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34 Katagiri, K. et al. (2000) Rap1 is a potent activation signal for leukocyte functionassociated antigen 1 distinct from protein kinase C and phosphatidylinositol-3-OH kinase. Mol. Cell. Biol. 20, 1956–1969 35 Reedquist, K.A. et al. (2000) The small GTPase, Rap1, mediates CD31-induced integrin adhesion. J. Cell Biol. 148, 1151–1158 36 Hashimoto, A. et al. (1998) Involvement of guanosine triphosphatases and phospholipase C-γ2 in extracellular signal-regulated kinase, c-Jun NH2-terminal kinase, and p38 mitogenactivated protein kinase activation by the B cell antigen receptor. J. Exp. Med. 188, 1287–1295 37 Doody, G.M. et al. (2000) Vav-2 controls NFAT-dependent transcription in B- but not T-lymphocytes. EMBO J. 19, 6173–6184 38 Lucas, P.C. et al. (2001) Bcl10 and MALT1, independent targets of chromosomal translocation in MALT lymphoma, cooperate in a novel NF-κB signaling pathway. J. Biol. Chem. 276, 19102–19019 39 Gold, M.R. et al. (1999) The B cell antigen receptor activates the Akt (protein kinase B)/glycogen synthase kinase-3 signaling pathway via phosphatidylinositol 3-kinase. J. Immunol. 163, 1894–1905 40 Swart, J.M. et al. (2000) Identification of a membrane Ig-induced p38 mitogen-activated protein kinase module that regulates cAMP response element binding protein phosphorylation and transcriptional activation in CH31 B lymphomas. J. Immunol. 164, 2311–2319

41 King, L.B. and Monroe, J.G. (2000) Immunobiology of the immature B cell: plasticity in the B-cell receptor-induced response fine tunes negative selection. Immunol. Rev. 176, 86–104 42 Healy, J.I. et al. (1997) Different nuclear signals are activated by the B cell receptor during positive versus negative signaling. Immunity 6, 419–428 43 Glynne, R. et al. (2000) How self-tolerance and the immunosuppressive drug FK506 prevent B-cell mitogenesis. Nature 403, 672–676 44 Glynne, R. et al. (2000) B-lymphocyte quiescence, tolerance, and activation as viewed by global gene expression profiling on microarrays. Immunol. Rev. 176, 216–246 45 Shaffer, A.L. et al. (2001) Signatures of the immune response. Immunity 15, 375–385 46 Sonenshein, G.E. (1997) Down-modulation of c-Myc expression induces apoptosis of B lymphocyte models of tolerance via clonal deletion. J. Immunol. 158, 1994–1997 47 Zhu, N. et al. (2002) CD40 signaling in B cells regulates the expression of the Pim-1 kinase via the NF-κB pathway. J. Immunol. 168, 744–754 48 Smith, K.G.C. and Fearon, D.T. (2000) Receptor modulators of B-cell receptor signaling–CD19/CD22. Curr. Top. Microbiol. Immunol. 245, 195–212 49 Tsubata, T. (1999) Co-receptors on B lymphocytes. Curr. Opin. Immunol. 11, 249–255 50 Cherukuri, A. et al. (2001) The CD19/CD21 complex functions to prolong B cell antigen

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Novel therapeutic strategies provide the real test for the amyloid hypothesis of Alzheimer’s disease Diana Ines Dominguez and Bart De Strooper The amyloid and tangle cascade hypothesis is the dominant explanation for the pathogenesis of Alzheimer’s disease (AD). A complete knowledge of the β) production and clearance in vivo metabolic pathways leading to β-amyloid (Aβ and of the pathological events that lead to fibril formation and deposition into plaques is crucial for the development of an ‘anti-amyloid’ therapeutic strategy. Important advances in this respect have been achieved recently, revealing new candidate drug targets. Among the most promising potential treatments are β- and γ-secretase inhibitors, Aβ β vaccination, Cu–Zn chelators, cholesterollowering drugs and non-steroidal anti-inflammatory drugs. Now, the major question is whether these drugs will work in the clinic.

Alzheimer’s disease (AD) is characterized by two types of protein aggregates, neurofibrillary tangles and amyloid plaques, distributed in regions of the CNS that are involved in learning and memory. The neurofibrillary tangles consist of twisted filaments http://tips.trends.com

containing hyperphosphorylated tau whereas the amyloid plaques contain mainly β-amyloid (Aβ) peptide fibrils. Incomplete knowledge of the molecular process that causes AD has hindered advances in drug development. The available cholinergic therapies target essentially late aspects of the disease, improving temporarily the performance of the undamaged neurones, but do not stop the progressive mental decline. In the past years, important progress has been made in the understanding of the pathogenic mechanism of AD, and new therapeutic targets have become available that should allow the underlying disease process to be tackled directly. In this respect, the ‘amyloid hypothesis’ has become the dominant theory in the field. It is believed that Aβ accumulation in plaques or as partial soluble filaments initiates a

0165-6147/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII: S0165-6147(02)02038-2

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a

Table 1. Anti-amyloid strategies for the treatment of Alzheimer’s disease Drug Class

Aim

Pros

Cons

β-Secretase inhibitors γ-Secretase inhibitors

Decrease Aβ synthesis

BACE1 knockout mice are normal γ-Secretase inhibitors decrease Aβ levels in the brain of a mouse model of AD

?

Activators of Aβ-degrading enzymes Metal chelators

Aβ vaccination

Statins

NSAIDs

Decrease Aβ synthesis

Increase Aβ degradation and clearance

In vivo (mice) demonstration that b neprilysin deficiency results in higher Aβ levels Solubilization of Aβ In mouse models of AD deposits and prevention metal chelators decrease of aggregate formation amyloid plaque area and by metal chelation improve general health Immune response against In mouse models of AD Aβ peptide Aβ vaccination decreases amyloid plaque area and total Aβ, and improves cognitive function Decrease Aβ production Reduced risk of by reducing cholesterol developing AD in patients levels (mechanism treated with statins; remains unknown) statins reduce cerebral Aβ load in guinea-pigs and mice Decrease inflammation in Reduced risk of the brain that contributes developing AD in patients to neuronal loss; treated with NSAIDs inhibition of Aβ1–42 generation

Inhibition of Notch signalling could affect haematopoiesis and lymphocyte differentiation; possible side-effects associated with lack of cleavage of other membrane proteins Unclear whether pharmacologically possible

Increase in soluble Aβ could potentially harm the brain; deficiency in vitamin B12, and SMON Risk of autoimmunity and brain inflammation; antibodies might not cross the blood–brain barrier in humans as they do in mice; immune response might not be efficient in older people ?

Side-effects, mostly at the level of the gastrointestinal tract following prolonged treatment

Abbreviations: Aβ, β-amyloid; AD, Alzheimer’s disease; BACE1, β-site APP-cleaving enzyme 1; NSAIDs, non-steroidal anti-inflammatory drugs; SMON, subacute myelo-optic neuropathy. Neprilysin (neutral endopeptidase) is a metallopeptidase that has been shown to degrade Aβ.

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b

Diana Ines Dominguez Bart De Strooper* Centre for Human Genetics, VIB4 and K.U. Leuven, Herestraat 49, 3000 Leuven, Belgium. *e-mail: Bart.Destrooper@ med.kuleuven.ac.be

pathological cascade leading to tangle formation [1,2], neuronal dysfunction and possibly inflammation and oxidative damage, with neurodegeneration and dementia as the final outcome [3]. Although evidence supporting this hypothesis is compelling, an important piece of the puzzle has not yet been fitted into place, namely the precise molecular link between Aβ and neuronal dysfunction. Aβ peptide has been implicated in several toxic processes [4] including disrupted Ca2+ homeostasis, apoptosis, direct intercalation into cellular membranes, activation of complement, generation of radicals and induction of tangle formation. The absence of a consensus probably indicates that the real mechanism of Aβ toxicity is not yet understood and that better models are needed to investigate this problem. Nevertheless, the considerable progress in this field of research now allows the final validation experiment of the amyloid hypothesis to be considered. It will be essential to demonstrate that decreasing the Aβ load in the brains of AD patients ameliorates the symptoms of this disorder. This could be achieved by: (1) decreasing Aβ production; (2) increasing Aβ clearance; or (3) interfering with Aβ aggregation and precipitation http://tips.trends.com

into fibrils or plaques. Each of these steps offers possibilities for therapeutic intervention. We will limit our discussion here to strategies that were developed in recent years and are mostly aimed at lowering Aβ burden (Table 1 and Fig. 1). β production: the secretases as therapeutic Lowering Aβ targets

Two enzymatic activities known as β- and γ-secretases cleave the Aβ precursor protein (APP) to yield Aβ peptide. β-Secretase, also called BACE (β-site APP-cleaving enzyme) [5], is a membrane-bound aspartic protease with broad tissue distribution but is specifically abundant in brain. Mice with inactivated genes encoding BACE are viable, show no obvious anatomical or physiological abnormalities and, as predicted, do not generate Aβ peptides [6–8]. Therefore, BACE appears to be an excellent drug target for AD. Nevertheless, further work is needed to establish the exact physiological function of BACE. Indeed, it cannot be excluded that BACE-deficient mice suffer from more subtle deficits that have, as yet, escaped detection. Furthermore, the apparent lack of phenotype in BACE knockout mice does not

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Brain

APP

Blood

+ 2 Aβ degradation

1 Aβ production

+ +

Cholesterol

Neurone

Neurotoxic?

3 Aβ aggregate formation Aβ vaccination?

Neurotoxic?

Neurotoxic? Other factors

Glutamate, cytokines, free radicals

4 Microglia activation

Release of 5 neurotoxic factors + Aβ clearance

+ Microglia Activated microglia

Aβ vaccination? TGF-β1 TRENDS in Pharmacological Sciences

Fig. 1. The amyloid hypothesis in Alzheimer’s disease (AD) and candidate targets for therapeutic intervention. AD is characterized by two types of protein aggregates, neurofibrillary tangles and β-amyloid (Aβ) plaques, distributed in regions of the CNS involved in learning and memory. Soluble Aβ (green cylinders) is formed following the cleavage of Aβ precursor protein (APP) by enzymatic activities known as β-secretase (purple scissors) and γ-secretase (cyan scissors) (1). Aβ formed is then degraded by enzymes (2). The balance between Aβ production and degradation can be disrupted leading to Aβ accumulation beyond pathological levels and, in turn, increased levels of Aβ aggregates (3; green) and deposits in the brain. Aggregates and Aβ fibrils could themselves be neurotoxic or could activate microglia (4), which can release neurotoxic factors as part of an inflammatory response (5). Several steps could be targeted pharmacologically to treat AD (indicated by red arrows). For example, inhibitors of β-secretase and γ-secretase or cholesterol-lowering drugs (blunt arrows) could be used to decrease the production of Aβ. It is possible that activators of Aβ-degrading enzymes could be developed to reduce Aβ levels and metal chelators could be used to dissolve amyloid plaques. Furthermore, Aβ vaccination is proposed to sequester Aβ in the blood, which in turn would induce a rapid efflux of Aβ from the brain. Microglia can also be activated by Aβ vaccination or by transforming growth factor β1 (TGF-β1), leading to increased Aβ clearance and neuroprotection.

necessarily imply that inhibition of BACE in adult brain will have no side-effects. For example, it is possible that compensatory mechanisms operating during embryogenesis cannot be activated in adulthood. Finally, the absence of toxic effects in mice does not prove absence of toxicity in humans. A homologue of BACE, BACE2, has been identified [5], and it is possible that this protease compensates for the absence of BACE function, an aspect that could become apparent in the double deficient BACE and BACE2 mouse model once it is generated. In the meantime, it seems advisable to develop BACE inhibitors that do not affect BACE2 function. Even with these theoretical concerns in mind, BACE remains probably the best drug target for http://tips.trends.com

AD treatment. Those searching for BACE inhibitors will certainly profit from the large amount of data available on other aspartic proteases and from the list of compounds that have been developed to inhibit these enzymes, particularly the highly investigated HIV-1 protease. Furthermore, the crystal structure of the BACE active domain bound to an inhibitor has been solved [9], opening the door to rational drug design. The second cleavage required to release Aβ from APP is performed by γ-secretase, a fascinating but poorly understood proteolytic system that cleaves proteins in their transmembrane domains. The polytopic membrane proteins presenilin 1 and 2 are part of the multimeric complex responsible for this proteolytic activity [10,11]. Another integral membrane protein, nicastrin, also participates in the cleavage reaction [12]. Both presenilin [13] and nicastrin [14] are absolute requirements for γ-secretase activity. Whether additional proteins are needed is not known at present. Inhibitors of γ-secretase have been developed and one effectively reduces Aβ levels in the brain of a mouse model of AD [15]. A major concern when considering γ-secretase inhibitors for the treatment of AD is their potential toxic side-effects. For example, γ-secretase participates not only in the cleavage of APP but also of other integral membrane proteins including Notch 1–4 [13], ErbB-4 receptor [16], CD44 receptor [17] and possibly telencephalin [18] and E- and N-cadherin [19]. The consequences of inhibiting the γ-secretase cleavage of the Notch receptors are best understood. In the absence of γ-secretase activity, mice die early in embryogenesis and their phenotype resembles that of mice with inactivated genes encoding Notch [13]. Notch

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signalling is, however, also important in adult tissues and its inhibition might affect haematopoiesis [20] and thymocyte maturation [21,22], causing immunosuppression in patients. One possible solution for the toxicity problem of γ-secretase inhibitors is to use doses that only partially inhibit the enzyme. It seems likely that a limited reduction of APP processing will suffice to limit amyloid precipitation in AD. The toxicity and efficacy of low concentrations of γ-secretase inhibitors must therefore be empirically tested. Another theoretical possibility is to target specifically γ-secretasemediated APP processing without affecting cleavage of Notch and other membrane proteins. In fact, some amino acid substitutions in presenilin have been shown to affect Notch and APP processing differentially [23]. One study even claimed that an inhibitor specifically affected γ-cleavage of APP but not cleavage of Notch [24]. Unfortunately, others could not reproduce these findings [25]. To advance strategies that target γ-secretase processing of APP it is important to develop a cell-free assay that allows reconstitution of γ-secretase activity in vitro, to define the minimal number of proteins needed for activity and to establish which component(s) of the complex contribute directly to the catalytic activity. Modulating cholesterol in the brain to decrease β production Aβ

Epidemiological studies show that high blood cholesterol levels correlate with a higher risk of developing AD [26–28] and, indeed, AD patients treated with cholesterol-lowering statins (but not with fibrates, cholestyramine or nicotinic acid) become protected against the disease [29,30]. Cell-based studies indicate that cholesterol modulates the proteolytic processing of APP. High cholesterol seems to favour processing of APP through the amyloidogenic β-secretase pathway [31,32], whereas low cholesterol increases the processing of APP by α-secretase [33–35]. α-Secretase cleaves APP within the Aβ sequence and is therefore believed to have a protective effect. The hypothesis that cholesterol modulates APP processing is supported by recent experiments in animal models. For example, transgenic mice engineered to develop Aβ amyloidosis and fed with a high-cholesterol diet exhibited significantly increased levels of Aβ in the CNS [36]. Similarly, mice [37] or guinea-pigs [32] treated with cholesterol-lowering statins displayed reduced levels of cerebral Aβ. Further work is needed to understand how cholesterol modulates the activities of the secretases, but a prospective study with statins in humans seems a logical next step. Recent insights [38] indicate that intracellular cholesteryl esters in particular are involved in the regulation of Aβ generation, providing a rationale for adding acyl-coenzyme A:cholesterol acyltransferase (ACAT) to the candidate list of AD drug targets. http://tips.trends.com

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β vaccination Aβ

A transgenic mouse model overexpressing a mutant form of human APP has been shown to be protected against amyloid plaque formation following intraperitoneal immunization with synthetic Aβ peptide [39]. Surprisingly, Aβ vaccination not only prevented amyloid deposition in young mice but also arrested and even cleared amyloidosis in older animals. Similar results were obtained using nasal administration of Aβ [40]. Moreover, in two other transgenic mouse models of AD, Aβ vaccination was able to protect the mice against age-dependent learning and memory deficits [41,42]. Passive immunization, by direct antibody injection, is sufficient to induce Aβ clearance [43,44]. Apparently the antibodies are able to cross the blood–brain barrier and probably trigger microglia to phagocytose Aβ via their Fc-receptors [43,45]. An alternative working mechanism has been proposed [44]: the antibodies sequester Aβ in the plasma, which in turn would induce a rapid efflux of Aβ from the brain. Analysis of the results of immunization experiments performed by Morgan et al. [41] and Janus et al. [42] further complicates the picture. The improvement in cognitive function in these models is not directly correlated to the decrease in Aβ plaques and it is therefore unclear at present what aspects of Aβ metabolism are affected by the vaccination procedure. Phase I clinical trials have been conducted with success. However, the Phase II trial being performed on 360 patients in USA and Europe has been temporarily suspended because of clinical signs of inflammation in the CNS of several patients. No clinical information has been made available concerning the exact problem, and it is therefore difficult to assess the consequences of these observations for further clinical trials using vaccination therapy. However, it is likely that improved vaccines with lower toxicity and higher immunogenicity [46] will become available for further testing and hopefully this approach can be continued. Non-steroidal anti-inflammatory drugs

Inflammation in AD is believed to be an important contributor to neuronal loss and is characterized by activated microglia, reactive astrocytes, cytokines and complement components in the vicinity of the plaques [47,48]. Epidemiological studies [49–51] and a pilot clinical trial [52] have shown reduced incidence and slower progression of AD in patients treated with non-steroidal anti-inflammatory drugs (NSAIDs). Furthermore, a recent prospective study provided strong evidence that the chronic use of NSAIDs significantly reduced the risk for AD [53]. Although it is believed that the anti-inflammatory activity of NSAIDs underlies these positive effects, some confusing issues remain. For example, other anti-inflammatory drugs such as hydroxychloroquine [54] or prednisone [55] do not display protective effects, indicating that suppression of inflammation is

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not the only issue involved. Moreover, the canonical targets of NSAIDs are cyclooxygenases (COX) 1 and 2, but specific inhibitors of COX-2 appear to display little effect in the first clinical trials [56]. Cell-based studies suggest that NSAIDs could function in AD through activation of the transcription factor peroxisome proliferator activated receptor γ (PPAR-γ), independently of COX-2 inhibition [57]. Conversely, inflammation could play a protective role in AD. This hypothesis is supported by the recent Aβ vaccination experiments (see above) and by the observation that transgenic mice expressing transforming growth factor β1 (TGF-β1) in astrocytes show strong activation of microglia and increased Aβ clearance [58]. Further work is therefore needed to identify the signalling pathways involved in the beneficial and toxic response of activated microglia, and the molecular targets of NSAIDs in the toxic response that could explain the beneficial effects of these drugs in AD. More recently, an exciting finding supports the idea that the beneficial effects of NSAIDs in AD are at least partially independent of their role in inflammation [59]. Treatment of cells with some NSAIDs (ibuprofen, sulindac sulfide and indomethacin) was shown to result unexpectedly in a selective inhibition of Aβ1–42 generation. This slightly longer Aβ peptide is more amyloidogenic than the more abundantly produced Aβ1–40. Therefore, NSAIDs could act at the root of the disease placing these compounds into the ivy league of anti-amyloidogenic drugs [60]. Suppression of plaque pathology and amyloid deposition was also demonstrated in a transgenic mouse model treated with ibuprofen [61]; therefore, the anti-amyloidogenic activity of certain NSAIDs seems to be relevant in vivo. Clearly, it is now an important challenge to understand the molecular mechanism behind the positive effects of NSAIDs in AD. Clinical trials have now begun with several of the existing compounds. This will allow evaluation of whether the benefits of chronic use of NSAIDs for the prevention of AD outweigh the risks of gastric bleeding and other side-effects associated with these drugs. An important question that also needs to be addressed is whether NSAIDs are useful in the treatment of patients after the disease process has been initiated. β clearance Aβ

Several mechanisms, including phagocytosis and intra- and extracellular proteolysis, probably contribute to Aβ catabolism in the brain in vivo. Age-related decreases in any of these processes might contribute to pathological Aβ accumulation. Several Aβ-degrading enzymes have been proposed. The evidence supporting the involvement of insulindegrading enzyme (IDE) is strong [62,63], but is based mainly on cell culture studies. Recently, a series of genetic markers in the vicinity of the IDE locus on chromosome 10 showed genetic linkage to (late onset) AD [64]. However, evidence supporting http://tips.trends.com

the contribution of IDE to Aβ removal in vivo is missing. Neprilysin, by contrast, was identified as the main enzyme responsible for the degradation of radioactively labelled Aβ peptides that were injected directly into rat brains [65]. There is an inverse correlation between vulnerability to Aβ deposition and neprilysin abundance in different brain regions [66]. More importantly, neprilysin-deficient mice exhibited defects in Aβ degradation that were gene-dose dependent [67]. Therefore, good evidence supports the claim that neprilysin is a major Aβ-degrading enzyme in brain. Finally, three other proteolytic systems, plasmin [68,69], angiotensin-converting enzyme (ACE) [70] and endothelin-converting enzyme 1 (ECE-1) [71], have also been implicated in Aβ degradation. Further work is needed to clarify to what extent IDE, plasmin, ECE-1 and ACE, together with neprilysin, are responsible for the removal of Aβ from the brain, and whether decreased activity of these enzymes is involved in the pathogenesis of AD. However, whether drugs can be developed that activate these proteolytic systems specifically in a way that is useful to treat patients remains to be determined. β fibrils Solubilization of Aβ

After Aβ levels exceed a certain threshold, insoluble fibrils arise that self-associate and deposit in the brain. The factors that contribute to the different stages of fibril formation are not completely understood but it is clear that interfering with this process might be beneficial for AD. Aβ can bind Cu and Zn in vitro, aggravating both its tendency to aggregate and its toxicity in certain assays [72,73]. Zn or Cu chelators can solubilize Aβ from AD brains [74] and thus a recent study directly addressed the potential benefits of using metal chelators to dissolve amyloid plaques in vivo in a mouse model of AD [75]. Animals treated with clioquinol, an antibioticum that binds Cu and is able to cross the blood–brain barrier, showed a substantial decrease in brain Aβ deposition and an improvement in general health, with no toxic side-effects. Clioquinol had been used as a medication in the past until it was withdrawn from the market in the early 1970s because of its association with subacute myelo-optic neuropathy (SMON). It is now believed that this complication can be prevented by co-administration of vitamin B12. Consequently, clioquinol in combination with vitamin B12 is currently in clinical trials for AD treatment [75]. Perspectives

Twenty years ago the cholinergic hypothesis of AD was prominent and pharmacological research was directed to develop acetylcholinesterase inhibitors to counterbalance the deficit in the neurotransmitter acetylcholine observed in AD brains. Indeed, several acetylcholinesterase inhibitors (e.g. galantamine, donepezil and rivastigmine) are available at present

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for the treatment of AD patients [76]. Unfortunately, these drugs act only at the symptomatic level, providing some improvement in cognition and social behaviour. Their effects remain modest and transient, however, probably because the cause of the disease is not targeted. The hope is now raised that novel therapies act at the root of the disease process and will be able to stop the progressive accumulation of Aβ in the brain. However, reducing Aβ production via inhibition of β-secretase, although a promising approach, is at present far from the clinic. β-Secretase itself has been known for less than three years and a BACE inhibitor has not been shown to function in vivo. Furthermore, the risk of toxicity associated with γ-secretase inhibition is hindering the use of such compounds for the treatment of AD patients. By contrast, the vaccination approach, even if temporarily suspended, remains promising as a result of behavioural improvements observed in mice and the possibility that the amyloid accumulated in the preclinical stages of the disease can be cleared following Aβ vaccination. References 1 Lewis, J. et al. (2001) Enhanced neurofibrillary degeneration in transgenic mice expressing mutant tau and APP. Science 293, 1487–1491 2 Gotz, J. et al. (2001) Formation of neurofibrillary tangles in P301l tau transgenic mice induced by Aβ 42 fibrils. Science 293, 1491–1495 3 Selkoe, D.J. (1999) Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature 399, A23–31 4 Small, D.H. et al. (2001) Alzheimer’s disease and Aβ toxicity: from top to bottom. Nat. Rev. Neurosci. 2, 595–598 5 Vassar, R. and Citron, M. (2000) Aβ-generating enzymes: recent advances in β- and γ-secretase research. Neuron 27, 419–422 6 Cai, H. et al. (2001) BACE1 is the major β-secretase for generation of Aβ peptides by neurons. Nat. Neurosci. 4, 233–234 7 Luo, Y. et al. (2001) Mice deficient in BACE1, the Alzheimer’s β-secretase, have normal phenotype and abolished β-amyloid generation. Nat. Neurosci. 4, 231–232 8 Roberds, S.L. et al. (2001) BACE knockout mice are healthy despite lacking the primary β secretase activity in brain: implications for Alzheimer’s disease therapeutics. Hum. Mol. Genet. 10, 1317–1324 9 Hong, L. et al. (2000) Structure of the protease domain of memapsin 2 (β-secretase) complexed with inhibitor. Science 290, 150–153 10 Wolfe, M.S. et al. (1999) Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and gamma-secretase activity. Nature 398, 513–517 11 De Strooper, B. et al. (1998) Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 391, 387–390 12 Yu, G. et al. (2000) Nicastrin modulates presenilin-mediated notch/glp-1 signal http://tips.trends.com

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To date, there is much experience of the clinical use of NSAIDs in the treatment of rheumatoid arthritis and statins in the treatment of hypercholesterolemie. Thus, it is possible to test these drugs rapidly in the treatment of AD. In this regard, it is possible that these drugs (acting on the disease process) can be combined with the cholinergic drugs (acting on the symptoms) to treat AD patients. The arguments in favour of performing clinical trials to test such combination therapy are, in our opinion, compelling. While awaiting the full development of secretase inhibitors and the vaccination protocols, optimism is already spreading among researchers and, in particular, among patients and their families that improved medication is at hand. However, caution is required: there is a huge difference between the cell culture and mice models that have been used to understand the disease process and the reality of the brain of an aged AD patient. The real challenge is still before us, and the amyloid hypothesis will only be validated after patients have benefited from one of the drugs discussed above.

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