here, too [5]. The gene predisposing to the familial form of Alzheimer's disease has been mapped to chromosome 21 [6], so it is noteworthy that long-lived individuals with Down's syndrome, caused by trisomy of chromosome 21, also develop Alzheimer pathology in their brains [7]. The plaques and tangles (Fig. 1) Alzheimer saw by light microscopy were revealed by contrasting the tissue with a silver stain and subsequently fluorescent dyes were used to fight up the pathology. Both stains pick
out, against a background of more normal neuropil, dense deposits of what proved on detailed electron microscopic examination to be fibrous aggregates. Thus the neurofibrillary tangles formed inside pyramidal cells were shown to consist principally of paired helical filaments (PHFs), whilst the extra-cellular neuritic plaques were found to be the sites of deposition of fibrils of fl-amyloid. Most recently, antibodies to the various abnormal deposits have been developed and these more sensitive probes have shown that the pathology is even more extensive than suggested by silver or fluorescent stains. In particular, the neurophil often appears disrupted by abnormal fine neuritic elements and extensive diffuse deposits of fl-amyloid may also be seen. Furthermore, the topographical relationships revealed by the antibodies suggest that the formation of tangles and the deposition of fl-amyloid might be linked. Some structural aspects of the different pathologies will now be described.
II. Structure of paired helical filaments PHFs form the principal fibrous component of neurofibrillary tangles. They were initially seen in electron micrographs of sectioned material [8], in which detailed ultrastructural analysis is difficult, though some tilting experiments were performed [9]. The PHFs appeared as double stranded structures, the images of which showed periodic variations in width between about 8 nm and 20
|
.
¢
;
.
Fig. 1. General view of neurofibrillary tangles and neuritic plaques. (a) Tangles form inside pyramidal cells, filling the cell body and apical dendrite. (b) Plaques represent extracellular deposits of fl-amyloid and often also have a neuritic element formed by dystrophic nerve terminals. Scale bar 100 um.
Fig. 2. Paired hefical filaments in various states of untwisting. (a) Native PHFs, showing characteristic width variation with a spacing between cross-overs of about 80 nm. (b-e) Successively more untwisted states, showing PHFs treated with 2% formic acid that have unwound into flat ribbons. In (d) and (e) each ribbon has a region (arrowed) where one of the two strands of subunits that constitute the ribbon is missing. Scale bar 100 nm.
nm with a spacing between cross-overs of about 80 nm. Subsequent studies of isolated negatively stained P H F s (Fig. 2a) showed that the P H F does indeed consist of two strands of structural subunits and that the whole P H F can be considered as a twisted ribbon [10]. Treatment of P H F s with acid leads to untwisting and production of long stretches of flat ribbon about 20 n m wide (Fig. 2 b - e ) . N o ribbons are seen in native untreated P H F preparations, though a similar untwisting effect can also be produced with alkali [10]. All stages of untwisting between standard P H F images with slightly lengthened cross-overs to completely untwisted flat ribbons can be found, indicating that that the ribbons really do arise from PHFs. The substructure of the ribbons shows four parallel stain-excluding lines of density, best seen by viewing the prints obliquely along the axis of the filament, when a double tram-track
appearance is clear. Such an appearance can also often be seen in the wide parts of P H F images. In rare instances one finds a region of a ribbon where one pair of lines is missing (Fig. 2d and e), leaving a single tram-track. However, regions exhibiting one or three of the usual four stain excluding white lines have never been observed. These half-ribbons presumably represent the untwisted form of the half-PHFs reported earlier [10]. The existence of such half structures, but not of structures containing one or three lines of density argues for an organization of the P H F based on two equivalent strands of subunits. Furthermore, the observation that breakage of P H F s leads to well-defined blunt ends and not to frayed ends suggests that the structural subunit, whatever it is, has relatively short axial extent, rather than itself being a long fibrous molecule. Mass measurements using scanning transmission electron microscopy of unstained specimens showed that pronase treatment of P H F s during their preparation leads to a loss of material, the mass per unit length being 79 k D a / n m without pronase treatment but 65 k D a / n m after pronase treatment [11]. The material removed appeared to be in the form of a morphogically ill-defined fuzzy coat around a core structure consisting of the double tram-track just described. Using computer image reconstruction techniques the cross-section through the P H F was shown to consist of two C-shaped features arranged base-to-base [12]. This elongated form of cross-section for the helical structure gives rise to the alternation in width between 8 and 20 nm, characteristic of the images of native PHFs. In the untwisted flat ribbons, each C-shaped unit gives rise to one of the tram-tracks, the penetration of stain into the cleft of the C-shaped unit and between the C-shaped units giving rise to the double tram-track appearance. In some images of native P H F s an axial spacing of 3 nm can be detected [12], though whether this really represents the axial extent of the subunit is not clear. Another form of abnormal filament, the so-called straight filament (SF), is also found in Alzheimer tangles [13] and similar looking filaments are found in other neurological disorders. The filaments are termed 'straight' because they do not exhibit the characteristic width variations shown by P H F images but appear rather to have a more or less uniform width of about 15 nm. SFs and P H F s share epitopes [14] and other observations indicate that they m a y well represent alternative forms of assembly of the same structural subunit (Crowther, R.A., unpublished results). III. Tau protein is a component of the P H F A monoclonal antibody (6/423) raised against a P H F preparation decorated the pronase-stripped P H F s more strongly than the fuzzy unstripped P H F s [11,15]. It proved to recognize protein fragments of about 10
k D a extracted by formic acid treatment of core PHFs. Protein sequence from the fragments corresponded to the repeat region of microtubule associated protein tan [15,16]. Thus the repeat region of tau, which is known to represent the microtubule binding domain of tau, forms part of the P H F core. Other parts of the tan molecule contribute to the fuzzy coat of the PHF, since some antibodies against tau decorate fuzzy P H F s but not stripped ones [11]. It is unlikely that P H F s are formed solely of tau protein. Although formic acid treatment of P H F cores released fragments of tau, the bulk of the material left after such treatment appears to be of very high molecular weight and does not enter the gel when subjected to polyacrylamide gel eletrophoresis. It is difficult to estimate what proportion of the core mass of the P H F is contributed by the repeat region of tan. If one assumes that the 3 nm axial spacing observed in some P H F images does indeed represent the axial extent of the C-shaped subunit, then that subunit would have a mass of 120 kDa before pronase treatment and 100 k D a after
pronase treatment. If the stoichiometry of tau is such that there is one tau molecule per 3 nm structural subunit, then the repeat region of tau would contribute only 10% of the mass of the core of the PHF. However, the repeating domain of each tau molecule could span more than one core subunit and the pronase treatment could be removing other material in addition to parts of the tan molecule. The identity of the remainder of the core is presently unknown. There are at least six different isoforms of human tau protein, which differ in having three or four repeats in the microtubule binding domain and by the presence or absence of additional inserts near the N-terminus of the protein [17]. The different isoforms arise from a single gene by alternative m R N A splicing. Antibodies raised against synthetic peptides corresponding to different parts of the longest tau isoform all detect neurofibrillary tangles by immunocytochemistry [17]. Protein sequencing also indicates the presence of three and four repeat regions in the P H F core [15]. It thus appears likely that all isoforms of tan protein are incorporated into PHFs.
Fig. 3. General view of a tangle in a specimen prepared by the quick-freeze deep-etch technique (reproduced with permission from Ref. 24). The nucleus (Nu) of the pyramidal cell is seen and dense bundles of PHFs push cellular organelles against the cytoplasmic membrane (arrows). Scale bar 1 urn.
N o differences in the levels of expression of mRNAs coresponding to the different isoforms could be detected in Alzheimer's disease compared with agedmatched normal controls [17,18]. Thus, if an abnormality of tau protein contributes to the assembly of PHFs in Alzheimer's disease, it is likely to be some form of post-translational modification, rather than the level of expression per se, which is operative. It does appear that abnormal forms of tau protein can be detected in Alzheimer's disease and the modification may reside in the phosphorylation state of the protein [19-22]. In this connection it is interesting to note that microtubule associated protein MAP2, which shows a high degree of homology to tau protein in its repeat region and which might therefore be expected to participate in the assembly of PHFs, appears not to do so [23], though the evidence on this point is not yet compelling.
IV. Structure of the tangle The structural studies described so far have concentrated on isolated PHFs. However, a recent study by quick-freezing and deep-etching of tissue has provided graphic images of tangles in situ [24]. An overall view of a tangle inside a pyramidal cell (Fig. 3) gives a vivid impression of dense bundles of PHFs forming in the cell body and pushing the cellular organelles up against the cytoplasmic membrane of the cell. At higher magnification (Fig. 4) the PHFs are visualized as twostranded ropes twisted in a left-handed sense, consistent with images of shadowed preparations of extracted filaments [10]. More interestingly, slender bridges about 6 nm thick can be seen linking neighbouring PHFs at intervals of about 26 nm. The chemical nature of these cross-bridges is unknown. However, it is likely that they
Fig. 4. Detailed views of a tangle in a specimen prepared by the quick-freezedeep-etch technique (reproduced with permission from Ref. 24). Many PHFs show anti-clockwise helical structure (arrows) and thin cross bridges (arrow heads) can be seen linking neighbouring PHFs. Scale bars 100 nm.
represent part of the fuzzy coat seen on extracted PHFs that have not been treated with proteinase. It is thus possible that they could be formed by tau protein, since this has been shown to form rather similar looking bridges between microtubules [25]. Neurofibrillary tangles also contain glycosaminoglycans (GAGs) [26,27], which are visualized in sectioned material, when stained with cationic dyes. Ruthenium red gives electron-dense granules associated with PHFs with a rough periodicity of 40-70 nm. Cuprolinic blue produces electron-dense tapered filaments, again associated with PHFs. The latter staining was abolished by high concentrations of magnesium chloride, suggesting that the GAGs in tangles are not highly sulphated [26]. How much the GAGs contribute to the three-dimensional organization of the tangle is unclear. Nor is it clear whether the GAGs act in some primary way during formation of the tangle or only secondarily become associated with the tangle after its formation. Possibly, as may also be the case with ubiquitination of the tangle [28,29], the decoration with GAGs represents an attempt by the body to turn over or mask the abnormal assembly. Tangles evolve from intracellular forms, such as that shown in Fig. 1, to extracellular fibrous masses. It appears that, as a tangle-bearing cell dies, the tangle becomes invaded by astrocytic processes [30]. At the light microscope level the morphology of the tangle changes from a compact flame-shaped structure to a more open, fibrous-looking expanded structure of variable shape. The immunocytochemical properties also change, in a way consistent with proteolysis of the outer fuzzy coat of the P H F [31], suggesting attempted removal of the abnormal material.
V. fl-Amyloid and its precursors The B-amyloid precursor proteins (BAPP) are a family of giycoproteins of presently unknown function. The first to be cloned corresponded to a protein of 695 amino acids, which contained putative N-terminal extracellular and C-terminal intracellular domains linked by a membrane spanning helix [32]. Subsequently variants containing an additional domain homologous to the Kunitz family of serine proteinase inhibitors and another smaller insert were found [33-35], respectively 751 and 770 amino acids long. In cultured cells BAPPs are tyrosine-sulphated, N- and O-glycosylated proteins, which appear transiently on the cell-surface [36]. A shorter, secreted, form lacking cytoplasmic and membrane-spanning regions has also been described [37]. The B-amyloid, which accumulates in neuritic plaques and in blood vessels in Alzheimer's disease, represents a small 4 kDa fragment of BAPP corresponding to the region of the extracellular domain adjacent to the membrane and part of the membrane spanning region (Fig.
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638
DAEFRItD SGYL'VItHQKLVFFAEDVGSN KC'~I IGLMVGGW'r A 1 42 Fig. 5. Relationship between /9-amyloid and its precursor protein. The sequence of the 42 amino-acid A4 protein derives from a region of the precursor just outside and within the putative membranespanning region.
5). It is presumed that the fragment, also known as A4 protein, arises by abnormal proteolytic processing but it is not known whether abnormal cleavage of normal BAPP is the primary event or whether malfunction of the normal mechanism for/3APP turnover leads to an abnormal configuration of /3APP which then gets cleaved. Whatever its mode of origin, the A4 fragment polymerizes to produce fibrils of B-amyloid which then seem to be resistant to further breakdown. In order to understand the nature of the polymerization of B-amyloid, various groups have studied synthetic peptides corresponding to different parts of the A4 sequence. What emerges very clearly is that quite different, non-overlapping, parts of the sequence can give rise to amyloid-like fibrils. Thus peptides corresponding to regions 1-28, 12-28 and 14-28 of A4, believed to lie outside the membrane in BAPP, all formed fibrils with a diameter of 8-10 nm, whereas peptides 16-28 and 18-28 formed sheets or ribbons [38,39]. X-ray diffraction patterns showed a sharp ring at a spacing of 0.47 nm and a more diffuse ring in the 1 nm region, which were taken as support for a cross-B structure, in which B-sheet strands run normal to the axis of the fibril. It was suggested that this region of the molecule in some way conferred amyloidogenic potential. However, regions 26-33 and 34-42 lying in the hydrophobic supposed membrane-spanning region of BAPP have also recently been shown to form fibrils [40] and the authors suggest that this part of the sequence is a major contributor to the formation of the fibrils. It is thus difficult to be sure that the assembly process of the complete A4 peptide and the factors that govern it in vivo are being realistically modelled by the studies of peptide fragments of A4. Equally, although small peptides may well adopt a cross-B conformation, especially when dried, it is not clear that this is the dominant secondary structure in the A4 fibrils. The critical molecular properties that drive assembly and make the resulting B-amyloid fibrils so stable and resistant to turnover still have to be identified.
VI. Homology of BAPP with other proteins The different kinds of /3APP arise by alternative m R N A splicing from a single large gene with many
exons [41]. However, it appears that the flAPP proteins are themselves members of a larger family of cell surface proteins. A protein from Drosophila that may be required for the development of the embryonic nervous system shows three regions of homology to flAPP, two regions being in the extracellular domain and the third corresponding to the whole of the cytoplasmic domain [42]. More recently a human sperm membrane protein has been shown to have a region homologous to the membrane segment and cytoplasmic domain of flAPP
[431. A further region of homology between flAPP and the fl-integrins is shown in Fig. 6. The homology in this region between flAPP and the fl-integrins is about the same as between the fl-integrins themselves. The fl-integrins provide one of the two polypeptide chains in a family of heterodimeric cell surface receptors involved in various aspects of cell adhesion (reviewed in Ref. 44). The cytoplasmic domain of the fl-integrins interacts with cytoskeletal components. However, although the region of homology corresponds to that part of the
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Fig. 6. Region of homology between fl-amyloid precursor and fl-integrins, flAPP - sequence starting from residue 302 of the 695 amino-acid flAPP [32]. fll - human fibronectin receptor from residue 752 [45]. f12 - human leukocyte adhesion protein from residue 716 [46]. f13 - human platelet glycoprotein l l l a from residue 742 [47]. Amino-acids underlined in the flAPP sequence occur in at least one of the fl-integrin sequences.
cytoplasmic domain closest to the membrane in the fl-integrins, in flAPP it lies in the extracellular domain. Thus the biological significance, if any, of the homology is unclear but it may possibly represent a common binding site or effector region for a presently unknown protein. Whatever the normal function of flAPP proves to be, the protein clearly contains regions homologous
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Fig. 7. Staining with antibodies against different parts of the A4 protein (See text). (a) Amyloid plaques stained by 4G8. (b) Plaque cores stained strongly but the remainder of the plaque less strongly by BR89. (c) Diffuse amyloid stained weakly by BR88. (d) Tangle-bearing cells stained by BR88. Scale bar 80 t~m.
to several other different types of protein and these homologies may give clues to that unknown function.
VII. Topographical relationships between fl-amyloid and tangles The dense deposits of fl-amyloid in plaques and blood vessels are intensely stained by Congo-red dye and the staining shows the apple-green birefringence, taken as the defining characteristic of all amyloids. Such staining does not show the more subtle and complex distribution of fl-amyloid revealed by antibodies raised against synthetic peptides corresponding to different parts of the A4 protein. A surprising feature of the antibody staining is that antibodies to different parts of something as small as the A4 protein can give radically different staining patterns. In particular, different antibodies stain different regions of plaques and some of the antibodies strongly stain neurofibrillary tanglebearing cells. The results [31,48,49] from three such antibodies will be briefly described: BR88 was raised against A4 1-12; 4G8 against A4 17-24 [50]; BR89 against A4 28-40 (see Fig. 5 for A4 numbering). Thus, 4G8 stains amyloid plaques and diffuse amyloid deposits (Fig. 7a). BR89 stains the cores of plaques intensely, the halo surrounding the core more weakly and diffuse deposits not at all (Fig. 7b). BR88 does not stain plaques, stains diffuse amyloid deposits weakly (Fig. 7c), but stains numerous tangle-beating cells strongly (Fig. 7d). From double labelling experiments, using an antibody against amyloid and an antibody against tau protein that labels tangles, it appears that the region of amyloid staining is always outside the region of tau protein staining [49]. Thus, although at first sight the appearance of tangle-bearing cells labelled with anti-amyloid is identical to those labelled with anti-tau, a more critical examination shows that different regions of the cell are being stained and that the amyloid staining is always peripheral to the tau staining. It is most likely that the anti-tau antibodies are labelling the PHFs, since they are known to do so in isolated preparations, but that the anti-amyloid antibodies are labelling regions apposed to the membrane in ceils with intracellular tangles and material on the surface of extracellular tangles. These observations argue for a complex processing and assembly pathway for fl-amyloid on its way from being part of a large precursor molecule to its final state as a dense mass of refractory fibrils. They suggest a model [48] in which the A4 sequence exists in different configurations and in different states of aggregation. Thus the BR88 staining of tangle bearing cells indicates that the flAPP has been cleaved to expose the Nterminus of what will become the A4 protein. Further proteolysis might then release the A4 protein in an uncondensed state in which it can be labelled by 4G8.
Finally, the A4 becomes a dense mass which can be stained by BR89 but in which the epitopes for BR88 and 4G8 are masked, either by altered folding of individual monomers or by assembly into fibrils or both. It does appear that some cells containing tangles have the amino-terminal region of B-amyloid exposed on their surface and this raises the possibility that the pathology in plaques and tangles, though biochemically distinct, might be related in its development.
VIII. Conclusions It is thus clear that there are many challenging structural problems still to be solved in connection with the pathology of Alzheimer's disease. These range in scale from the level of cells and tissue to the level of atomic detail. We need to know much more about the sites of appearance of abnormal molecules and how they get transported to their sites of final deposition. We need to establish the identity of all the molecules involved in the pathology, to determine the reasons for their abnormal assembly and to discover why the assembled structures are so stable. But most crucially we need to discover the primary cause that triggers the cascade of molecular pathology associated with Alzheimer's disease.
Acknowledgements I am grateful to Dr. K. Ohtsubo for providing Figs. 3 and 4 and to M.G. Spillantini for providing Figs. 1 and 7.
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