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Secretases as targets for the treatment of Alzheimer’s disease
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Secretases as targets for the treatment of Alzheimer’s disease Martin Citron
Alzheimer’s disease (AD) is the major cause of dementia in most developed countries. Treatment to modify this disease is currently unavailable, but needed urgently. The amyloid-cascade hypothesis proposes that amyloid b-peptide (Ab), found in the plaques characteristic of AD, plays an early, critical role in the disease process. It follows that preventing the generation of Ab could be therapeutically useful in all cases of AD. Inhibition of the secretases that produce Ab from a large precursor protein is the main approach to achieve this goal. ALZHEIMER’S disease (AD) accounts for about 50–70% of typical, late-onset cases of dementia. It is characterized clinically by a global decline in cognitive function that progresses slowly and leaves endstage patients bedridden, incontinent and dependent on custodial care. Most cases are diagnosed after the age of 65 (hence the term, lateonset) with death occurring, on average, nine years later. The major risk factor for AD is increasing age and in the USA alone there are currently over four million patients with AD with the direct and indirect costs of the disease approaching US$ 90 billion annually1. The medications currently approved for mild to moderate AD enhance cognition by inhibiting acetylcholinesterase activity. However, these drugs are of limited benefit and do not address the pathogenic mechanism of the disease. Thus, identifying the key steps in pathogenesis of AD, and developing therapeutics to block these, is the central goal in the study of AD.
Amyloid as a target for the treatment of AD The first clues to the cause of AD came from pathology studies carried out by Alois Alzheimer at the beginning of the last century. His studies revealed the two neuropathological lesions, senile plaques and neurofibrillary tangles, which still provide a definitive diagnosis of AD after the patient’s death. The biochemical characterization of both lesions has proved difficult, but, over the last 20 years, the major constituents of both plaques and tangles have been determined. The intraneuronal tangles consist primarily of aggregates of a hyperphosphorylated form of the neuronal protein, tau. The extracellular amyloid plaques Martin Citron Senior Scientist Dept of Neuroscience, M/S 29-2-B, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA. Tel: +1 805 447 4520 Fax: +1 805 480 1347 e-mail: [email protected]
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consist of aggregates of amyloid b-peptide (Ab) isoforms. These are 39–42 residue peptides that are proteolytically derived from the large amyloid precursor protein (APP) by two proteases, b-secretase and g-secretase, and secreted by all cells2,3. The two major isoforms of Ab are Ab40 and Ab42, which have identical N-termini but Ab42 is two residues longer. Although cells secrete more Ab40 than Ab42, Ab42 is less soluble and forms the major component of the amyloid plaques3. While pathology studies support an involvement of both tau and Ab in the pathogenesis of AD, it has been suggested that the plaques and/or tangles might not themselves cause AD, rather they are just tombstones of, or reactions to, an unrelated, underlying disease process. Indeed, mutations in tau have been identified in other neurological diseases, where they cause dementia and tangles without deposition of Ab. Although this supports the proposal that plaques, not tangles, drive the disease process, it does not rule out the possibility that tangles are critical to developing the clinical picture of AD (Ref. 4). In contrast, all known genetic factors predisposing to AD are related to the amyloid phenotype. Patients with trisomy of chromosome 21 (to which APP maps) develop classical AD neuropathology by the age of 50. These patients have an increased APP gene dosage that leads to overproduction of Ab and deposition of amyloid at a young age. It is clearly demonstrated that amyloid deposition precedes tangle formation in the brains of these individuals. However, the possibility that other genes on chromosome 21, in addition to APP, contribute to the pathology cannot be formally excluded.
The genes The most convincing evidence for a causal role of Ab in AD comes from studies of familial AD, in particular, a rare form of familial, autosomal-dominant AD with early disease onset (before age 65 years). Although the onset of disease is unusually early in these families, the clinical course and pathology is so similar to the more common, sporadic AD that they have been used to map disease genes and identify causative mutations. In the last ten years, point mutations in three different genes have been identified which cause AD in these families.
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Apolipoprotein E4
Glossary Amyloid b-peptide (Ab) – a peptide of 39–42 amino acid residues that is the major component of the amyloid plaques in the brains of Alzheimer’s disease (AD) patients. Amyloid precursor protein (APP) – a large transmembrane protein from which Ab is released by the secretase enzymes. b site APP cleaving enzyme (BACE) – a novel aspartic transmembrane protease, with all known properties of b-secretase. Presenilins – two proteins, presenilin 1 and presenilin 2, point mutations in which cause early onset AD, encoded by PSEN1 and PSEN2 respectively. Secretases – proteases that cleave APP and lead to the release of secreted metabolites. Cleavage of APP by b- and g-secretase is required to generate Ab.
APP
In addition to the rare mutations that cause familial early-onset AD, a major risk factor for late-onset AD has been identified. The apolipoprotein E (APOE) allele has three isoforms, APOE*2, APOE*3 and APOE*4, and numerous studies show that there is a dose-dependent increase in the likelihood that carriers of the APOE*4 allele will both develop AD and that the disease will become apparent at an earlier age8. The mechanism of apoE4 action in AD remains controversial. While apoE4 does not increase Ab production, apoE4 carriers have increased levels of both parenchymal and vascular Ab deposits, and a transgenic mouse model of Ab formation suggests that apoE has a critical, isoform-specific role in influencing Ab deposition and structure in vivo9.
The amyloid-cascade hypothesis The finding that all known mutations that cause early onset AD enhance production of Ab42, plus evidence from pathology studies, has led to the amyloid-cascade hypothesis in which Ab42 production plays an early, critical role in AD pathogenesis10 (Fig. 1). In early-onset AD, overproduction of Ab42 directly triggers the pathogenic cascade, whereas in sporadic cases Ab42 production is not necessarily increased. In this model, Ab42 produced in excessive quantities, cleared too slowly, or in contact with aggregation factors, would form aggregates. This could lead to the formation of senile plaques and initiate immunological and neurotoxicological cascades resulting in the pathologic and clinical
The first of these to be implicated was the APP gene. Interestingly, all disease-causing mutations identified are substitutions within or near the region containing Ab (Ref. 5), even though this forms only a small fraction of the large APP molecule. This suggests that these mutations affect the generation or the properties of Ab, rather than alter aspects of APP function unrelated to Ab. Subsequent studies show that each mutation (1) Overproduction, decreased clearance or enhanced aggregation of Aβ42 enhances the generation of Ab42. Increases in Ab42 have been observed in transfection experiments (2) Deposition of aggregated Aβ42 as diffuse plaques and transgenic models, as well as in peripheral tissues of young, presymptomatic mutation carriers, clearly demonstrating that the increase (3) Aggregation of Aβ40 onto diffuse Aβ42 plaques in Ab42 precedes AD (Ref. 3).
Presenilins Mutations in two other closely related genes, presenilin 1 (PSEN1) and presenilin 2 (PSEN2), also cause early-onset, familial AD. Presenilin 1 and presenilin 2 are eight transmembrane proteins with no sequence similarity to known proteases or receptors. Several studies show that presenilins control the proteolytic step that leads to release of the intracellular domain of Notch6. They are, thus, required for Notch signaling, which is important for cell-fate decisions in embryogenesis, neuronal differentiation and hematopoiesis. Presenilins might also regulate the cleavage of other integral membrane proteins, including APLP-1, a homolog of APP, and Ire1 (Refs 6 and 7). The mechanism by which mutant presenilins cause AD is unknown. However, as with mutations in the APP gene, in transfection experiments, transgenic models and peripheral tissues of young, presymptomatic mutation carriers5, it has been demonstrated that the AD-linked mutant presenilin proteins affect APP processing and increase Ab 42 production.
(4) Inflammatory response: microglial activation and cytokine release, astrocytosis and acute-phase protein release
(5) Progressive neuritic injury within amyloid plaques, disruption of neuronal metabolic homeostasis, oxidative injury
(6) Altered kinase/phosphatase activities, tau hyperphosphorylation and tangle formation
(7) Widespread neuronal dysfuction and death in hippocampus and cortex with progressive neurotransmitter deficits
(8) Dementia
Molecular Medicine Today
Figure 1. The amyloid-cascade hypothesis (modified from Ref. 2). The key steps of the hypothetical pathogenic cascade are shown. The cascade is initiated by the formation of Ab42 aggregates. This can result from overproduction of Ab42, as demonstrated by familial early-onset AD, or hypothetically, by lack of clearance or enhanced aggregation (1). While there is evidence for each step in the cascade and also the temporal sequence of events, it is not clear whether every step from (2) to (7) is necessary for pathogenesis. In addition, the causal relationship between steps (5) and (6) remains to be shown. Most importantly, the mechanism by which amyloid pathology causes tangle pathology remains to be demonstrated. The steps indicated in red still have to be demonstrated in transgenic animal models of AD pathology.
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manifestations of AD (Ref. 3). It follows that pharmacological intervention at various steps of the amyloid cascade could be used to halt or, possibly, even reverse the pathogenesis of AD. Various therapeutic strategies can be suggested to block the progression of this cascade. However, the further downstream in the cascade a given target appears, the less certainty there is about its relative importance in the overall process. For example, it is clear that neurons die in AD, so an antineurotoxic strategy should be useful. But what exactly kills the neurons? Is it the fibrillar amyloid in the plaques, the less-compacted amyloid fibrils, the tangles that have formed as a reaction to the amyloid or the inflammatory cascade triggered by the amyloid? Furthermore, do the neurons die by apoptosis and would it be therapeutically useful to merely delay the death of dysfunctional, tangle-bearing neurons, rather than prevent this completely? As long as such questions remain, selection of a specific target for any antineurotoxic strategy is difficult. In contrast, the situation at the top of the cascade is quite clear: formation of Ab42, its aggregation and clearance are critical, and preventing these should be beneficial. In principle, a clearance approach is interesting, in particular because of speculation that Ab clearance is impaired in many late-onset AD patients. However, establishing the exact mechanisms of Ab clearance in the brain is difficult. A recent study demonstrates that neprilysin is required for Ab clearance in vivo11. However, from a practical perspective, specific activation of proteases is more difficult
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to accomplish than specific inhibition. Therefore, an ‘activation of clearance’ approach might not be feasible. Anti-aggregation approaches have been discussed recently in this journal12. Here we focus on inhibiting production of Ab42.
The secretases
The identification of b-secretase in 1999 and the progress in the characterization of the still unknown g-secretase over the last two years has provided new opportunities to develop therapies to inhibit secretase activity. At the same time, the challenges associated with this therapeutic approach have become more clearly defined. Blocking Ab42 production could be accomplished either by reducing formation of APP or by inhibiting proteolysis of APP to Ab42. Blocking APP production appears the less attractive option, as APP and/or some of its non-Ab derivatives could have important biological functions. Moreover, from a practical perspective, preventing transcription of one specific target gene is difficult. Therefore, blocking the proteolytic machinery that produces Ab42 is the preferred strategy. The principles of this proteolytic process are outlined in Fig. 2. The APP molecule is large (<700 amino acids) and membrane bound. Following synthesis, it moves through the secretory pathway to the cell surface and from there to endosomes. On its way to the cell surface, two different proteases, a- and b-secretase can cleave at different positions within the APP molecule leading to the release of the large soluble N-terminal fragments, a-APPs and b-APPs, respectively. Cleavage by a-secretase occurs within the region containing Ab and, thus, precludes formation of Ab. In conAPP trast, b-secretase cleavage generates the free N-terminus of Ab and is, therefore, considered the first critical step in amyloid formation. Both cleavage events produce a membrane-bound C-terminal fragment. These fragments differ in their N-termini and are named C99 and C83 according to the number of amino acids they contain (generated by band a-secretase respectively). Both C99 and C83 can be further cleaved by g-secretase, within the APP transmembrane domain, α-secretase β-secretase which leads to the release and secretion of Ab from C99 and of p3, a shortened, presumably nonpathogenic, Ab form from C83. The majority of the g-secretase cleavage occurs at α-APPs C83 β-APPs C99 one of two positions, after either residue 40 or 42, leading to the formation of Ab40 and Ab42 γ-secretase γ-secretase from C99, and p340 and p342 from C83. Because the therapeutic goal is reduction of Ab production, three strategies can be pursued: (1) stimulation of a-secretase p3 Aβ cleavage to divert substrate from the AbMolecular Medicine Today forming b-secretase pathway; (2) inhibition of b-secretase; and (3) inhibition of g-secreFigure 2. Schematic of the amyloid precursor protein (APP) and its metabolites relevant to Alzheimer’s distase. The first strategy is the most demanding ease (not drawn to scale). APP can be processed along two major pathways, the a-secretase pathway and the amyloid forming b-secretase pathway. In the a-secretase pathway, a-secretase cleaves in the midas it requires stimulation rather than inhibidle of the Ab region to release a large soluble APP fragment, a-APPs. The C-terminal C83 peptide is tion of a proteolytic enzyme. However, metabolised to p340 and p342 by g-secretase. In the amyloid forming b-secretase pathway, b-secretase various studies suggest that activation of releases a large soluble fragment, b-APPs. The C-terminal C99 peptide is then metabolised to Ab40 and neurotransmitter receptors coupled to the Ab42 by g-secretase. b-secretase inhibitors block the formation of b-APPs and C99; g-secretase inhibitors block the formation of p3 and Ab. protein kinase C signaling cascade stimulate the a-secretase pathway at the expense
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of the b-secretase pathway13. While this pharmacological approach can be used to decrease Ab42 production indirectly, it does create two concerns. One is that the activation of neurotransmitter receptors affects systems other than APP processing. In addition, not every experimental system demonstrates a mutually exclusive relationship between cleavage by a- and b-secretase14. Therefore, the major emphasis of research into anti-amyloid therapeutics is currently based on strategies (2) and (3).
Inhibition of secretase activity as a therapeutic strategy To the author, inhibition of secretase activity appears to be the most promising strategy at this point. The general pros and cons are the same for inhibition of both b- and g-secretase. Amyloid plays a central role in AD and blocking amyloid production should prevent the development of AD. The theoretical specificity and tractability of protease targets suggests that it should be possible to generate secretase-specific protease inhibitors that penetrate the blood–brain barrier. At this point, there is no evidence for additional functions of Ab, so there are no serious concerns about reduction of this metabolite. As the pathway that produces Ab consumes only a minor proportion of all APP molecules, concerns about disrupting APP processing with b- or g-secretase inhibitors are also minimal. However, there are two serious concerns with this approach. First, both secretases are present in many different cells in the body and it is reasonable to assume that they have substrates in addition to APP. Therefore, complete inhibition of one of these enzymes might result in toxicity problems, especially under the conditions of chronic treatment that would presumably be required. However, even if one or both of the secretases are vital enzymes, partial inhibition could still be useful therapeutically. It is remarkable that most of the mutations identified in early-onset AD only increase Ab42 levels by a factor of two; therefore even a 30–50% reduction of Ab levels could have a significant impact3. In this context it should also be remembered that although the best-selling cholesterol-lowering agents also inhibit a vital enzyme, HMG CoA (3-hydroxy-3-methylglutaryl co-enzyme A) reductase, partial, chronic inhibition lowers cholesterol safely. Obviously, concerns about toxicity need to be addressed in extensive animal studies. The second concern stems from the fact that amyloid production is at the top of the amyloid cascade and dementia is at the bottom (see Fig. 1). Thus, one could argue that by the time a patient presents with symptoms, the amyloid burden is already high and the disease too far advanced to be treatable with anti-amyloid drugs. However, plaques are dynamic structures that can be cleared15 and anti-amyloid therapy would allow clearance mechanisms to remove the existing amyloid load. Such an approach is effective in peripheral amyloidoses, where blocking the production of the amyloid-forming protein leads to clearance of preexisting deposits and therapeutic improvement16.
Inhibition of b- or g-secretase?
As Figure 2 shows, complete inhibition of either b- or g-secretase alone should be sufficient to block Ab production completely. This situation is reminiscent of preventing the production of angiotensin by either inhibiting angiotensin-converting enzyme or renin activity17. However, the consequences of b- and g-secretase inhibition for the production of APP metabolites are slightly different. They could be biologically important, but whether they really matter in the actual treatment situation remains to be seen. Inhibiting b-secretase would stop the production of the immediate b-secretase cleavage products b-APPs and C99 and, thus, the production of both Ab40
and Ab42 (Fig. 2). It is possible that under these conditions alternative, b-secretase-like cleavages could be enhanced, although such effects have been observed primarily in peripheral cells and not in neurons. The production of all a-secretase metabolites, including p3 42 would not be decreased. This could be problematic if p342 had a role in disease pathogenesis. The effect of blocking g-secretase, especially of inhibiting both 40 and 42 cleavage would be more striking; production of both p3 and Ab would stop and C83 and C99 would accumulate (Fig. 2). This raises concerns because massive overproduction of C99 is toxic in some experimental paradigms. However, pre-clinical studies of g-secretase inhibitors suggest that C99 is cleared by other mechanisms and does not necessarily harm cells18. Apart from these theoretical concerns, the choice of whether to inhibit b- or g-secretase is based on practical considerations. Until last year, there were no published reports on the identity of either b- or g-secretase, therefore development of inhibitors proceeded without well-characterized molecular targets. Usually, cells that endogenously produce the secretase enzymes were used in cellbased screening assays to identify inhibitors of Ab secretion. Compounds were validated in secondary assays to identify inhibitors of either the b- or g-secretase pathway, as judged by the accumulation of certain APP metabolites. Using this kind of approach, various groups identified relatively potent inhibitors of the g-secretase pathway. Why this approach has identified more g- than b-secretase inhibitor leads, is currently unknown, as the primary screen data have generally not been published.
Identification of the secretase enzymes is important If potent secretase inhibitor leads have been generated while the secretases remained unknown, then is it necessary to identify the secretase enzymes for drug discovery? The generation of a specific protease inhibitor that penetrates the blood–brain barrier is a challenge for medicinal chemists and this task is made more difficult if the enzyme is unknown. Although you can identify inhibitors of the b- and g-secretase pathways without pure enzyme, the molecular mechanism of the inhibitor remains unknown and it is not possible to prove that the inhibitor targets the active site of the enzyme. This is a somewhat uncomfortable concept on which to base a large drug-development program. More importantly, structural information on the enzyme cannot be used to guide optimization of the leads. Such rational approaches have been critical for the development of HIV-protease inhibitors17. Finally, without identifying the secretases it is impossible to systematically address the potential mechanism based-toxicity directly. For these reasons, and because the
The outstanding questions presenilin g-secretase? • IsIf presenilin is g-secretase, how can the spatial paradox be re• solved and what other proteins does this enzyme cleave? If it
• • •
is not, what is the molecular nature of g-secretase and how does it interact with presenilin? Will inhibition of secretase activity stop the cognitive decline in AD patients? Is b- or g-secretase the better target for AD therapy? What toxicity will b- and g-secretase inhibitors have?
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identification of b- and g-secretase is a major academic challenge, numerous groups in academia and industry have tried to identify b- and g-secretase, and major progress in this area was published in 199919–23. The identification of a novel aspartic transmembrane protease, termed b site APP cleaving enzyme (BACE, Fig. 3), with all the known properties of b-secretase was first reported by Vassar et al.19 They showed that overexpression of BACE leads to increased b-secretase cleavage at the expected positions, that preventing the expression of BACE decreases b-secretase activity and that the purified enzyme cleaves APP-derived substrates with the right specificity. Four subsequent, independent publications using different methods identified the same enzyme20–22,24. At this point, there appears to be broad consensus that BACE is the major b-secretase in human brain cells. At the mRNA level, BACE is expressed widely in human brain. Expression is also high in pancreas, although enzymatic activity in this tissue is low22. Apart from APP cleavage, we do not know what other activity BACE possesses and so it is too early to predict what toxicity b-secretase inhibitors may have. However, it is widely assumed that BACE
N
D
D
C Molecular Medicine Today
Figure 3. Schematic of the BACE protein (b-secretase). BACE is a type I transmembrane domain protein with an N-terminal propeptide (black), two active sites containing the catalytic aspartate residues (D) and three intramolecular disulfide bonds29. The ectodomain is sufficient for protease activity19. BACE and its homolog (BACE2) are the only known membrane-bound aspartic proteases. APP is also a transmembrane protein and it is oriented in the same way as BACE so that the proteolytically active ectodomain of BACE can access the b-secretase cleavage site.
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did not evolve only to cleave APP. Analysis of knockout mice will help to shed some light on this question, although the final answer might require the production of conditional knockouts if embryonic lethality is a problem. Studies using this new knowledge to identify b-secretase inhibitors by screening or rational design approaches are already underway. As for g-secretase, the situation is less clear. A definitive identification of g-secretase has not yet been reported and, at this point, it is not clear whether there is one g-secretase that cleaves at Ab40 and Ab42, or whether there are multiple isoforms involved. Clearly, mutations near the C-terminus of Ab that cause AD somehow enhance generation of Ab42, and the same holds for mutations in the PSEN1 and PSEN2 genes. The finding that PSEN1-knockout mice have an 80% reduction g-secretase activity25 demonstrates that presenilin 1 is involved in cleavage of g-secretase. A subsequent study has reported that two aspartic acid residues within transmembrane domains six and seven of presenilin are critical for generation of Ab. Based primarily on this finding, and the data from the knockout mice, it has been hypothesized that presenilin 1 itself is g-secretase, a novel transmembrane aspartic protease in which the two critical Asp residues form an unusual active site23. Consistent with this idea, a recent biochemical study identified presenilin 1 in a large protein complex with g-secretase activity26. However, the critical role of only one, but not the other, Asp residues for Ab generation has been confirmed27, which does not support the protease model. Moreover, there is a spatial paradox. At the subcellular level, presenilins appear to be restricted to early-transport compartments, whereas g-secretase is active primarily in late-transport compartments and the endosomal pathway6. Furthermore, presenilin has no similarity to known proteases and it has no known proteolytic activity. Notwithstanding these concerns, at the present time, data from the PSEN-knockout mice, combined with the fact that, despite intense efforts over the last decade, no better candidates for g-secretase have been identified are consistent with the proposed role of presenilin-1 as g-secretase, although it is possible that it could be a critical cofactor for the enzyme. This question is not only of academic interest. Presenilin regulates not only cleavage of APP by g-secretase, but also the intracellular cleavage of Notch (Ref. 28) and possibly several other proteins and complete inhibition would also block cleavage of all presenilin substrates. Notch is required for hematopoiesis6 in the adult, and Ire1, which might also be controlled by presenilin7, might be critical for the unfolded protein response. If presenilin controls the unknown g-secretase and other, as yet unidentified, enzymes that cleave Notch and Ire1, it could be possible to distinguish these proteases pharmacologically and so avoid effects on Notch and Ire1 signalling. From a practical perspective, the identification of the unknown g-secretase as presenilin would not make the target much more tractable, as it shows no homology to known aspartic proteases and, thus, cannot be modeled easily. A simple in vitro assay using purified components does not exist and crystallization of this protein to determine the three-dimensional structure to guide rational drug design is also expected to be difficult. But even if presenilin turns out to be a critical cofactor of a more conventional g-secretase enzyme, the biochemistry of this system will be challenging.
Concluding remarks
The definitive identification of g-secretase remains the biggest scientific challenge in this field. The development of inhibitors of both b- and g-secretase promises progress towards a treatment to
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modify the disease. Although current animal models can predict the efficacy of secretase inhibitors in Ab generation and plaque formation, only a clinical trial using AD patients will show if these compounds block the cognitive decline caused by the underlying pathology. Assuming that the required cognitive effects are demonstrated, the toxicity of the compounds will determine how broadly they can be used. If there are no major side effects associated with treatment with secretase inhibitors, their use could be extended from therapy of symptomatic AD patients to include prevention of the disease in carriers of risk factors or even the general population above a certain age. References 1 Davis, K.L. and Samuels, S.C. (1998) Dementia and delirium. In Pharmacological management of neurological and psychiatric disorders (Enna, S.J. and Coyle, J.T., eds), pp. 267–316, McGraw-Hill 2 Selkoe, D.J. (1998) The cell biology of b-amyloid precursor protein and presenilin in Alzheimer’s disease. Trends Cell Biol. 8, 447–453 3 Selkoe, D.J. (1999) Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature 399, A23–A31 4 Goedert, M. et al. (1998) Tau mutations cause frontotemporal dementias. Neuron 21, 955–958 5 Hardy, J. (1997) Amyloid, the presenilins and Alzheimer’s disease. Trends Neurosci. 20, 154–159 6 Haass, C. and De Strooper, B. (1999) The presenilins in Alzheimer’s disease proteolysis holds the key. Science 286, 916–919 7 Niwa, M. et al. (1999) A role for presenilin-1 in nuclear accumulation of Ire1 fragments and induction of the mammalian unfolded protein response. Cell 99, 691–702 8 Roses, A. (1996) Apolipoprotein E alleles as risk factors in Alzheimer’s disease. Annu. Rev. Med. 47, 387–400 9 Holtzman, D.M. et al. (2000) Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 97, 2892–2897 10 Hardy, J. and Allsop, D. (1991) Amyloid deposition as the central event in the aetiology of Alzheimer’s disease. Trends Pharmacol. Sci. 12, 383–388 11 Iwata, N. et al. (2000) Identification of the major Ab1-42-degrading catabolic pathway in brain parenchyma: suppression leads to biochemical and pathological deposition. Nat. Med. 6, 143–150
12 Soto, C. (1999) Plaque busters: strategies to inhibit amyloid formation in Alzheimer’s disease. Mol. Med. Today 5, 343–350 13 Buxbaum, J.D. et al. (1993) Protein phosphorylation inhibits production of Alzheimer amyloid b/A4 peptide. Proc. Natl. Acad. Sci. U. S. A. 90, 9195–9198 14 Sinha, S. and Lieberburg, I. (1999) Cellular mechanisms of b-amyloid production and secretion. Proc. Natl. Acad. Sci. U. S. A. 96, 11049–11053 15 Urbanc, B. et al. (1999) Dynamics of plaque formation in Alzheimer’s disease. Biophys. J. 76, 1330–1334 16 Tan, S.Y. et al. (1995) Treatment of amyloidosis. Am. J. Kidney Dis. 26, 267–285 17 Leung, D. et al. (2000) Protease inhibitors: current status and future prospects. J. Med. Chem. 43, 305–341 18 Higaki, J. et al. (1995) Inhibition of b-amyloid formation identifies proteolytic precursors and subcellular site of catabolism. Neuron 14, 651–659 19 Vassar, R. et al. (1999) b-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286, 735–741 20 Hussain, I. et al. (1999) Identification of a novel aspartic protease (Asp2) as b-secretase. Mol. Cell. Neurosci. 14, 419–427 21 Yan, R. et al. (1999) Membrane-anchored aspartyl protease with Alzheimer’s disease b-secretase specificity. Nature 402, 533–537 22 Sinha, S. et al. (1999) Purification and cloning of amyloid precursor protein b-secretase from human brain. Nature 402, 537–540 23 Wolfe, M.S. et al. (1999) Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and g-secretase activity. Nature 398, 513–517 24 Lin, X. et al. (2000) Human aspartic protease memapsin 2 cleaves the bsecretase site of b-amyloid precursor protein. Proc. Natl. Acad. Sci. U. S. A. 97, 1456–1460 25 De Strooper, B. et al. (1998) Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 391, 387–391 26 Li, Y-M. et al. (2000) Presenilin 1 is linked with g-secretase activity in the detergent solubilized state. Proc. Natl. Acad. Sci. U. S. A. 97, 6138–6143 27 Capell, A. et al. (2000) Presenilin-1 differentially facilitates endoproteolysis of the b-amyloid precursor protein and Notch. Nat. Cell Biol. 2, 205–211 28 Struhl, G. and Greenwald, I. (1999) Presenilin is required for activity and nuclear access of Notch in Drosophila. Nature 398, 522-525 29 Haniu, M. et al. Characterization of Alzheimer’s b-secretase protein BACE. A pepsin family member with unusual properties. J. Biol. Chem. (in press)
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Secretases as targets for the treatment of Alzheimer’s disease Martin Citron
Alzheimer’s disease (AD) is the major cause of dementia in most developed countries. Treatment to modify this disease is currently unavailable, but needed urgently. The amyloid-cascade hypothesis proposes that amyloid b-peptide (Ab), found in the plaques characteristic of AD, plays an early, critical role in the disease process. It follows that preventing the generation of Ab could be therapeutically useful in all cases of AD. Inhibition of the secretases that produce Ab from a large precursor protein is the main approach to achieve this goal. ALZHEIMER’S disease (AD) accounts for about 50–70% of typical, late-onset cases of dementia. It is characterized clinically by a global decline in cognitive function that progresses slowly and leaves endstage patients bedridden, incontinent and dependent on custodial care. Most cases are diagnosed after the age of 65 (hence the term, lateonset) with death occurring, on average, nine years later. The major risk factor for AD is increasing age and in the USA alone there are currently over four million patients with AD with the direct and indirect costs of the disease approaching US$ 90 billion annually1. The medications currently approved for mild to moderate AD enhance cognition by inhibiting acetylcholinesterase activity. However, these drugs are of limited benefit and do not address the pathogenic mechanism of the disease. Thus, identifying the key steps in pathogenesis of AD, and developing therapeutics to block these, is the central goal in the study of AD.
Amyloid as a target for the treatment of AD The first clues to the cause of AD came from pathology studies carried out by Alois Alzheimer at the beginning of the last century. His studies revealed the two neuropathological lesions, senile plaques and neurofibrillary tangles, which still provide a definitive diagnosis of AD after the patient’s death. The biochemical characterization of both lesions has proved difficult, but, over the last 20 years, the major constituents of both plaques and tangles have been determined. The intraneuronal tangles consist primarily of aggregates of a hyperphosphorylated form of the neuronal protein, tau. The extracellular amyloid plaques Martin Citron Senior Scientist Dept of Neuroscience, M/S 29-2-B, Amgen Inc., One Amgen Center Drive, Thousand Oaks, CA 91320, USA. Tel: +1 805 447 4520 Fax: +1 805 480 1347 e-mail: [email protected]
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consist of aggregates of amyloid b-peptide (Ab) isoforms. These are 39–42 residue peptides that are proteolytically derived from the large amyloid precursor protein (APP) by two proteases, b-secretase and g-secretase, and secreted by all cells2,3. The two major isoforms of Ab are Ab40 and Ab42, which have identical N-termini but Ab42 is two residues longer. Although cells secrete more Ab40 than Ab42, Ab42 is less soluble and forms the major component of the amyloid plaques3. While pathology studies support an involvement of both tau and Ab in the pathogenesis of AD, it has been suggested that the plaques and/or tangles might not themselves cause AD, rather they are just tombstones of, or reactions to, an unrelated, underlying disease process. Indeed, mutations in tau have been identified in other neurological diseases, where they cause dementia and tangles without deposition of Ab. Although this supports the proposal that plaques, not tangles, drive the disease process, it does not rule out the possibility that tangles are critical to developing the clinical picture of AD (Ref. 4). In contrast, all known genetic factors predisposing to AD are related to the amyloid phenotype. Patients with trisomy of chromosome 21 (to which APP maps) develop classical AD neuropathology by the age of 50. These patients have an increased APP gene dosage that leads to overproduction of Ab and deposition of amyloid at a young age. It is clearly demonstrated that amyloid deposition precedes tangle formation in the brains of these individuals. However, the possibility that other genes on chromosome 21, in addition to APP, contribute to the pathology cannot be formally excluded.
The genes The most convincing evidence for a causal role of Ab in AD comes from studies of familial AD, in particular, a rare form of familial, autosomal-dominant AD with early disease onset (before age 65 years). Although the onset of disease is unusually early in these families, the clinical course and pathology is so similar to the more common, sporadic AD that they have been used to map disease genes and identify causative mutations. In the last ten years, point mutations in three different genes have been identified which cause AD in these families.
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Apolipoprotein E4
Glossary Amyloid b-peptide (Ab) – a peptide of 39–42 amino acid residues that is the major component of the amyloid plaques in the brains of Alzheimer’s disease (AD) patients. Amyloid precursor protein (APP) – a large transmembrane protein from which Ab is released by the secretase enzymes. b site APP cleaving enzyme (BACE) – a novel aspartic transmembrane protease, with all known properties of b-secretase. Presenilins – two proteins, presenilin 1 and presenilin 2, point mutations in which cause early onset AD, encoded by PSEN1 and PSEN2 respectively. Secretases – proteases that cleave APP and lead to the release of secreted metabolites. Cleavage of APP by b- and g-secretase is required to generate Ab.
APP
In addition to the rare mutations that cause familial early-onset AD, a major risk factor for late-onset AD has been identified. The apolipoprotein E (APOE) allele has three isoforms, APOE*2, APOE*3 and APOE*4, and numerous studies show that there is a dose-dependent increase in the likelihood that carriers of the APOE*4 allele will both develop AD and that the disease will become apparent at an earlier age8. The mechanism of apoE4 action in AD remains controversial. While apoE4 does not increase Ab production, apoE4 carriers have increased levels of both parenchymal and vascular Ab deposits, and a transgenic mouse model of Ab formation suggests that apoE has a critical, isoform-specific role in influencing Ab deposition and structure in vivo9.
The amyloid-cascade hypothesis The finding that all known mutations that cause early onset AD enhance production of Ab42, plus evidence from pathology studies, has led to the amyloid-cascade hypothesis in which Ab42 production plays an early, critical role in AD pathogenesis10 (Fig. 1). In early-onset AD, overproduction of Ab42 directly triggers the pathogenic cascade, whereas in sporadic cases Ab42 production is not necessarily increased. In this model, Ab42 produced in excessive quantities, cleared too slowly, or in contact with aggregation factors, would form aggregates. This could lead to the formation of senile plaques and initiate immunological and neurotoxicological cascades resulting in the pathologic and clinical
The first of these to be implicated was the APP gene. Interestingly, all disease-causing mutations identified are substitutions within or near the region containing Ab (Ref. 5), even though this forms only a small fraction of the large APP molecule. This suggests that these mutations affect the generation or the properties of Ab, rather than alter aspects of APP function unrelated to Ab. Subsequent studies show that each mutation (1) Overproduction, decreased clearance or enhanced aggregation of Aβ42 enhances the generation of Ab42. Increases in Ab42 have been observed in transfection experiments (2) Deposition of aggregated Aβ42 as diffuse plaques and transgenic models, as well as in peripheral tissues of young, presymptomatic mutation carriers, clearly demonstrating that the increase (3) Aggregation of Aβ40 onto diffuse Aβ42 plaques in Ab42 precedes AD (Ref. 3).
Presenilins Mutations in two other closely related genes, presenilin 1 (PSEN1) and presenilin 2 (PSEN2), also cause early-onset, familial AD. Presenilin 1 and presenilin 2 are eight transmembrane proteins with no sequence similarity to known proteases or receptors. Several studies show that presenilins control the proteolytic step that leads to release of the intracellular domain of Notch6. They are, thus, required for Notch signaling, which is important for cell-fate decisions in embryogenesis, neuronal differentiation and hematopoiesis. Presenilins might also regulate the cleavage of other integral membrane proteins, including APLP-1, a homolog of APP, and Ire1 (Refs 6 and 7). The mechanism by which mutant presenilins cause AD is unknown. However, as with mutations in the APP gene, in transfection experiments, transgenic models and peripheral tissues of young, presymptomatic mutation carriers5, it has been demonstrated that the AD-linked mutant presenilin proteins affect APP processing and increase Ab 42 production.
(4) Inflammatory response: microglial activation and cytokine release, astrocytosis and acute-phase protein release
(5) Progressive neuritic injury within amyloid plaques, disruption of neuronal metabolic homeostasis, oxidative injury
(6) Altered kinase/phosphatase activities, tau hyperphosphorylation and tangle formation
(7) Widespread neuronal dysfuction and death in hippocampus and cortex with progressive neurotransmitter deficits
(8) Dementia
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Figure 1. The amyloid-cascade hypothesis (modified from Ref. 2). The key steps of the hypothetical pathogenic cascade are shown. The cascade is initiated by the formation of Ab42 aggregates. This can result from overproduction of Ab42, as demonstrated by familial early-onset AD, or hypothetically, by lack of clearance or enhanced aggregation (1). While there is evidence for each step in the cascade and also the temporal sequence of events, it is not clear whether every step from (2) to (7) is necessary for pathogenesis. In addition, the causal relationship between steps (5) and (6) remains to be shown. Most importantly, the mechanism by which amyloid pathology causes tangle pathology remains to be demonstrated. The steps indicated in red still have to be demonstrated in transgenic animal models of AD pathology.
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manifestations of AD (Ref. 3). It follows that pharmacological intervention at various steps of the amyloid cascade could be used to halt or, possibly, even reverse the pathogenesis of AD. Various therapeutic strategies can be suggested to block the progression of this cascade. However, the further downstream in the cascade a given target appears, the less certainty there is about its relative importance in the overall process. For example, it is clear that neurons die in AD, so an antineurotoxic strategy should be useful. But what exactly kills the neurons? Is it the fibrillar amyloid in the plaques, the less-compacted amyloid fibrils, the tangles that have formed as a reaction to the amyloid or the inflammatory cascade triggered by the amyloid? Furthermore, do the neurons die by apoptosis and would it be therapeutically useful to merely delay the death of dysfunctional, tangle-bearing neurons, rather than prevent this completely? As long as such questions remain, selection of a specific target for any antineurotoxic strategy is difficult. In contrast, the situation at the top of the cascade is quite clear: formation of Ab42, its aggregation and clearance are critical, and preventing these should be beneficial. In principle, a clearance approach is interesting, in particular because of speculation that Ab clearance is impaired in many late-onset AD patients. However, establishing the exact mechanisms of Ab clearance in the brain is difficult. A recent study demonstrates that neprilysin is required for Ab clearance in vivo11. However, from a practical perspective, specific activation of proteases is more difficult
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to accomplish than specific inhibition. Therefore, an ‘activation of clearance’ approach might not be feasible. Anti-aggregation approaches have been discussed recently in this journal12. Here we focus on inhibiting production of Ab42.
The secretases
The identification of b-secretase in 1999 and the progress in the characterization of the still unknown g-secretase over the last two years has provided new opportunities to develop therapies to inhibit secretase activity. At the same time, the challenges associated with this therapeutic approach have become more clearly defined. Blocking Ab42 production could be accomplished either by reducing formation of APP or by inhibiting proteolysis of APP to Ab42. Blocking APP production appears the less attractive option, as APP and/or some of its non-Ab derivatives could have important biological functions. Moreover, from a practical perspective, preventing transcription of one specific target gene is difficult. Therefore, blocking the proteolytic machinery that produces Ab42 is the preferred strategy. The principles of this proteolytic process are outlined in Fig. 2. The APP molecule is large (<700 amino acids) and membrane bound. Following synthesis, it moves through the secretory pathway to the cell surface and from there to endosomes. On its way to the cell surface, two different proteases, a- and b-secretase can cleave at different positions within the APP molecule leading to the release of the large soluble N-terminal fragments, a-APPs and b-APPs, respectively. Cleavage by a-secretase occurs within the region containing Ab and, thus, precludes formation of Ab. In conAPP trast, b-secretase cleavage generates the free N-terminus of Ab and is, therefore, considered the first critical step in amyloid formation. Both cleavage events produce a membrane-bound C-terminal fragment. These fragments differ in their N-termini and are named C99 and C83 according to the number of amino acids they contain (generated by band a-secretase respectively). Both C99 and C83 can be further cleaved by g-secretase, within the APP transmembrane domain, α-secretase β-secretase which leads to the release and secretion of Ab from C99 and of p3, a shortened, presumably nonpathogenic, Ab form from C83. The majority of the g-secretase cleavage occurs at α-APPs C83 β-APPs C99 one of two positions, after either residue 40 or 42, leading to the formation of Ab40 and Ab42 γ-secretase γ-secretase from C99, and p340 and p342 from C83. Because the therapeutic goal is reduction of Ab production, three strategies can be pursued: (1) stimulation of a-secretase p3 Aβ cleavage to divert substrate from the AbMolecular Medicine Today forming b-secretase pathway; (2) inhibition of b-secretase; and (3) inhibition of g-secreFigure 2. Schematic of the amyloid precursor protein (APP) and its metabolites relevant to Alzheimer’s distase. The first strategy is the most demanding ease (not drawn to scale). APP can be processed along two major pathways, the a-secretase pathway and the amyloid forming b-secretase pathway. In the a-secretase pathway, a-secretase cleaves in the midas it requires stimulation rather than inhibidle of the Ab region to release a large soluble APP fragment, a-APPs. The C-terminal C83 peptide is tion of a proteolytic enzyme. However, metabolised to p340 and p342 by g-secretase. In the amyloid forming b-secretase pathway, b-secretase various studies suggest that activation of releases a large soluble fragment, b-APPs. The C-terminal C99 peptide is then metabolised to Ab40 and neurotransmitter receptors coupled to the Ab42 by g-secretase. b-secretase inhibitors block the formation of b-APPs and C99; g-secretase inhibitors block the formation of p3 and Ab. protein kinase C signaling cascade stimulate the a-secretase pathway at the expense
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of the b-secretase pathway13. While this pharmacological approach can be used to decrease Ab42 production indirectly, it does create two concerns. One is that the activation of neurotransmitter receptors affects systems other than APP processing. In addition, not every experimental system demonstrates a mutually exclusive relationship between cleavage by a- and b-secretase14. Therefore, the major emphasis of research into anti-amyloid therapeutics is currently based on strategies (2) and (3).
Inhibition of secretase activity as a therapeutic strategy To the author, inhibition of secretase activity appears to be the most promising strategy at this point. The general pros and cons are the same for inhibition of both b- and g-secretase. Amyloid plays a central role in AD and blocking amyloid production should prevent the development of AD. The theoretical specificity and tractability of protease targets suggests that it should be possible to generate secretase-specific protease inhibitors that penetrate the blood–brain barrier. At this point, there is no evidence for additional functions of Ab, so there are no serious concerns about reduction of this metabolite. As the pathway that produces Ab consumes only a minor proportion of all APP molecules, concerns about disrupting APP processing with b- or g-secretase inhibitors are also minimal. However, there are two serious concerns with this approach. First, both secretases are present in many different cells in the body and it is reasonable to assume that they have substrates in addition to APP. Therefore, complete inhibition of one of these enzymes might result in toxicity problems, especially under the conditions of chronic treatment that would presumably be required. However, even if one or both of the secretases are vital enzymes, partial inhibition could still be useful therapeutically. It is remarkable that most of the mutations identified in early-onset AD only increase Ab42 levels by a factor of two; therefore even a 30–50% reduction of Ab levels could have a significant impact3. In this context it should also be remembered that although the best-selling cholesterol-lowering agents also inhibit a vital enzyme, HMG CoA (3-hydroxy-3-methylglutaryl co-enzyme A) reductase, partial, chronic inhibition lowers cholesterol safely. Obviously, concerns about toxicity need to be addressed in extensive animal studies. The second concern stems from the fact that amyloid production is at the top of the amyloid cascade and dementia is at the bottom (see Fig. 1). Thus, one could argue that by the time a patient presents with symptoms, the amyloid burden is already high and the disease too far advanced to be treatable with anti-amyloid drugs. However, plaques are dynamic structures that can be cleared15 and anti-amyloid therapy would allow clearance mechanisms to remove the existing amyloid load. Such an approach is effective in peripheral amyloidoses, where blocking the production of the amyloid-forming protein leads to clearance of preexisting deposits and therapeutic improvement16.
Inhibition of b- or g-secretase?
As Figure 2 shows, complete inhibition of either b- or g-secretase alone should be sufficient to block Ab production completely. This situation is reminiscent of preventing the production of angiotensin by either inhibiting angiotensin-converting enzyme or renin activity17. However, the consequences of b- and g-secretase inhibition for the production of APP metabolites are slightly different. They could be biologically important, but whether they really matter in the actual treatment situation remains to be seen. Inhibiting b-secretase would stop the production of the immediate b-secretase cleavage products b-APPs and C99 and, thus, the production of both Ab40
and Ab42 (Fig. 2). It is possible that under these conditions alternative, b-secretase-like cleavages could be enhanced, although such effects have been observed primarily in peripheral cells and not in neurons. The production of all a-secretase metabolites, including p3 42 would not be decreased. This could be problematic if p342 had a role in disease pathogenesis. The effect of blocking g-secretase, especially of inhibiting both 40 and 42 cleavage would be more striking; production of both p3 and Ab would stop and C83 and C99 would accumulate (Fig. 2). This raises concerns because massive overproduction of C99 is toxic in some experimental paradigms. However, pre-clinical studies of g-secretase inhibitors suggest that C99 is cleared by other mechanisms and does not necessarily harm cells18. Apart from these theoretical concerns, the choice of whether to inhibit b- or g-secretase is based on practical considerations. Until last year, there were no published reports on the identity of either b- or g-secretase, therefore development of inhibitors proceeded without well-characterized molecular targets. Usually, cells that endogenously produce the secretase enzymes were used in cellbased screening assays to identify inhibitors of Ab secretion. Compounds were validated in secondary assays to identify inhibitors of either the b- or g-secretase pathway, as judged by the accumulation of certain APP metabolites. Using this kind of approach, various groups identified relatively potent inhibitors of the g-secretase pathway. Why this approach has identified more g- than b-secretase inhibitor leads, is currently unknown, as the primary screen data have generally not been published.
Identification of the secretase enzymes is important If potent secretase inhibitor leads have been generated while the secretases remained unknown, then is it necessary to identify the secretase enzymes for drug discovery? The generation of a specific protease inhibitor that penetrates the blood–brain barrier is a challenge for medicinal chemists and this task is made more difficult if the enzyme is unknown. Although you can identify inhibitors of the b- and g-secretase pathways without pure enzyme, the molecular mechanism of the inhibitor remains unknown and it is not possible to prove that the inhibitor targets the active site of the enzyme. This is a somewhat uncomfortable concept on which to base a large drug-development program. More importantly, structural information on the enzyme cannot be used to guide optimization of the leads. Such rational approaches have been critical for the development of HIV-protease inhibitors17. Finally, without identifying the secretases it is impossible to systematically address the potential mechanism based-toxicity directly. For these reasons, and because the
The outstanding questions presenilin g-secretase? • IsIf presenilin is g-secretase, how can the spatial paradox be re• solved and what other proteins does this enzyme cleave? If it
• • •
is not, what is the molecular nature of g-secretase and how does it interact with presenilin? Will inhibition of secretase activity stop the cognitive decline in AD patients? Is b- or g-secretase the better target for AD therapy? What toxicity will b- and g-secretase inhibitors have?
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identification of b- and g-secretase is a major academic challenge, numerous groups in academia and industry have tried to identify b- and g-secretase, and major progress in this area was published in 199919–23. The identification of a novel aspartic transmembrane protease, termed b site APP cleaving enzyme (BACE, Fig. 3), with all the known properties of b-secretase was first reported by Vassar et al.19 They showed that overexpression of BACE leads to increased b-secretase cleavage at the expected positions, that preventing the expression of BACE decreases b-secretase activity and that the purified enzyme cleaves APP-derived substrates with the right specificity. Four subsequent, independent publications using different methods identified the same enzyme20–22,24. At this point, there appears to be broad consensus that BACE is the major b-secretase in human brain cells. At the mRNA level, BACE is expressed widely in human brain. Expression is also high in pancreas, although enzymatic activity in this tissue is low22. Apart from APP cleavage, we do not know what other activity BACE possesses and so it is too early to predict what toxicity b-secretase inhibitors may have. However, it is widely assumed that BACE
N
D
D
C Molecular Medicine Today
Figure 3. Schematic of the BACE protein (b-secretase). BACE is a type I transmembrane domain protein with an N-terminal propeptide (black), two active sites containing the catalytic aspartate residues (D) and three intramolecular disulfide bonds29. The ectodomain is sufficient for protease activity19. BACE and its homolog (BACE2) are the only known membrane-bound aspartic proteases. APP is also a transmembrane protein and it is oriented in the same way as BACE so that the proteolytically active ectodomain of BACE can access the b-secretase cleavage site.
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did not evolve only to cleave APP. Analysis of knockout mice will help to shed some light on this question, although the final answer might require the production of conditional knockouts if embryonic lethality is a problem. Studies using this new knowledge to identify b-secretase inhibitors by screening or rational design approaches are already underway. As for g-secretase, the situation is less clear. A definitive identification of g-secretase has not yet been reported and, at this point, it is not clear whether there is one g-secretase that cleaves at Ab40 and Ab42, or whether there are multiple isoforms involved. Clearly, mutations near the C-terminus of Ab that cause AD somehow enhance generation of Ab42, and the same holds for mutations in the PSEN1 and PSEN2 genes. The finding that PSEN1-knockout mice have an 80% reduction g-secretase activity25 demonstrates that presenilin 1 is involved in cleavage of g-secretase. A subsequent study has reported that two aspartic acid residues within transmembrane domains six and seven of presenilin are critical for generation of Ab. Based primarily on this finding, and the data from the knockout mice, it has been hypothesized that presenilin 1 itself is g-secretase, a novel transmembrane aspartic protease in which the two critical Asp residues form an unusual active site23. Consistent with this idea, a recent biochemical study identified presenilin 1 in a large protein complex with g-secretase activity26. However, the critical role of only one, but not the other, Asp residues for Ab generation has been confirmed27, which does not support the protease model. Moreover, there is a spatial paradox. At the subcellular level, presenilins appear to be restricted to early-transport compartments, whereas g-secretase is active primarily in late-transport compartments and the endosomal pathway6. Furthermore, presenilin has no similarity to known proteases and it has no known proteolytic activity. Notwithstanding these concerns, at the present time, data from the PSEN-knockout mice, combined with the fact that, despite intense efforts over the last decade, no better candidates for g-secretase have been identified are consistent with the proposed role of presenilin-1 as g-secretase, although it is possible that it could be a critical cofactor for the enzyme. This question is not only of academic interest. Presenilin regulates not only cleavage of APP by g-secretase, but also the intracellular cleavage of Notch (Ref. 28) and possibly several other proteins and complete inhibition would also block cleavage of all presenilin substrates. Notch is required for hematopoiesis6 in the adult, and Ire1, which might also be controlled by presenilin7, might be critical for the unfolded protein response. If presenilin controls the unknown g-secretase and other, as yet unidentified, enzymes that cleave Notch and Ire1, it could be possible to distinguish these proteases pharmacologically and so avoid effects on Notch and Ire1 signalling. From a practical perspective, the identification of the unknown g-secretase as presenilin would not make the target much more tractable, as it shows no homology to known aspartic proteases and, thus, cannot be modeled easily. A simple in vitro assay using purified components does not exist and crystallization of this protein to determine the three-dimensional structure to guide rational drug design is also expected to be difficult. But even if presenilin turns out to be a critical cofactor of a more conventional g-secretase enzyme, the biochemistry of this system will be challenging.
Concluding remarks
The definitive identification of g-secretase remains the biggest scientific challenge in this field. The development of inhibitors of both b- and g-secretase promises progress towards a treatment to
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modify the disease. Although current animal models can predict the efficacy of secretase inhibitors in Ab generation and plaque formation, only a clinical trial using AD patients will show if these compounds block the cognitive decline caused by the underlying pathology. Assuming that the required cognitive effects are demonstrated, the toxicity of the compounds will determine how broadly they can be used. If there are no major side effects associated with treatment with secretase inhibitors, their use could be extended from therapy of symptomatic AD patients to include prevention of the disease in carriers of risk factors or even the general population above a certain age. References 1 Davis, K.L. and Samuels, S.C. (1998) Dementia and delirium. In Pharmacological management of neurological and psychiatric disorders (Enna, S.J. and Coyle, J.T., eds), pp. 267–316, McGraw-Hill 2 Selkoe, D.J. (1998) The cell biology of b-amyloid precursor protein and presenilin in Alzheimer’s disease. Trends Cell Biol. 8, 447–453 3 Selkoe, D.J. (1999) Translating cell biology into therapeutic advances in Alzheimer’s disease. Nature 399, A23–A31 4 Goedert, M. et al. (1998) Tau mutations cause frontotemporal dementias. Neuron 21, 955–958 5 Hardy, J. (1997) Amyloid, the presenilins and Alzheimer’s disease. Trends Neurosci. 20, 154–159 6 Haass, C. and De Strooper, B. (1999) The presenilins in Alzheimer’s disease proteolysis holds the key. Science 286, 916–919 7 Niwa, M. et al. (1999) A role for presenilin-1 in nuclear accumulation of Ire1 fragments and induction of the mammalian unfolded protein response. Cell 99, 691–702 8 Roses, A. (1996) Apolipoprotein E alleles as risk factors in Alzheimer’s disease. Annu. Rev. Med. 47, 387–400 9 Holtzman, D.M. et al. (2000) Apolipoprotein E isoform-dependent amyloid deposition and neuritic degeneration in a mouse model of Alzheimer’s disease. Proc. Natl. Acad. Sci. U. S. A. 97, 2892–2897 10 Hardy, J. and Allsop, D. (1991) Amyloid deposition as the central event in the aetiology of Alzheimer’s disease. Trends Pharmacol. Sci. 12, 383–388 11 Iwata, N. et al. (2000) Identification of the major Ab1-42-degrading catabolic pathway in brain parenchyma: suppression leads to biochemical and pathological deposition. Nat. Med. 6, 143–150
12 Soto, C. (1999) Plaque busters: strategies to inhibit amyloid formation in Alzheimer’s disease. Mol. Med. Today 5, 343–350 13 Buxbaum, J.D. et al. (1993) Protein phosphorylation inhibits production of Alzheimer amyloid b/A4 peptide. Proc. Natl. Acad. Sci. U. S. A. 90, 9195–9198 14 Sinha, S. and Lieberburg, I. (1999) Cellular mechanisms of b-amyloid production and secretion. Proc. Natl. Acad. Sci. U. S. A. 96, 11049–11053 15 Urbanc, B. et al. (1999) Dynamics of plaque formation in Alzheimer’s disease. Biophys. J. 76, 1330–1334 16 Tan, S.Y. et al. (1995) Treatment of amyloidosis. Am. J. Kidney Dis. 26, 267–285 17 Leung, D. et al. (2000) Protease inhibitors: current status and future prospects. J. Med. Chem. 43, 305–341 18 Higaki, J. et al. (1995) Inhibition of b-amyloid formation identifies proteolytic precursors and subcellular site of catabolism. Neuron 14, 651–659 19 Vassar, R. et al. (1999) b-secretase cleavage of Alzheimer’s amyloid precursor protein by the transmembrane aspartic protease BACE. Science 286, 735–741 20 Hussain, I. et al. (1999) Identification of a novel aspartic protease (Asp2) as b-secretase. Mol. Cell. Neurosci. 14, 419–427 21 Yan, R. et al. (1999) Membrane-anchored aspartyl protease with Alzheimer’s disease b-secretase specificity. Nature 402, 533–537 22 Sinha, S. et al. (1999) Purification and cloning of amyloid precursor protein b-secretase from human brain. Nature 402, 537–540 23 Wolfe, M.S. et al. (1999) Two transmembrane aspartates in presenilin-1 required for presenilin endoproteolysis and g-secretase activity. Nature 398, 513–517 24 Lin, X. et al. (2000) Human aspartic protease memapsin 2 cleaves the bsecretase site of b-amyloid precursor protein. Proc. Natl. Acad. Sci. U. S. A. 97, 1456–1460 25 De Strooper, B. et al. (1998) Deficiency of presenilin-1 inhibits the normal cleavage of amyloid precursor protein. Nature 391, 387–391 26 Li, Y-M. et al. (2000) Presenilin 1 is linked with g-secretase activity in the detergent solubilized state. Proc. Natl. Acad. Sci. U. S. A. 97, 6138–6143 27 Capell, A. et al. (2000) Presenilin-1 differentially facilitates endoproteolysis of the b-amyloid precursor protein and Notch. Nat. Cell Biol. 2, 205–211 28 Struhl, G. and Greenwald, I. (1999) Presenilin is required for activity and nuclear access of Notch in Drosophila. Nature 398, 522-525 29 Haniu, M. et al. Characterization of Alzheimer’s b-secretase protein BACE. A pepsin family member with unusual properties. J. Biol. Chem. (in press)
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