Apr 2, 1998 - P-amyloid peptide (AD) are thought by many to be early and key pathological events in the development of Alzheimer's disease [ 1-31. This.
Suspect Proteins in Neurodegeneration Protein and Peptide Science Group Colloquium Organized and Edited by B. Austen (Department of Surgery, St. George’s Hospital Medical School). 665th Meeting held at Southampton, 3 I March-2 April 1998.
Fibrillogenesis of P-amyloid D. Allsop, D. Howlett, G. Christie and E. Karran Neurosciences Research, SmithKline Beecham Pharmaceuticals, N e w Frontiers Science Park, Harlow, Essex CM I 9 SAW, U.K.
T h e formation and deposition in the brain of the P-amyloid peptide (AD) are thought by many to be early and key pathological events in the development of Alzheimer’s disease [ 1-31. This applies in particular to the longer and more hydrophobic/fibrillogenic 1-42 form of AD (A&) rather than the shorter forms such as Afl40. T h e main pieces of evidence pointing to this are: (i) mutations in all three genes known to cause familial Alzheimer’s disease (APP, PS-1, PS-2) have been shown to increase A042 production relative to AD40 [4-111; (ii) AD42 is the predominant amyloid species found in early senile plaques (which develop before neurofibrillary tangles in Down’s syndrome) [12]; (iii) synthetic AP42 peptide aggregates more readily in vitro than AD4,, into a neurotoxic and fibrillar form [ 131; (iv) transgenic mice that overexpress mutant forms of the amyloid precursor protein (AF’P) develop fibrillar plaques that contain predominantly AD42 and also show many of the major pathological features of Alzheimer’s disease [ 14-18]. These and other observations have tended to reinforce the ‘amyloid cascade’ hypothesis of disease progression [ 1,3]. Naturally, it follows from this that drugs designed to inhibit the formation of AD (particularly AP42) or its aggregation into an insoluble neurotoxic form, should be potential therapeutic agents. T h e recent observation that appears to be formed intracellularly within the endoplasmic reticulum of neuronal cells [19,20] gives the added consideration that such drugs may need to accumulate to an active concentration in this Abbreviations used: AP, P-amyloid peptide; APP, amyloid precursor protein; CLIP, chymotrypsin-like activity of proteasome.
subcellular compartment of neuronal cells in the brain. Here we summarize briefly the progress that has been made towards identification of inhibitors of either AP formation or aggregation.
Inhibitors of A/?formation A straightforward means to prevent formation of AP would be to inhibit the proteolytic enzymes ( /I-secretase and y-secretase) that are involved in its release from the APP. However, despite the fact that for the last 10 years these two particular enzyme activities have been associated with the production of A/?, their identity still remains unknown. Numerous candidate p- and y-secretases have been proposed but they remain unsubstantiated or disproved. As far as p-secretase is concerned, the multicatalytic proteinase or ‘proteasome’ has been implicated [21,22], as have several chymotrypinlike serine proteinases, including cathepsin G and ‘clipsin’ [23-251. T h e metallopeptidase thimet (EC 3.4.24.15) has been proposed [26] (despite the fact that it appears not to tolerate large protein substrates such as AF’P) but has now been disproved [27]. Cathepsin D (an aspartyl proteinase) has received considerable attention as a potential D-secretase due to its ability to cleave peptide substrates containing the APP ‘Swedish mutant’ sequence at a much faster rate than the normal sequence [28]. However, the fact that cathepsin D knockout mice still produce AP [29] indicates that this enzyme cannot be p-secretase. In the case of y-secretase, there is the additional complication that there may be separate enzymes responsible for generation of AD4” and
I998
Biochemical Society Transactions
460
A[&2 [30,31], although this question has not been clearly resolved. Candidates for 7-secretase include the proteasome [22,32], prolylendopeptidase [33], and a cathepsin D-like proteinase [28,34]. However, for none of the various enzymes mentioned here is there strong evidence that they are actually [j- or 7-secretase. Given lack of progress in the identification of the proteinases involved in AB production, an alternative strategy has been to test compounds for the ability to inhibit A[j formation by whole cells in culture. This assumes little knowledge of the precise molecular target of such inhibitors. Table 1 summarizes compounds identified in this way. A number of small peptide aldehydes of the type known to inhibit both cysteine and
serine proteinases have been shown to inhibit A[) formation, probably through inhibition of the p-secretase pathway. There is limited evidence to suggest that they are more potent inhibitors of A/&,, than of [30,31]. Our own experiments have confirmed the activity of these compounds [36], but we have also shown that they are reasonably potent inhibitors of the chymotrypsinlike activity of the proteasome (CLIP). In order to evaluate more fully the role of the proteasome in Alzheimer's disease amyloidogenesis, we have tested the effects on AP formation of a wide range of peptide-based inhibitors of CLIP. We have found that the ability of the inhibitors to suppress A[) formation by cells correlates very well with potency of CLIP inhibition (D. Allsop
Table I Inhibitors of A[] formation
Compound
Structure
Target
Refs.
Cbz-Val-Phe-H MDL-28 I70
7 -secretase?
[30,35,361
Cbz-Leu-Leu-Leu-H
y-secretase?
[3 11
Cbz-Leu-Leu-Nle-H
1,-secretase?
~361
C bz- Leu- Nle-H Calpeptin
21-secretase?
[3 1,361
vATPase
[381
'Substrate-based difluoroketone'
/
Bafilomycin A and related compounds \
Volume 26
Suspect Proteins in Neurodegeneration
and G. Christie, unpublished work). Thus, we conclude that the proteasome (or an alternative proteinase activity with close similarity to CLIP) may be involved either directly or indirectly in AP secretion. Binding of A0 to proteasome [39], and a potential role of proteasome in APP asecretase activity [40] and turnover of presenilins [41], have been reported by others. Compounds that inhibit proteasome activity may exhibit toxicity/side-effect problems and so are unlikely to be viable therapeutic agents for chronic disease. T h e latter also applies to vATPase inhibitors (bafilomycins and related compounds) which have been shown to inhibit AP formation [38], presumably through effects on intracellular pH, and are highly toxic in vivo.
Inhibitors of AD aggregation T h e last few years have seen the emergence of a number of compounds that inhibit aggregation of AD in vztro (Figure 1). B-Peptide aggregation leads to formation of a cytotoxic form of amyloid, so many of these compounds, such as the benzofuran SKF-74652 [44], also reduce the neurotoxic effects of AD in cell lines. Congo red, which is well known as a histochemical dye for the detection of amyloid, also protects against AD-mediated cellular toxicity [45] but is not a potent anti-aggregant; the structurally related chrysamine-G, however, is reported to block AP aggregation [46]. Of particular interest is the compound IDOX, which has been reported to
result in the resorption of amyloid deposits in patients with AL-type (immunoglobulin-related) systemic amyloidosis 1431. Similar claims for systemic amyloid deposits in mice have been made for a series of highly charged sulphonates [47]. These studies demonstrate that anti-aggregatory drugs can be effective in vivo. A major stumbling block in the discovery of anti-aggregatory compounds has been a lack of understanding of the form of fl-peptide that is responsible for neurotoxicity and is thus the target for inhibitor interaction. If monomeric AD is the target, then a 1:l stoichiometry between inhibitor and peptide may be required. However, if binding to the growing face of a fibre is sufficient to prevent the formation of a toxic form, then a much lower inhibitor to peptide ratio may suffice. Although it is well established that neurotoxicity is dependent upon the aggregation state of the peptide, the identity of the toxic form is unclear. Both mature fibrils [48] and dimers [49] have been implicated. T h e recent identification [50] of a protofibrillar intermediate in P-amyloid fibril formation may eventually shed light on this matter. It has been known for some time that the aggregation of AB is pH dependent [51]. In our own studies (D. Howlett, unpublished work), we have compared the pH profile of AD4" aggregation, as assessed by Congo red binding assay and neurotoxicity in IMR-32 cells [43]. What emerges from these studies is that the pH optimum for formation of a Congo red binding form is around
Figure I
Inhibitors of A/3 fibrillization
U
,
n
N
Me \\
0
I
Rifampicin [42]
IDOX [43] Me
f N %
6
c'moM. Qo
LNEI,
SKF-74652 [44]
SKF-64346 [44]
I998
46 I
Biochemical Society Transactions
5.8 (close to the PI), but peptide aggregates formed at this pH are not neurotoxic. T h e pH 462
optimum for formation of a neurotoxic form is close to 7.4. T h i s suggests that the Congo redpositive A/3 deposits observed in Alzheimer’s disease brain tissue sections are not necessarily neurotoxic.
Conclusion Despite the large amount of effort in this area of research in many laboratories worldwide, it is clear that a number of key questions need to be answered before further genuine progress can be and - y-secretases has made. T h e identity of the ,I? still not been determined, and the question of whether there are separate y-secretases responsible for formation of A/340and AP4*has not been resolved. Furthermore, it has not been possible to determine the precise oligomeric or multimeric form of AP that is neurotoxic, or the binding site with which the known inhibitors of aggregation interact. T h e ‘anti-amyloid’ compounds identified to date all appear to have low in uitro affinity or undesirable pharmacokinetic or potential clinical profiles which render them unsuitable as therapeutic agents. Nevertheless, their discovery should at least allow us to test, in animal models, the concept that inhibition of AD formation or aggregation prevents Alzheimer-like pathology.
1 Hardy, J. and Allsop, D. (1991) Trends Pharmacol. Sci. 12, 383-388 2 Sisodia, S. S. and Price, D. L. (1995) FASEB J. 9, 366-370 3 Hardy, J. (1997) Trends Neurosci. 20, 154-159 4 Citron, M., Oltersdorf, T., Haass, C., McConlogue, L., Hung, A. Y., Seubert, P., Vigro-Pelfrey, C., Lieberburg, I. and Selkoe, D. J. (1992) Nature 360, 672-674 5 Cai, X.-D., Golde, T. E. and Younkin, S. G. (1993) Science 259, 514-516 6 Haass, C., Hung, A. Y., Selkoe, D. J. and Teplow, D. B. (1994) J. Biol. Chem. 269, 17741-17748 7 Suzuki, N., Cheung, T. T., Cai, X.-D., Odaka, A., Otvos, Jr., L., Eckman, C., Golde, T. E. and Younkin, S. G. (1994) Science 264, 1336-1340 8 Citron, M., Vigo-Pelfrey, C., Teplow, D. B., Miller, C., Schenk, D., Johnston, J., Winblad, B., Venizelos, N., Lannfelt, L. and Selkoe, D. J. (1994) Proc. Natl. Acad. Sci. U.S.A. 91, 11993-1 1997 9 Scheuner, D., Eckman, C., Jensen, M., Song, X., Citron, M., Suzuki, N., Bird, T. D., Hardy, J.,
Volume 26
Hutton, M., Kukull, W. et al. Nature Med. 2, 864-870 10 Borchelt, D. R., Thinakaran, G., Eckman, C. B., Lee, M. K., Davenport, F., Ratovitsky, T., Prada, C. M., Kim, G., Seekins, S., Yager, D. et al. (1996) Neuron 17, 1005-1013 11 Duff, K., Eckman, C., Zehr, C., Yu, X., Prada, C. M., Perez-tur, J., Hutton, M., Buee, L., Harigaya, Y., Yager, D. et al. (1996) Nature (London) 383, 710-713 12 Iwatsubo, T., Odaka, A., Suzuki, N., Mizusawa, H., Nukina, N. and Ihara, Y. (1994) Neuron 13, 45-53 13 Burdick, D., Soreghan, B., Kwon, M., Kosmoski, J., Knauer, M., Henschen, A., Yates, J., Cotman, C. and Glabe, C. (1992) J. Biol. Chem. 267, 546-554 14 Games, D., Adams, D., Alessandrini, R., Barbour, R., Berthelette, P., Blackwell, C., Carr, T., Clemens, J., Donaldson, T. and Gillespie, F. (1995) Nature (London) 373, 523-527 15 Masliah, E., Sisk, A., Mallory, M., Mucke, L., Schenk, D. and Games, D. (1995) J. Neurosci. 16, 5795-581 1 16 Johnson-wood, K., Lee, M., Motter, R., Hu, K., Gordon, G., Barbour, R., Khan, K., Gordon, M., Tan, H., Games, D. et al. (1997) Proc. Natl. Acad. Sci. U.S.A. 94, 1550-1555 17 Hsiao, K., Chapman, P., Nilsen, S., Eckman, C., Harigaya, Y., Younkin, S. G., Yang, F. and Cole, G. (1996) Science 274,99-102 18 Frautschy, S. A., Yang, F., Irrizarry, M., Hyman, B., Saido, T. C., Hsiao, K. and Cole, G. M. (1998) Am. J. Pathol. 152, 307-317 19 Hartmann, T., Bieger, S. C., Bruhl, B., Tienari, P. J., Ida, N., Allsop, D., Roberts, G. W., Masters, C. L., Dotti, C. G., Unsicker, K. and Beyreuther, K. (1997) Nature Med. 3, 1016-1020 20 Cook, D. G., Forman, M. S., Sung, J. C., Leight, S., Kolson, D. L., Iwatsubo, T., Lee, V. M. and Doms, R. W. (1997) Nature Med. 3, 1021-1023 21 Ishiura, S., Tsukahara, T., Tabira, T. and Sugita, H. (1989) FEBS Lett. 257, 388-392 22 Kojima, S. I. and Omori, M. (1992) FEBS Lett. 304,57-60 23 Nelson, R. B., Siman, R., Iqbal, M. A. and Potter, H. (1993) J. Neurochem. 61, 567-577 24 Sahasrabudhe, S. R., Brown, A. M., Hulmes, J. D., Jacobsen, J. S., Vitek, M. P., Blume, A. J. and Sonnenberg, J. L. (1993) J. Biol. Chem. 268, 16699-16705 25 Savage, M. J., Iqbal, M., Loh, T., Trusko, S. P., Scott, R. and Siman, R. (1994) Neuroscience 60, 607-619 26 McDermott, J. R., Biggins, J. A. and Gibson, A. M. (1992) Biochem. Biophys. Res. Commun. 185, 746-752 27 Chevallier, N., Jiracek, J., Vincent, B., Bauer, C. P., Spillantini, M. G., Goedert, M., Dive, V. and Checler, F. (1997) Br. J. Pharmacol. 121, 556-562
Suspect Proteins in Neurodegeneration
28 Ladror, U. S., Snyder, S. W., Wang, G. T., Holzman, T. F. and Krafft, G. A. (1994) J. Biol. Chem. 269, 18422-18428 29 Saftig, P., Peters, C., von Figura, K., Craessaerts, K., van Leuven, F. and de Strooper, B. (1996) J. Biol. Chem. 271,27241-27244 30 Citron, M., Diehl, T. S., Gordon, G., Biere, A. L., Seubert, P. and Selkoe, D. J. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 13170-13175 31 Klafki, H., Abramowski, D., Swoboda, R., Paganetti, P. A. and Staufenbiel, M. (1996) J. Biol. Chem. 271,28655-28659 32 Mundy, D. I. (1994) Biochem. Biophys. Res. Commun. 204,333-341 33 Ishiura, S. (1991) J. Neurochem. 56, 363-369 34 Evin, G., Cappai, R., Li, Q. X., Culvenor, J. G., Small, D. H., Beyreuther, K. and Masters, C. L. (1995) Biochemistry 34, 14185-14192 35 Higaki, H., Quon, D., Zhong, 2. and Cordell, B. (1995) Neuron 14,65 1-659 36 Allsop, D., Christie, G., Gray, C., Holmes, S., Markwell, R., Owen, D., Smith, L., Wadsworth, H., Ward, R. V., Hartmann, T. et al. (1997) in Alzheimer’s Disease: Biology, Diagnostics and Therapeutics (Iqbal, K., Winblad, B., Nishimura, T., Takeda, M. and Wisniewski, H. M., eds.), pp. 717-727, John Wiley, Chichester 37 Wolfe, M. S., Citron, M., Diehl, T. S., Xia, W., Donkor, I. 0. and Selkoe, D. J. (1998) J. Med. Chem. 41,6-9 38 Haass, C., Capell, A., Citron, M., Teplow, D. B. and Selkoe, D. J. (1995) J. Biol. Chem. 17, 6186-6192 39 Gregori, L., Hainfeld, J. F., Simon, M. N. and Goldgaber, D. (19979) J. Biol. Chem. 272, 58-62 40 Marambaud, P., Chevallier, N., Barelli, H., Wilk, S. and Checler, F. (1997) J. Neurochem. 68,698-703
41 Kim, T. W., Pettingell, W. H., Hallmark, 0. G., Moir, R. D., Wasco, W. and Tanzi, R. E. (1997) J. Biol. Chem. 272, 11006-11010 42 Tomiyama, T., Shoji, A., Kataoka, K., Suwa, Y., Asano, S., Kaneko, H. and Endo, N. (1996) J. Biol. Chem. 271,6839-6844 43 Merlini, G., Ascari, E., Amboldi, N., Bellotti, V., Arbustini, E., Perfetti, V., Ferrari, M., Zorzoli, O., Marinone, M. G. and Garini, P. (1995) Proc. Natl. Acad. Sci. U.S.A. 92,2959-2963 44 Howlett, D. R., Markwell, R. E. and Wood, S. J. (1997) Presented at the British Pharmacology Society Meeting, Harrogate, UK, 9-12 Dec. Abstract C27 45 Pollack, S. J., Sadler, I. I., Hawtin, S. R., Tailor, V. J. and Shearman, M. S. (1995) Neurosci. Lett. 197,211-214 46 Klunk, W. E., Debnath, M. L. and Pettegrew, J. E. (1994) Neurobiol. Aging 15, 691-698 47 Kisilevsky, R., Lemieux, L. J., Fraser, P. E., Kong, X., Hultin, P. G. and Szarek, W. A. (1995) Nature Med. 1, 143-148 48 Harper, J. D., Wong, S. S., Lieber, C. M. and Lansbury, P. T. (1997) Chem. Biol. 4, 119-125 49 Roher, A. E., Chaney, M. O., Kuo, Y. M., Webster, S. D., Stine, W. B., Haverkamp, L. J., Woods, A. S., Cotter, R. J., Tuohy, J. M., Krafft, G. A. et al. (1996) J. Biol. Chem. 271, 20631-20635 50 Walsh, D. M., Lomakin, A., Benedek, G. B., Condron, M. M. and Teplow, D. B. (1997) J. Biol. Chem. 272,22364-22372 51 Mantyh, P. W., Ghilardi, J. R., Rogers, S., DeMaster, E., Allen, C. J., Stimson, E. R. and Maggio, J. E. (1993) J. Neurochem. 61, 1171-1174
Received 16 April 1998
lntraneuronal filamentous tau protein and a-synuclein deposits in neurodegenerative diseases M. Goedert’, R. Jakes, R. A. Crowther, M. Hasegawa, M. J. Smith and M. G. Spillantini* MRC Laboratory of Molecular Biology, Hills Road, Cambridge CB2 2QH, U.K., and *MRC Centre for Brain Repair and Department of Neurology, Robinson Way, Cambridge CB2 2PY, U.K.
Alzheimer’s disease and Parkinson’s disease are the most common neurodegenerative disorders of the human brain. They are characterized by the presence of ordered filamentous assemblies which gradually develop in a small number of nerve cell types. I n Alzheimer’s disease, vulnerAbbreviations used: NFT, neurofibrillary tangle; PHF, paired helical filament; SF, straight filament. ‘ T o whom correspondence should be addressed.
able nerve cells develop neurofibrillary tangles, neuropil threads and abnormal neurites, whereas in Parkinson’s disease they develop Lewy bodies and Lewy neurites (reviewed in [1,2]). Alzheimer’s disease is characterized by the additional presence of extracellular deposits in the form of amyloid plaques. Over recent years, it has become clear that the intraneuronal filamentous deposits of Alzheimer’s disease and Parkinson’s disease are composed of tau protein and a-synu-
I998
463
