Tau protein and the neurofibrillary pathology of Alzheimer's disease

41 Meldrum, B. S. etaL (1992) Brain Res. 593, 1-6 42 Prenen, G. H. M., Go, K. G., Postema, F., Zuiderveen, F. and Koff, J. (1988) Exp. Neurol. 99, 118-132 43 Yamasaki, Y., Kogure, K., Hara, H., Ban, H. and Akaika, N. (1991) Neurosci. Lett. 121,251-254 44 Boening, J. A., Kass, I. S., Cottrell, J. E. and Chambers, G. (1989) Neuroscience 33, 263-268 45 Tasker, R. C., Coyle, J. T. and Vornov, J. J. (1992) J. Neurosci. 12, 4298-4308 46 Burke, S. P. and Taylor, C. P. (1991) Soc. Neurosci. Abstr. 17,

1267 47 Hansen, A. J. (1985) PhysioL Rev. 65, 101-148 48 Nedergaard, M. and Astrup, J. (1986) J. Cerebr. Blood Flow Metab. 6, 607-615 49 Gill, R., Andin6, P., Hillered, L., Persson, L. and Hagberg, H. (1992) J. Cerebr. Blood Flow Metab. 12, 371-379 50 Kass, I. S., Abramowicz, A. E., Cottrell, J. E. and Chambers, G. (1992) Neuroscience 49, 537-543 51 Haigney, M. C. P., Miyata, H., Lakatta, E. G., Stern, M. D. and Silverman, H. S. (1992) Circ. Res. 71,547-557

Tauprotein and the neurol brillarypathologyofAIzheimer's disease Michel Goedert

Michel Goedertis at the MedicalResearch Council,Laboratory of MolecularBiology, HillsRoad, Cambridge, UK CB22QH.

Abundant neurofibrillary tangles, neuropil threads and senile plaque neurites constitute the neurofibrillary pathology of Alzheimer's disease. They form in the nerve cells that undergo degeneration in the disease, in which their regional distribution correlates with the degree of dementia. Each lesion contains the paired helical filament (PHF) as its major fibrous component. Recent work has shown that PHFs are composed of the microtubule-associated protein tau in an abnormally phosphorylated state. PHF-tau is hyperphosphorylated on all six adult brain isoforms. As a consequence, tau is unable to bind to microtubules and is believed to selfassemble into the PHF. Current evidence suggests that protein kinases or protein phosphatases with a specificity for serine/threonine-proline residues are involved in the abnormal phosphorylation of tau. Alzheimer's disease is characterized clinically by a progressive loss of memory and other cognitive functions, resulting in a profound dementia. The intellectual decline is accompanied by the progressive

Fig.

1. Neurofibrillary pathology in the entorhinal cortex. The section was stained with an anti-tau antiserum. Abbreviations: NFT, neurofibrillary tangle; NT, neuropil threads; NP, neuritic plaque. Scale bar, lO0t~m. (Reproduced, with permission, from Ref. 643 460

© 1993, ElsevierSciencePublishersLtd,(UK)

accumulation in the brain of insoluble fibrous material, both extracellularly and within nerve cells. Extracellular deposits are made of beta-amyloid protein A[~1'2. Initial deposits are non-fibrillar but are progressively transformed into fibrils, giving rise to the characteristic amyloid plaques. Neurofibrillary lesions constitute the intraneuronal deposits. They are found in cell bodies and apical dendrites as neurofibrillary tangles, in distal dendrites as neuropil threads and in the abnormal neurites that are associated with some amyloid plaques (neuritic plaques) (Fig. 1). Ultrastructurally, all three lesions contain abnormal paired helical filaments (PHFs) as their major fibrous components and straight filaments (SFs) as their minor fibrous components (Fig. 2)3. NeurofibriUary lesions develop in the nerve cells that undergo degeneration in Alzheimer's disease. Their relative insolubility enables them to survive after the death of the affected nerve cells as extracellular tangles (or ghost tangles) that accumulate in the neuropil. These are then engulfed by astrocytes and are probably slowly degraded. Over the past five years significant progress has been made in unravelling the molecular composition of PHFs and in deducing possible mechanisms that may lead to their assembly. Current evidence strongly suggests that they are made entirely of the microtubule-associated protein tan in an abnormally phosphorylated state. Moreover, earlier results 4 indicating that the extent and topographical distribution of neurofibrillary lesions provide a reliable pathological correlate of the degree of dementia have been confirmed and extended 5'6.

Neuropathological stages of Alzheimer's disease The development of the neurofibrillary lesions is not random but follows a stereotyped pattern with regard to affected cell types, cellular layers and brain regions, with little individual variation. This has recently been used to define six neuropathological stages of Alzheimer's disease (Fig. 3) 2. The very first nerve cells in the brain to develop neurofibrillary lesions are located in layer pre-alpha of the trans-entorhinal region, thus defining stage I. Stage II shows a more severe involvement of this region, as well as a mild involvement of the pre-alpha TINS, VoL 16, NO. 11, 1993

layer of the entorhinal cortex. Patients with this pathology are cognitively unimpaired, indicating that stages I and II may represent clinically silent stages of Alzheimer's disease. Mild impairments of cognitive function become apparent in stages III and IV. Stage III is characterized by severe neurofibrillary lesions in the pre-alpha layers of both entorhinal and transentorhinal regions. The vast majority of nerve cells shows neurofibrillary tangles and dendritic neuropil threads. The first extracellular tangles also appear during stage III. In stage IV the deep pre-alpha layer develops extensive neurofibrillary lesions. During stages III and IV mild changes are also seen in layer I of Ammon's horn of the hippocampus and in a number of subcortical nuclei, such as the basal forebrain magnocellular nuclei and the anterodorsal tbalamic nucleus. The major feature of stages V and VI is the massive development of neurofibrillary lesions in isocortical association areas. They meet the criteria for the neuropathological diagnosis of Alzheimer's disease and are found in patients who were severely demented at the time of death. The stereotyped nature of the temporal and spatial development of neurofibrillary lesions contrasts with the development of A[3 deposits. They show a density and distribution pattern that are subject to great individual variation, precluding their use for the neuropathological staging of Alzheimer's diseaseS. In general, the first A[3 deposits occur in isocortical areas of the frontal, temporal and occipital lobes. This contrasts with the neurofibrillary lesions which first appear in the trans-entorhinal region. Moreover, A[3 deposits develop relatively late in the fascia dentata of the hippocampus, the major termination area of the pre-alpha layer cells of the entorhinal cortex. It follows that neurofibrillary changes in the pre-alpha layer can develop with no parts of these cells or their processes in contact with A[3 deposits. These findings are inconsistent with the view that the neurofibrillary pathology develops as a mere consequence of the neurotoxic action of A[37.

Fig. 2. Electron micrographs of negatively stained abnormal filaments from the brain of an Alzheimer's disease patient. (A) Low-power view showing predominantly paired helical filaments but with a few straight filaments (arrows). (B,C) High-power view of a paired hefical filament (B) and a straight filament (C). Scale bars: (A), 200nm; (B,C), lOOnm. (Reproduced, with permission, from Ref. Co4.)

units, corresponding to the two strands of the PHF, arranged in a base-to-base manner8. When dispersed filaments are treated with pronase under the same conditions they are completely degraded13, illustrating the differing protease sensitivities of tangle fragment PHFs and of dispersed PHFs. Straight filaments represent a minority species, both in tangle fragment and dispersed filament preparations. Images of SFs show approximately the same apparent periodicity as PHFs but a much less marked modulation in width (Fig. 2) 14. Straight filaments and PHFs share tau epitopes and behave in a similar manner when treated with pronase. Rarely, hybrid filaments are observed which show a sharp transition from a segment of PHF into a segment of SF. This Structure of the PHF indicates that PHFs and SFs contain identical or The PHF, as its name suggests, consists of two closely related subunits that are arranged differently strands of subunits which twist around one another in in the two types of filament. This is supported by the a helical fashion (Fig. 2). When viewed in the electron computed cross-section of the SF which shows two Cmicroscope, the helical twist and relative disposition shaped subunits very similar to those seen in the PHF of the two strands give rise to images in which the but arranged back-to-back rather than base-to-base 14. width alternates between about 8 and 20 nm, with an The SF is thus a structural variant of the PHF, in that apparent period of 80 nm (Ref. 8). both contain two strands of closely related or possibly PHFs can be isolated either in the form of tangle identical subunits but the relative arrangement of the fragments 9'1° or in the form of dispersed fila- two strands differs in the two kinds of filament. ments 11'12. The two types of PHFs have tau epitopes in common but differ in their solubility in strong Tau protein denaturing agents. While a majority of dispersed Multiple tau isoforms are produced from a single PHFs is soluble in gnanidine or sodium dodecyl- gene through alternative mRNA-splicing 15. In adult sulphate, a majority of tangle fragment PHFs is human brain six isoforms are found, which range from insoluble in these reagents. Both types of PHFs differ 352 to 441 amino acids and differ from each other by also in their sensitivity to proteases. Pronase treat- the presence or absence of three inserts (Fig. 4A) 16. ment of tangle preparations removes a fuzzy coat The most striking feature of the tau sequences is the from the PHF and leaves behind a pronase-resistant presence of three or four tandem repeats of 31 or 32 core. The morphology of the core is similar to that of amino acids located in the carboxy-terminus half. untreated PHFs but structural details are seen more Experiments with recombinant tau proteins show that clearly because the disordered coat has been re- the repeats constitute microtubule-binding domains 17. moved. From electron micrographs it is possible to Microtubules assembled in the presence of tau show compute a map of the cross-sectional density in the arms projecting from the surfaceTM. Tan thus consists core. Such maps show two C-shaped morphological of a carboxy-terminus microtubule-binding domain and

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461

Trans-entorhinal I-II

Limbic Ill-IV

Isocortical V-VI

proline-directed protein kinase phosphorylate tau on at least some of these residues in vitro 27-31. Prolonged incubation with MAP kinase phosphorylates up to 12-14 residues in recombinant tau 27'2s, while glycogen synthase kinase-3 phosphorylates four residues 29'3°, as does proline-directed protein kinase 31. The phosphorylation state of a protein is the result of a balance between protein kinase and protein phosphatase activities. Of the major brain phosphatase activities tau phosphorylated by MAP kinase is dephosphorylated only by protein phosphatase 2A (Ref. 28). This enzyme exists in vivo as a 36 kDa catalytic subunit that is complexed to a 60kDa A-subunit, which in turn is complexed to one of several B-subunits a2. The latter exist as Fig. B. Six stages of the neurofibrillary pathology of Alzheimer's disease. Stages I and II show several isoforms produced from changes that are largely confined to the trans-entorhinal region (trans-entorhinal I-II). Stages III distinct genes, at least one of and IV show severe changes in the pre-alpha layer of the entorhinal and trans-entorhinal regions which is specific for the nervous (limbic stages Ill-IV). Stages V and Vl show additional changes throughout the isocortex (isocortical stages V-Vl). Increasing density of shading indicates the increasing severity of the neurofibrillary system. The catalytic subunit of protein phosphatase 2A dephospathology. (Taken, with permission, from Ref. 5.) phorylates tau very poorly, whereas the trimeric form of the enzyme an amino-terminus projection domain. Besides being is much more effective than the dimeric form consisting distinguished by the presence of three or four tandem of the catalytic subunit and the A-subunit. This raises repeats, some tau isoforms contain 29 or 58 amino the possibility that a B-subunit may be instrumental acid inserts located near the amino terminus (Fig. in the dephosphorylation of tau phosphorylated at 4A). Isoforms with a large additional insert in the serine/threonine-proline sites. amino-terminus half have recently been described in The largest human brain tan isoform contains 17 the PNS 19'2°. serine/threonine-proline sites (Fig. 5) 16. They show Tau is subject to developmentally regulated alterna- an intriguing distribution in that they are distributed tive mRNA-splicing in that in immature brain only the throughout the sequence, with the notable exception transcript encoding the shortest isoform with three of the microtubule-binding tandem repeat region. In repeats is expressed 16. The developmental shift of tau particular, a cluster of serine/threonine-proline sites bands from a simple foetal pattern to a more complex is present in a proline-fich region of the protein adult pattern thus involves the transition from the located upstream of the repeats and a smaller cluster expression of the isoform with three repeats and no at the carboxy terminus end. inserts to the expression of all six isoforms. Tau is a phosphoprotein 21 and phosphorylation is Tau functions also developmentally regulated. Thus, tau from imEver since its discovery tau has been known to be a mature brain is phosphorylated at more sites than is potent promoter of tubulin polymerization in vitro33. tau from adult brain, implying selective dephosphoryl- The binding of tau to microtubules reduces their ation of the shortest isoform during brain maturation. dynamic instability a4. Analysis of the dynamics of the In immature brain tau is phosphorylated at six to eight growth of individual microtubules indicates that tau sites on the shortest isoform, whereas in adult brain it increases the rate of association and decreases the is phosphorylated at two or three sites on all six rate of dissociation of tubulin molecules at the growing isoforms22. Several phosphorylation sites have been end and inhibits the transition to the catastrophic identified through the use of mass spectrometry and shortening phase. Binding studies indicate that the of phosphorylation-dependent antibodies 23-26. Thus, affinity of the 31 or 32 amino acid-repeat region for in foetal brain, a significant fraction of tau is phos- microtubules is concentrated in 18 amino acid-binding phorylated at serine residues 202, 396 and 404 elements that are separated by flexible linker se(according to the numbering of the largest human quences 17. Tau may have a similar function in vivo; brain tau isoform); in adult brain, serine 404 is still when microinjected into fibroblasts it produces an phosphorylated, whereas serines 202 and 396 are no increase in microtubule mass and an increased resistlonger phosphorylated. All these serine residues are ance of microtubules to depolymerizing agents 35. followed by a proline, suggesting that protein kinases Several laboratories have transfected various tau with a specificity for seryl-proline and threonyl- constructs into cells that do not normally express proline are responsible for the phosphorylation of tau tau 25'a~'-4°. In the transfected cells tau binds to in normal brain. Accordingly, mitogen-activated pro- microtubules, enhances microtubule stability and, in tein (MAP) kinase, glycogen synthase kinase-3 and some cases, induces microtubule bundling. The 462

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bundle thickness is i0-i00 times that of normal microtubules, with an average distance of about 20 nm between adjacent microtubules. Bundling is probably a consequence of the microtubule stabilization effected by tau. At present it is unclear whether microtubule bundling has a physiological correlate or whether it results from the non-physiological overexpression of tan in some cell types. Experiments with truncated tau proteins have confirmed the critical role played by the tandem repeat region in microtubule binding; however, the inclusion of sequences flanking the repeats appears to be essential for optimal binding and bundling 38'4°. In particular, the proline-rich region upstream of the repeats appears to be essential for tubulin binding in vivo4°; this is the region with a cluster of serine/threonine--proline phosphorylation sites. In Sf9 insect cells infected with a baculovirus construct, the overexpression of tau results not only 1

352

A 1

381

1

B

410

C 1

383

1

D

412

E 441

F

in microtubule bundles, but also in the extension of long and thin neurite-like extensions37. However, overexpression of tan does not invariably lead to the extension of neurite-like processes. This might be due to different levels of expression and phosphorylation in the various cell types. Tensile forces exerted by the cortical actin network are likely to represent another factor. Thus, cells transfected with the microtubule-associated protein MAP2c only grow processes when treated with the actin depolymerizing drug cytochalasin B (Ref. 41). The introduction into developing cerebellar nerve cells of anti-sense oligonucleotides to block tau expression prevents the differentiation of the short neurites, one of which would otherwise have developed into an axon 42. Similarly, tan anti-sense oligonucleotides produce a retraction of neurites in PC12 cells treated with nerve growth f a c t o r 43. These results suggest that axonal morphology might be dependent on tau expression, although it is unclear whether tan plays a critical role in the establishment of neuronal polarity. Phosphorylation negatively regulates the ability of tan to bind to microtubules. Tan phosphorylated by MAP kinase has one-tenth the ability of non-phosphorylated tau to bind to microtubules34. Little is known about the relative contributions made by individual phosphorylated residues. A recent study using transfected cells has shown that phosphoserine residue 396 makes a significant contribution towards the reduced ability of tau to bind to microtubuleszt. This approach should permit the identification of the other sites involved.

Ser404 Ser396

,m t~ O to °~ t~o

E o

Ser235 Thr231

Thr217 Tau protein and the PHF Thr212 Protein chemistry and molecular cloning directly established that tan forms an integral component Ser199 of the PHF 9'1°'44, thereby putting more indirect immunohistochemical studies on a solid basis. The tau T h r l 8 1 ~ sequences obtained from pronase-treated PHFs have shown that both three and four repeats contribute to Thr175 the core of the PHF but that a length of protein containing only three repeats is protected45. This indicates that the amino-terminus half of tau is lost by C + F phosphorylated proteolysis either in situ because of endogenous B + E phosphorylated proteases or during tangle purification because of A + D phosphorylated added proteases. In Alzheimer's disease the aminoterminus half and part of the carboxy terminus of tau are lost during the transition from intra- to extracellular tangles46. The above results were obtained using PHFs extracted from tangle fragments. Because of the insolubility of these filaments it was not possible to Fig. 4. (Top) Schematic representation of the six human conclude that tau is the only component of the PHF or brain tau isoforms. The region common to all isoforms is to show whether there are other components. This stippled and the inserts that distinguish them are shown in situation has changed with the development of exTkr69 white. The three or four tandem repeats are shown by traction techniques for dispersed PHFs 11' 1 2 . A study black bars. Each isoform is identified by letter and the involving purification of these filaments to apparent number of amino acids is indicated. Isoform A is expressed homogeneity has provided strong evidence that tau in foetal brain, whereas all six isoforms (A-F) are forms the PHF 12. 1

2

expressed in adult brain. (Bottom) A comparison of recombinant human brain tau isoforms with PHF-tau. Lane 1, mixture of recombinant human brain tau isoforms, with each band identified by letter. Lane 2, the three major PHF-tau bands, with the isoform composition of each band indicated by letter. After electrophoresis on a 10% SDS--PAGEgel the tau isoforms were identified by immunoblotting. TINS, Vol. 16, No. 11, 1993

Serz

Fig. 5. (Right) Diagram of the largest human-brain tau isoform showing the distribution of potential phosphorylation sites of the 5er/Thr-Pro type. Heavy stipple indicates the repeat region and light stipple the alternatively spliced exons near the amino terminus. 463

Fig. 6. Paired helical filament (PHF) from the brain of an Alzheimer's disease patient and filaments assembled in vitro from a non-phosphorylated 99-amino acid fragment of tau encompassing three tandem repeats. (A) Pronasetreated Alzheimer's disease PHF. (B-E) Filaments resembling PHFs assembled from expressed tau protein. The arrows indicate regions of filament where the characteristic pattern of four white stain-excluding lines parallel to the filament axis can be seen. Scale bar, 100nm.

In addition, it has recently been shown that paired helical-like filaments can be assembled in vitro from bacterially expressed non-phosphorylated threerepeat or four-repeat fragments of tau (Fig. 6)47'48. Filaments similar to PHFs were produced by dialysis or by hanging-drop equilibration of the fragments against high concentrations of Tris at acidic pH. A series of filaments made from three repeats of tau is shown in Fig. 6B-E, compared with a pronase-treated PHF from an Alzheimer tangle preparation (Fig. 6A). The dimensions of the artificial filaments are very much like those of Alzheimer PHFs (Fig. 6B-E). A characteristic pattern of four longitudinal, white, stain-excluding lines can also be seen. The production of artificial paired helical-like filaments lends strong support to the view that tau is the only component necessary to form the PHF. Unlike tangle fragment PHFs, a large proportion of dispersed PHFs contains the whole of tau 12'1a'49'5°. This indicates that dispersed filaments constitute an earlier stage of PHFs than the bulk of filaments isolated from tangle fragments. A further difference between tangle-fragment and dispersed PHFs is that the majority of tangle-fragment PHFs is ubiquitin a t e d51' 52 . A recent study has identified several lysine residues in the repeat region of tau that ubiquitin becomes attached to 53, indicating that these residues are located on the surface of the PHF. The natural history of the PHF after assembly thus leads from an initial guanidine-soluble intracellular form to a very insoluble, proteolysed, ubiquitinated and cross-linked extracelIular form, with a multitude of intermediate states. Tau protein extracted from PHFs runs as three bands of 60, 64 and 68kDa apparent molecular weight, with a variable amount of background smear (Fig. 4) 11-1a'49'5°'54. The latter results from partially 464

proteolysed and cross-linked PHFs. These PHF-tau bands (also known as Tau-PHF, A68 or Alzheimer's disease-associated proteins) run more slowly on gels than normal or recombinant tau. They contain the whole of tau, as they stain with antibodies directed against the amino- and carboxy-termini of tan. After treatment with alkaline phosphatase at high temperature the three PHF-tau bands become six bands which align with the recombinant tan isofoITnS13'55. The relative proportions of the six bands are the same as those of soluble tau from adult brain. Thus, PHFs contain all six adult brain tau isoforms in an abnormally phosphorylated state. As expected, brain regions with abundant neurofibrillary lesions contain large amounts of PHF-tau ~'sT. Moreover, the phosphorylationdependent tau antibodies stain neurofibrillary lesions in a phosphorylation-dependent manner 12's8'~9. The exact number of phosphorylation sites in PHFtau is not known, with average estimates ranging from six to eight 22. PHF-tau has a greatly reduced ability to bind to rnicrotubules2S; this functional impairment results entirely from abnormal phosphorylation, since dephosphorylated PHF-tau binds as well to microtubules as does normal tan. The reduced binding of PHF-tau to microtubules 25 coupled with reduced levels of normal tau s6 probably destabilizes microtubules in Alzheimer's disease, resulting in the impairment of vital cellular processes, such as rapid axonal transport, and leading to the degeneration of the affected nerve cells. Abnormal phosphorylation means that PHF-tau is phosphorylated at more sites than normal adult tau. Several sites that are phosphorylated in PHF-tau have been identified through mass spectrometry and the use of phosphorylation-dependent antibodies; they include serine 202, threonine 231 and serines 235, 262 and 396 (Refs 12, 24, 60). Unexpectedly, some of the phosphorylation-dependent antibodies recognize not only PHF-tau, but also tan from immature brain 23-25. This indicates that in Alzheimer's disease all six adult brain tau isoforms are abnormally phosphorylated in a way similar to the normal phosphorylation of the single foetal tau isoform. At present, it is unknown whether this is true of all the sites that are abnormally phosphorylated in PHF-tau. Some of the mechanisms underlying the phosphorylation of tau during development are thus reactivated in Alzheimer's disease. With the exception of serine 262, which is phosphorylated only in a fraction of PHF-tau 6°, the known phosphorylation sites are serine and threonine residues followed by a proline. The picture that emerges is that in normal adult brain tan is phosphorylated at only a few of the 17 serine/threonine-proline sites, whereas in PHF-tau it is phosphorylated at a large number of these sites. Abnormal phosphorylation thus constitutes an exaggeration of normal phosphorylation, implying the deregulation of some phosphorylation-dephosphorylation mechanisms in Alzheimer's disease. MAP kinase, glycogen synthase kinase-3 and prolinedirected protein kinase all phosphorylate at least some of the abnormal phosphorylation sites in tau in vitro 27-al. Moreover, three protein kinases that have been purified by virtue of their ability to phosphorylate tau to a PHF-like state in vitro are probably identical with the above61'62. However, it is unclear whether these kinases are the ones that cause the abnormal TINS, VoL 16, No. 11, 1993

phosphorylation of tan in Alzheimer's disease. It is not even known whether the defect results from an increased protein kinase activity or a decreased protein phosphatase activity or from a combination of both. Future experiments involving measurements of protein kinase and protein phosphatase activities in Alzheimer's disease brains will address these issues. Whatever the mechanisms involved, it appears unlikely that tau is the only protein that is abnormally phosphorylated. In affected nerve cells PHFs probably represent the most visible manifestation of the deregulation of phosphorylation--dephosphorylation at serine/threonine-proline sites. In dividing cells such a deregulation is likely to result in cell transformation, since constitutive activation of MAP kinase occurs in response to a variety of oncogenes 63. Since brain cells cannot undergo cell division, the consequence of sustained MAP kinase activation would not be cell transformation. Instead normal brain function might be disrupted by the abnormal phosphorylation of proteins that are not normally phosphorylated to such an extent in vivo. The degree of phosphorylation is the only known difference between PHF-tau and normal adult tau, with the hyperphosphorylated sites located outside the tandem repeat region. As a consequence, tau is prevented from binding to microtubules and is believed to self-assemble through the tandem repeat region into the PHF. If this view is correct it should be possible to form PHFs from recombinant whole tau phosphorylated in vitro. It might also be possible to induce the formation of PHFs in nerve cells of transgenic animals over-expressing protein kinases that phosphorylate tau at serine/threonine-protine sites. This could lead to an animal model for the neurofibrillary lesions, an essential prerequisite for the testing of compounds aimed at halting or preventing the intracellular pathology of Alzheimer's disease.

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Acknowledgements / am gratefu/to Dr H. Braakfor Fig. 3, I thank Dr R. A. Crowther and Dr A. K/ug for helpful commentson the manuscript.

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