Physiol Rev 90: 465– 494, 2010;
doi:10.1152/physrev.00023.2009.
Proteases and Proteolysis in Alzheimer Disease:
A Multifactorial View on the Disease Process
BART DE STROOPER
Center for Human Genetics, K.U.Leuven and Department for Molecular and Developmental Genetics, VIB,
Leuven, Belgium
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I. The Alzheimer Disease Puzzle
A. The amyloid cascade hypothesis
B. Moving towards more comprehensive theories for AD
C. The importance of tau
II. Proteolysis Involved in Multiple Scenarios for Alzheimer Disease
A. Proteolytic processing of the APP
B. General disturbances of the endolysosomal-phagocytotic system in AD neurons
III. Heterogeneity of the Amyloid A␤ Peptide and the Toxic Oligomer Hypothesis
A. A␤ toxicity in Alzheimer disease
B. Heterogeneity of the A␤ peptide in vivo and potential contribution to overall toxicity
IV. ␣-Secretase and the Physiologically Most Relevant Proteolysis of APP
A. ␣-Secretase activity and the generation of APPs␣ and p3
B. Members of the ADAM family exert ␣-secretase activity
C. Other ␣-secretases
D. ␣-Secretase as a Drug Target for AD
V. ␤-Secretase
A. BACE1 is the major ␤-secretase
B. Molecular and cellular biology of BACE1
C. Deregulation of BACE1 expression in AD
D. BACE1 physiological functions
E. BACE1 as a drug target for AD
VI. ␥-Secretases
A. The different ␥-secretase complexes
B. Molecular and cellular biology of the ␥-secretase
C. ␥-Secretases proteolytic functions
D. Role of ␥-secretase in the generation of carboxy-terminal heterogeneity of A␤
E. ␥-Secretase as a drug target for AD
VII. Neurofibrillar Tangles and Tau
A. Tangles and the importance of nuclei formation
B. Hyperphosphorylation of tau
C. Proteolysis of tau and relevance for AD
D. Tau as a drug target in AD
VIII. Proteolytic Degradation of the Amyloid A␤ Peptide
A. Clearance of A␤ from the brain
B. Neprilysin
C. Endothelin converting enzymes 1 and 2
D. Angiotensin converting enzyme
E. Insulin degrading enzyme
F. Matrix metalloproteases
G. Plasmin
H. Cathepsin B and cystatin C
IX. Conclusion
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De Strooper B. Proteases and Proteolysis in Alzheimer Disease: A Multifactorial View on the Disease Process.
Physiol Rev 90: 465– 494, 2010; doi:10.1152/physrev.00023.2009.—Alzheimer disease is characterized by the accumulation of abnormally folded protein fragments, i.e., amyloid beta peptide (A␤) and tau that precipitate in amyloid
plaques and neuronal tangles, respectively. In this review we discuss the complicated proteolytic pathways that are
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BART DE STROOPER
responsible for the generation and clearance of these fragments, and how disturbances in these pathways interact
and provide a background for a novel understanding of Alzheimer disease as a multifactorial disorder. Recent
insights evolve from the static view that the morphologically defined plaques and tangles are disease driving towards
a more dynamic, biochemical view in which the intermediary soluble A␤ oligomers and soluble tau fragments are
considered as the main mediators of neurotoxicity. The relevance of proteolytic pathways, centered on the
generation and clearance of toxic A␤, on the cleavage and nucleation of tau, and on the general proteostasis of the
neurons, then becomes obvious. Blocking or stimulating these pathways provide, or have the potential to provide,
interesting drug targets, which raises the hope that we will be able to provide a cure for this dreadful disorder.
I. THE ALZHEIMER DISEASE PUZZLE
A. The Amyloid Cascade Hypothesis
The modern era of AD research began with the identification of missense mutations in the amyloid beta precursor protein (APP) (43) and presenilin (PS) genes (175,
246, 277), providing important clues in the advancement
of the understanding of (rare) early-onset familial forms
of AD. Mutations in the three genes affect the production
and/or aggregation of the A␤ peptide (263). In 1998, it
became clear that PS is an essential component of the
proteolytic activity that cleaves APP to generate the A␤
peptide (58). Thus both mutations in the substrate (APP)
and the protease (PS) responsible for generation of A␤
are sufficient to cause all of the symptoms of classical AD,
including the neuronal tangle pathology. The initial version of the amyloid cascade hypothesis proposed that
amyloid fibrils and plaques in the brain were the drivers of
the disease, while more recent variations of the hypothesis focus on small soluble aggregates of A␤ peptide as the
primary impetus of disease progression. Toxicity is also
more specifically associated with certain variants of the
A␤ peptide, which displays amino- and carboxy-terminal
heterogeneity (discussed in section III) (99). Especially
the A␤1– 42 is considered pathogenic because mutations
causing AD in APP (296) or in PS (65, 263) increase the
relative production of this peptide.1 This variant is also
abundant in amyloid plaques of sporadic and familial AD
1
We use subscripts to indicate the number of amino acids present in
the A␤ peptide. If not indicated otherwise, the peptide starts at amino acid
residue Asp-1 and ends at Val-40. A␤42 ends at Ala-42. A␤11– 42 starts at
Glu-11 and ends at Ala-42 (see Fig. 1C for the amino acid sequence of A␤).
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B. Moving Towards More Comprehensive Theories
for AD
Multifactorial diseases are very common in the elderly population, and treatment of these diseases usually
requires a combination of different approaches. Indeed,
treatment of A␤ alone would probably not suffice to effectively cure all forms of sporadic AD as likely part of
the damage in the brain becomes irreversible (119). Thus
we need to establish a more comprehensive, albeit more
complex, view of the different factors that underlie the
pathogenesis of AD. Decreased blood circulation and oxygenation of the brain, oxidative stress, or changes that
affect housekeeping functions or survival mechanisms
[proteostasis (11), the microRNA rheostat (109), and others] have to be taken into account to understand why the
aging brain reacts differently from the young brain to A␤
peptides (87). These changes might not only increase
levels of A␤, but also the sensitivity of the brain to A␤
toxicity and compromise (via parallel pathways) tau func-
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Alzheimer disease (AD) is one of the major health
problems in the world. The disease is characterized by
the accumulation of proteins and protein fragments in the
brain, progressive neuronal loss, inflammation, and the
progressive and inexorable decline of memory and cognition. Major landmarks in AD research include the isolation and sequencing of the amyloid peptide (A␤) from
amyloid plaques and amyloid angiopathy in the brain and
the demonstration of abnormal tau phosphorylation in
neuronal tangles.
patients (138, 190), and it provides a nidus for amyloid
formation (141).
This novel version of the amyloid hypothesis accounts for several discrepancies in human and mouse
studies, e.g., abundant amyloid deposition does not necessarily correlate with memory deficits or neuronal toxicity. However, the implications of the hypothesis remain
the same: tangle formation and neurodegeneration are the
consequence of abnormal A␤ metabolism, and a drug that
inhibits production or facilitates removal of A␤ from the
brain should significantly improve, cure, or prevent disease progression. Since such a drug is not available yet,
the amyloid cascade hypothesis remains unproven. The
amyloid cascade hypothesis provides, however, a very
good explanation for the development of familial earlyonset forms of AD. More than 30 mutations in APP and
more than 180 mutations in the two PS genes have been
identified (http://www.molgen.ua.ac.be/ADMutations).
Nevertheless, these missense mutations account for
⬍0.5% of all AD cases (37). The other, much more
frequent forms of AD have been called somewhat paradoxically “sporadic” (as opposed to familial) AD and
are caused by a combination of environmental and
genetic factors.
PROTEASES AND PROTEOLYSIS IN ALZHEIMER DISEASE
467
tion, inflammation, synaptic connectivity, and/or neuronal
survival. This sets the scene for a multifactorial view on
AD and indicates how, depending on the relative contribution of these different causes to the disease, different
therapeutic options will have to be considered. Abnormal
proteolysis is an important part of the emerging multifactorial view on the pathogenesis of AD.
C. The Importance of Tau
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Missense mutations in the tau gene are sufficient to
cause dementia in frontotemporal dementia with Parkinsonism (FTDP-17) (88), demonstrating that tau phosphorylation and tangle formation is a self-propagating disease.
Thus it is logical to assume that once tangle pathology is
initiated in AD, further decline occurs independently of
the presence of A␤. Preliminary evidence from a follow-up study of one of the A␤ vaccination trials supports
this possibility given that two patients, who were almost
entirely devoid of A␤ deposition, displayed signs of severe
and progressed tangle pathology (Braak stage VI). Importantly, the intellectual capacities of these patients were
severely affected, and clinical decline was not significantly different from nontreated AD patients (119).
II. PROTEOLYSIS INVOLVED IN MULTIPLE
SCENARIOS FOR ALZHEIMER DISEASE
Amyloid-centered research focuses on abnormal generation of the A␤ peptide and how changes in production
or clearance affect assembly of toxic A␤ oligomers and
neuronal toxicity. Alternative approaches focus on disturbances in lysosomal, autophagosomal (217), apoptotic
(61, 348), ubiquitin-proteasome, or other proteolytic systems in the brain and the effect that disruption of these
pathways has on the global health of neurons. In this field
of study, A␤ production is usually considered to be a
consequence or part of a feed-forward cycle, aggravating
the underlying disease process, which results in AD.
A. Proteolytic Processing of the APP
The most prevalent area of research in AD studies the
proteolytic generation of A␤ from APP. The ␤-secretase
and ␥-secretase cleave APP in the so-called amyloidogenic pathway (Fig. 1A). ␤-Secretase releases the ectodomain APPs␤, and the remaining APP carboxy-terminal
fragment (APP-CTF␤) is subsequently cleaved by the
␥-secretase liberating the (secreted) A␤ peptide(s) and
the APP intracellular domain (AICD). The biological
functions of APPs␤, A␤, and the AICD remain rather
elusive, although A␤ release is associated with synaptic
activity, depressing excitatory synaptic transmission onto
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FIG. 1. Proteolysis of the amyloid precursor protein (APP).
A: full-length APP is a type I integral membrane protein. Cleavage sites
of ␣-, ␤-, and ␥-secretase are indicated. Cleavage by ␣-secretase yields a
soluble ectodomain of APP (APPs␣) and a membrane-bound carboxyterminal fragment (APP-CTF␣) which after cleavage by ␥-secretase
yields p3 and the APP intracellular domain (AICD). ␤-Secretase cleavage
yields APPs␤ and APP-CTF␤, which is then cleaved by ␥-secretase into
A␤ and AICD. B: A␤ monomers can aggregate into various oligomeric
assemblies or larger aggregates that have different conformations and
different names according to the way they have been isolated or generated. Small nuclei of such aggregates can induce protofibril and amyloid
fibril formation. Most of the toxicity associated with A␤ is probably
exerted by these soluble species. C: the amino acid sequence of A␤ is
indicated in the one-letter code. The different cleavage sites identified
for ␤-, ␣-, and ␥-secretases are indicated. Also, the positions of the amino
acid residues undergoing pyroglutamate or isoaspartyl conversion are
indicated. See text for further discussion.
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BART DE STROOPER
B. General Disturbances of the
Endolysosomal-Phagocytotic System in
AD Neurons
More general perturbations of the proteolytic degradation machinery in neurons have been implicated in the
pathogenesis of AD and may occur downstream and even
upstream of A␤ generation. A striking morphological alteration in neurons in AD brains is the accumulation of
autophagosomes, autolysosomes, and lysosomal dense
bodies in dystrophic neurites (30, 40, 216). Similar alterations are also observed in APP and/or PS transgenic AD
mouse models (358), even before amyloid deposition is
observed. However, these mice generate a large amount
of the A␤ peptide. Thus (soluble) A␤ toxicity probably
precedes the morphological alterations. Interestingly, PS
loss-of-function mice also display disturbances in autophagocytosis, suggesting a poorly understood role for PS in
this process (72, 342). When autophagy is compromised in
the brain of an APP overexpressing AD mouse model
following inactivation of the Beclin gene (Beclin⫹/⫺
mice), enhanced A␤ deposition and synapse and neuronal
loss are observed, which suggests that decreased autophagy could aggravate or independently contribute to the
overall neurodegenerative process (232). Interestingly,
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expression of Beclin using lentiviral vectors attenuated
the amyloid load in the injected mouse brain areas.
III. HETEROGENEITY OF THE AMYLOID A␤
PEPTIDE AND THE TOXIC
OLIGOMER HYPOTHESIS
A. A␤ Toxicity in Alzheimer Disease
Several studies have suggested that A␤ or A␤ aggregates can induce tau phosphorylation and tangle formation or interfere with synaptic function as measured by
decreased long-term potentiation (LTP), and can cause
changes in dendritic spines and altered memory function
in rats and mice. A␤ has been shown to kill neurons in cell
culture and in the brain in vivo (164, 173, 329, 354, 355).
Nevertheless, the nature and physiological relevance of
A␤ toxicity has been criticized for a long time given the
high micromolar concentrations of A␤ peptides required
to observe an effect.
In AD mouse models and in the normal aging human
brain, large amounts of amyloid plaques have been observed with minor neuronal alterations, indicating that
the relationship between A␤ accumulation and A␤ toxicity is not straightforward (1, 74, 233). A␤ in its two extreme forms, i.e., soluble as a single monomer or insoluble
when trapped in amyloid fibrils, displays an insignificant
degree of toxicity (195). Over the last decade, the concept
of “A␤-derived diffusible ligands” (ADDL) (164) or “soluble toxic oligomers” (164, 173, 329) has advanced. Several
different oligomeric assemblies of the A␤ peptide have
been described, generated in vitro (45), or isolated from
transfected CHO cells as stable dimers, trimers, and multimers (234) or from transgenic mouse brains as a 56-kDa
oligomer (173). Various oligomeric species have also been
isolated from the brains of AD patients; the smallest toxic
isolate was reported to be comprised of a dimeric structure (275). It is quite likely that different oligomeric species are in dynamic equilibrium (Fig. 1B) with each other,
single peptides, and inert fibrils (250), a balance which
may be influenced by the presence of lipids (195). Currently, no consensus exists with regard to which toxic A␤
assembly is most relevant in vivo, and it is not unlikely
that various conformations of toxic aggregates exist next
to each other. Indeed, A␤ aggregates behave quite differently under different experimental conditions, explaining
part of the controversy in the literature (115).
The second question is obviously what parameters
determine the equilibrium between less toxic and more
toxic assemblies of A␤. Apart from differences in the
experimental conditions, a major parameter seems to be
the biophysical nature of the peptide itself. A␤40 for instance appears to be much less toxic, and even protective
compared with A␤42 (199). This might be explained by the
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neurons (146). The AICD has been proposed to be a
nuclear signaling molecule (4, 38, 52), but this remains
controversial (112). A recent study suggests that APPs␤ is
further processed by an unknown protease, generating a
⬃35-kDa amino-terminal domain fragment that serves as
a ligand for the death receptor DR6. Binding to DR6
triggers activation of caspase-6 and mediates axonal pruning during embryogenesis (215). Although it would be
intriguing to speculate that this pathway contributes to
AD pathogenesis, no patient data are available to support
this possibility.
The quantitatively and functionally most important
proteolytic cleavage of APP is mediated by the ␣-secretase, which releases the APPs␣ ectodomain. The resulting
carboxy-terminal APP-CTF␣ is further processed by the
␥-secretase and generates a small p3 fragment and the
AICD (Fig. 1A). APPs␣ has been suggested to exhibit
neuroprotective and synapse-promoting activities (13),
but elucidation of a mechanism or identification of a
receptor mediating these effects has not been achieved
yet. Nevertheless, a study utilizing a knock-in of a stop
codon in the mouse APP gene at the ␣-secretase cleavage
site elegantly demonstrated that mice that only produce
APPs␣ do not exhibit the various phenotypes caused by a
full APP knockout, including disturbed LTP and memory
function. Thus APPs␣ probably mediates the majority of
the functions of the APP holoprotein. (242). The structure-function of APP is reviewed elsewhere (238).
PROTEASES AND PROTEOLYSIS IN ALZHEIMER DISEASE
influence of A␤42 on the relative equilibrium between
toxic and inert assemblies of A␤.
B. Heterogeneity of the A␤ Peptide In Vivo and
Potential Contribution to Overall Toxicity
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tide backbone. This reaction is part of the natural aging
process of many proteins (240). As this reaction cannot
occur on the amino-terminal Asp, the iso-Asp1 modification described in A␤ peptides isolated from brain must
occur in the full-length APP protein (28). Interestingly,
␤-secretase cannot cleave sequences with an iso-Asp
modification; however, cathepsin B has been shown to
cleave this sequence in vitro. Thus cathepsin B is speculated to be an alternative ␤-secretase (123). It should be
mentioned that the issue regarding the extent to which
cathepsin B displays a quantitative contribution to (toxic)
A␤ generation is highly controversial, particularly since
cathepsin B is also involved in A␤ turnover as we will see
below.
Both pGlu and iso-Asp modifications have profound
effects on the biophysical properties of the A␤ peptide.
These modifications are apparently a frequent event in AD
(161, 231, 248, 253, 279), but only recent research has
begun to indicate that these alternative pathways might
provide valuable drug targets for the treatment of AD. For
instance, compounds that specifically inhibit glutaminyl
cyclase showed benefits in a transgenic AD mouse model
by decreasing the amyloid plaque load. The mice also
appeared to exhibit (weakly statistically significant) improvement in memory performance (266). Obviously, further work is urgently needed to evaluate the extent to
which these modifications affect the equilibrium between
monomeric, oligomeric, and fibrillized A␤ species, and to
what extent these modifications affect the overall toxicity
of the A␤ mixtures observed in AD patients.
IV. ␣-SECRETASE AND THE PHYSIOLOGICALLY
MOST RELEVANT PROTEOLYSIS OF APP
A. ␣-Secretase Activity and the Generation of
APPs␣ and p3
The major APP proteolytic process in nonneuronal
cells actually prevents further generation of the A␤ peptide (285). This proteolytic activity is called ␣-secretase
(95) and cleaves APP between Lys-16-Leu-17 in the A␤
sequence (Fig. 1C). The membrane-bound carboxy-terminal fragment (APP-CTF␣) is further processed by the
␥-secretase (95) (Fig. 1A). The p317-40 and p317-42 fragments have been identified in the brains of AD patients.
These fragments could be considered to be part of the A␤
peptide family given that they are highly hydrophobic and
tend to aggregate. They have been found in diffuse amyloid plaques in AD brains (93, 118) but seem a very minor
component of the classical dense-cored plaques (139,
254). In fact, very little is known about the toxicity or
function of these p3 fragments. Nevertheless, the majority
of research suggests that the ␣-secretase-mediated cleavage of APP is the nonamyloidogenic pathway, and it is
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A large variety of A␤ peptides has been isolated from
the brains of sporadic AD patients (Fig. 1C). This diversity
can be explained by proteolysis and additional enzymatic
modifications of A␤, but also by chemical reactions that
occur slowly during the many years that A␤ peptides
reside in amyloid plaques in the brain. The former are of
particular interest as they might provide targets for drug
development.
Much of the carboxy-terminal heterogeneity of A␤ is
readily demonstrated in cell culture, suggesting that this
is an inherent part of the production of A␤ by the ␥-secretase (see further discussion in the appropriate section). In
contrast, A␤ peptides isolated from cell cultures contain a
relatively homogeneous amino terminus that usually begins with an Asp-1 residue. In contrast, A␤ peptides isolated from AD brains display a variety of modifications at
the amino terminus. The most prominent alterations
are amino-terminal truncations, cyclized Glu residues,
i.e., pyro-Glu-3 or pyro-Glu-11 (Fig. 1C) (205, 253), and
isomerization of Asp (iso-Asp) residues (160, 248, 279).
Such modifications make the peptide more resistant to
proteolytic degradation and/or more hydrophobic as a
result of the loss of the amino-terminal charge (251).
Interestingly, both in vitro and in vivo evidence in mice
indicates that pGlu derivatives are particularly neurotoxic
(251, 266). Glutaminyl cyclase catalyzes the modification
of Glu to pGlu (264) by apparently reacting with glutamate residues at low pH (265). Given that the majority of
A␤ peptides are generated in the acidic endosomal pathway, modification of the A␤ peptide by glutaminyl cyclase
may be physiologically relevant; however, it remains unclear whether glutaminyl cyclase can act intracellularly,
or only later, when the A␤ peptide has been secreted. One
of the issues of debate focuses on generation of the free
amino-terminal Glu residue required for this reaction.
Amino-terminal Glu-11 is an alternative cleavage product
of authentic ␤-secretase activity (49, 170, 182, 319), but
Glu-3 becomes only available after aminopeptidases (273)
have removed Asp-1 and Ala-2. Alternatively, dipeptidyl
peptidases or acyl amino acid releasing enzyme could
remove iso-Asp-1 (27).
Additional factors that are linked to heterogeneity of
the A␤ peptide include spontaneous reactions such as
oxidation, hydrolysis, racemization, and disulfide bond or
ketoamine formation. A spontaneous succinimide-mediated rearrangement of L-aspartate (L-Asp) (and L-asparagine) to L/D-isoAsp or D-Asp residues results in the incorporation of an extra methylene group in the A␤ polypep-
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BART DE STROOPER
generally believed that increasing ␣-secretase activity
could be beneficial in AD (13), although proof for this is
lacking.
B. Members of the ADAM Family Exert
␣-Secretase Activity
1. ADAM10
Overexpression of ADAM10 in the brain lowers the
amyloid plaque load and leads to an improvement of
cognitive performance in a mouse AD model (236). Dominant negative ADAM10 or highly selective inhibitors of
ADAM10 also block ␣-secretase cleavage of APP (165,
269), which suggests that endogenously expressed
ADAM10 can cleave APP as well. ADAM10 has many
other substrates and is crucial in several signaling pathways. Both N-cadherin (239) and E-cadherin (192) ectodomain shedding is mediated by ADAM10. Remarkably, this
influences not only intercellular adhesion, but also the
amount of ␤-catenin at the cell surface and in the nucleus,
and thereby regulates to a certain extent ␤-catenin signalPhysiol Rev • VOL
2. ADAM9
The ADAM9 knockout mouse display no major phenotypical alterations (340) but show decreased pathological retinal neovascularization after oxygen- or laser-induced retinopathy (94). In primary cultures of Adam9⫺/⫺
neurons, a reduction in APP cleavage, as determined by
p3 release, was not observed (340).
It is surprising that the knockout of ADAM9 displays
such a minor phenotype in light of the widespread distribution of ADAM9 mRNA in embryonic and adult tissues.
This contrasts with the severe phenotypes observed in
ADAM10 (101) and ADAM17 knockout mice (230), which
suggests that these two ADAMs could potentially compensate for the loss of ADAM9 in the brain with regard to
␣-secretase activity.
3. ADAM17
ADAM17 has a crucial role in the release of a series of
membrane-bound proteins, including transforming growth
factor-␣ (TGF-␣), tumor necrosis factor-␣ (TNF-␣), L-selectin, p75 tumor necrosis factor receptor (p75TNFR),
and others (230). ADAM17 is also called TACE (TNF-␣
converting enzyme), and deficiency leads to perinatal and
early postnatal lethality in mice with defects in diverse
epithelial tissues, including skin, intestine, lung, and several glands (25). Given that the phenotype is clearly different from ADAM10 knockout mouse, these studies indicate that both ADAM proteases have different functions, although they can cleave similar substrates in cell
culture (168). These differential effects are probably due
to specific expression patterns and to different activation
pathways that regulate the two proteases (168). The evidence to support a role for ADAM17 in ␣-secretase processing of APP is not conclusive given that experiments in
ADAM17 brain or primary neurons have not been published thus far, for similar reasons as ADAM10. However,
in fibroblasts derived from ADAM17 knockout mice, constitutive ␣-secretase activity is maintained, while induced
␣-secretase activity (by phorbol myristate acetate) was
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The protease(s) responsible for ␣-secretase activity
are membrane-bound metalloproteases, and their activity
is regulated by an intricate network of secondary messengers (13). These proteases are fairly promiscuous with
regard to their cleavage specificity as demonstrated by
extensive mutagenesis of the amino acid sequence around
the cleavage site in APP (284). Several members of the “A
disintegrin and metalloprotease” or ADAM family have
been implicated as ␣-secretases. Downregulation or overexpression of ADAM9, -10, -17, and -19 in various cell lines
leads to decreased or increased APPs␣ generation, respectively (9, 35, 154, 165, 301). Given the high structural and
functional homology among the ADAMs (in particular the
8 other ADAM proteins that also contain the catalytic
consensus sequence HEXXH, i.e., ADAM8, -12, -15, -20,
-21, -28, -30, and -33), it is likely that additional APP
␣-secretases will be identified in specific cellular contexts. Interestingly, ADAM12 and -15 do not cleave APP
directly but are involved in the processing of ADAM10,
the major candidate ␣-secretase, potentially liberating a
soluble form of the ␣-secretase (311) and indirectly regulating ␣-secretase activity. An emerging concept is that
the ADAMs can substitute for each other in various shedding events (101, 168) as ADAMs in general appear to be
fairly nonselective with regard to membrane topology and
sequence specificity of their substrates.
Thus, while cellular assays may provide a hint with
regard to the proteases involved, only in vivo experiments
using loss of functions paradigms can really demonstrate
the physiological relevance of cell culture observations.
Three ADAM proteases have been intensively investigated
for their role in APP processing.
ing (192, 239). ADAM10 is also responsible for the regulated ectodomain cleavage of Notch, initiating further
processing by the ␥-secretase (see below) and the formation of the Notch intracellular domain. The latter transmits Notch signals to the nucleus. Given that ADAM10
knock-out (KO) mice display an embryonic lethal phenotype, probably due to the predominant role of Notch in
vascularization during embryogenesis (101), the ultimate
genetic experiment, demonstrating that lack of ADAM10
expression blocks ␣-secretase processing of APP in the
adult brain, has not been performed. Thus, based on
overexpression and inhibitor studies, the current research
indicates that ADAM10 is a major constitutive ␣-secretase
in many cell types.
PROTEASES AND PROTEOLYSIS IN ALZHEIMER DISEASE
inactivated (35), suggesting that another ␣-secretase
activity mediates constitutive APP processing, e.g.,
ADAM10, and that ADAM17 is responsible for the regulated fraction of the cleavage process. Nevertheless, the
extent to which this simple scheme holds true in the brain
in vivo remains unclear. In this regard, conflicting data
pertaining to the expression of ADAM17 in neuronal cells
has been published (191, 286), whereas infusion of a
highly specific TACE inhibitor in the mouse brain led to a
significant reduction of APPs␣, strongly arguing for the
importance of TACE in ␣-secretase processing in vivo in
the brain (151).
Over the years, several other proteases have been
proposed to contribute to ␣-secretase processing of APP.
Some positive, but also negative, reports have discussed
the potential involvement of the prohormone convertase
family members (57, 183, 184). This family of proteases,
which are responsible for the removal of propeptides
from hormones and proteolytic maturation of proteases
and other proteins, indirectly influences ␣-secretase activity (as they actually also do on ␤-secretase; Ref. 49)
by catalyzing removal of the prodomain from certain
ADAMs. Furin and other convertases have been implicated in the removal of the propeptide sequences of
ADAM10 and ADAM17, rendering these protease catalytically active (3, 135, 184).
Interestingly, BACE2, a homolog of BACE1 (␤-secretase see below), has also been suggested to act as an
alternate ␣-secretase because it cleaves between Phe-19
and Phe-20 (Fig. 1B), close to the classical ␣-secretase site
(14, 79, 81, 352). Selective knockdown of endogenous
BACE2 in human embryonic kidney cells using siRNA
elevates A␤ secretion (14), further arguing that BACE2
functions in the “nonamyloidogenic” pathway. However,
results from experiments in cell lines are not always
predictive for the in vivo situation. BACE2 is apparently
not expressed in neurons, and knockdown of BACE2 in
primary glia cell cultures paradoxically results in decreased
A␤ generation, suggesting that BACE2 in glia cells might
promote A␤ generation (62). Additional in vitro experiments
have shown that BACE2 can cleave at the Asp-1 site in APP
to a certain extent (79).
D. ␣-Secretase as a Drug Target for AD
Numerous studies have suggested that the ␣-secretase is an interesting alternative for the development of
anti-amyloidogenic drugs (reviewed in Ref. 13). ␣-Secretase activity appears to be regulated via protein kinase C,
tyrosine kinase, mitogen-activated protein (MAP) kinase,
and Ca2⫹-mediated pathways, providing ample opportuPhysiol Rev • VOL
nities for drug development. Stimulating ␣-secretase activity should decrease A␤ generation and increase p3 and
APPs␣ secretion, which has been proposed to have neuroprotective effects. However, the available data are insufficient to support these two assumptions: it remains
unclear whether p3 is indeed an innocent by-product of
␣-secretase processing and whether the neuroprotective
effect of APP has real clinical relevance.
In this regard, the ADAM10 protease has been studied
most intensively. A transgene construct driving ADAM10
overexpression in brain was shown to have protective effects in an AD mouse model, lowering amyloid plaque load
and improving several cognitive parameters (236). However,
regulated ␣-secretase activity is not mediated by ADAM10
but by ADAM17/TACE. Therefore, it remains unclear what
therapeutic strategy can be followed to specifically stimulate
ADAM10-mediated ␣-secretase activity in the brain. One
possibility is to target transcription of ADAM10 with retinoic
acid derivatives (306).
Stimulating regulated ␣-secretase activity via ADAM17
would provide an alternative strategy. However, ADAM17 is
also involved in the release of the proinflammatory TNF-␣,
which might provoke inflammation in the AD brain. A
recent report determined that specific inhibition of ADAM17 in
the brain results in a decrease in sAPP␣ release; however, A␤
steady-state levels remain unaltered (151).
It is clear that further work is needed to fully understand the balance between the ␣-secretase- and ␤-secretase-mediated pathways of APP processing and their relative importance for AD pathogenesis. Such studies
should focus on the in vivo situation in adult brain and
explain not only the relative contribution of both pathways to amyloid plaque generation but also address the
biological relevance of these processing events, and
whether APPs␣ and APPs␤ have different functions
in vivo. These questions will not be easy to address, given
the various and not well understood phenotypes of the
different genetic mouse models of APP and its homologs
(108, 242).
V. ␤-SECRETASE
A. BACE1 is the Major ␤-Secretase
Cleavage of APP by ␤-site APP cleaving enzyme 1
(BACE1) is the rate-limiting step in the generation of the
A␤ peptides. The protease was identified in 1999 by five
different groups using different methodologies (133, 181,
283, 319, 351). BACE1 is a membrane-bound aspartyl
protease that is optimally active at a slightly acidic pH. It
primarily resides in the Golgi and in endosomes but
briefly transits to the cell surface to locations where APP
can also be found (156). BACE1 has a rather broad tissue
distribution, but it is enriched in brain and in neuronal
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C. Other ␣-Secretases
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B. Molecular and Cellular Biology of BACE1
BACE1 and its homolog BACE2 (see the section on
␣-secretase for further discussion of BACE2) are members of the pepsin-like family of aspartic proteases (cathepsin D and E, pepsin A and C, renin, napsin A). They
display a typical bilobal structure with the catalytic site
located at the interface between the amino- and the carboxy-terminal lobe (121, 226). BACE1 and -2 are anchored
to the cell membrane via a transmembrane domain (Fig. 2),
which, together with several unique amino acid stretches
and the arrangement of the three disulfide bridges (98),
sets BACE apart from the rest of the pepsin family and
facilitates the generation of fairly specific inhibitors for
BACE1 and -2. Moreover, the fact that BACE1 is mem-
brane-anchored has been exploited to increase the efficacy of BACE1 inhibitors using a lipid anchor to target the
inhibitor to the cell and endosomal membranes (237).
BACE1 and -2 are generated as prepropeptides (Fig.
2). BACE1 is N-glycosylated, sulfated, phosphorylated,
and palmitoylated (21, 39, 49, 80, 131, 330), and the
propeptide of BACE1 is removed by a member of the furin
family (21, 49), generating a mature protease. A fraction
of BACE1 is also shed from the cell surface after cleavage
by a metalloprotease, probably ADAM10 (132), but the
significance of shed BACE1 is unknown. Most BACE1
remains membrane-bound and is internalized from the
cell surface to late endosomal vesicles, cycling further to
the lysosomes or to the trans-Golgi network (TGN) (131,
330). BACE1 has a relatively long half-life (⬃16 h compared with ⬃3 h for its substrate APP) and a fast recycling
rate, suggesting that BACE1 might undergo multiple cycles of transport from the endosomes to the cell surface
and back.
A DxxLL dileucine motif in the carboxy terminus of
BACE1 (Fig. 2) is a characteristic binding site for Golgilocalized ␥-ear containing ARF binding (GGA) proteins
(106). A similar acidic dileucine motif is also found in
sortilin and the mannose-6-phosphate receptor. The GGA
adaptor proteins are involved in sorting between the TGN
and endosomes (31). Phosphorylation of Ser-498, next to
this dileucine motif (Fig. 2), enhances the affinity of GGA
proteins for BACE1 (278) and a redistribution of BACE1
in the cell. In contrast, deletion of the dileucine motif,
overexpression of dominant forms of GGA1, or mutation
of Ser-498 impair the transport of BACE1 from the endosomes to the TGN, resulting in accumulation of BACE1 in
early endosomes (107, 324, 326). This accumulation is
associated with increased A␤ generation, indicating that
alterations in the subcellular localization of BACE1 have
an effect on the amyloidogenic processing of APP. Some
observations showed decreased GGA expression in the
AD brain, suggesting that this interaction might be relevant for the disease (304, 326).
C. Deregulation of BACE1 Expression in AD
FIG. 2. Schematic representation of BACE1. BACE1 is a type I
integral membrane protein. It contains a signal peptide and a prepropeptide that are removed during the maturation of BACE1. The two catalytic
aspartates and the consensus sequences are indicated. The cytoplasmic
domain contains a DxxLL motif that binds GGA proteins that regulate
the trafficking of BACE1 (see text). Three cysteines that are modified by
palmitoylation are indicated with small arrows.
Physiol Rev • VOL
Several reports have documented increased BACE1
protein and activity in the brain of AD patients (83, 120,
177, 353). It should be noticed, however, that it is unclear
whether BACE1 expression in healthy people is rate limiting for A␤ generation. Therefore, it remains uncertain
whether the recorded changes are sufficient to affect A␤
generation in a quantitative significant way. BACE1 expression is affected by many stress factors associated
with aging or neurodegenerative disease such as oxidative stress (300), energy inhibition (321), ischemia (337),
hypoxia (360), and traumatic brain injury (26). Insight in
these regulatory mechanisms would be important to un-
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cells, in particular. Genetic inactivation of BACE1 results
in a dose-dependent decrease in A␤ generation and is
sufficient to block A␤ deposition in different APP-overexpressing mouse AD models (163, 185, 198, 222). Recently,
a brain-penetrant BACE1 inhibitor was used to decrease
A␤ levels in the cerebrospinal fluid of monkeys (256).
Overall, there is little doubt that the A␤ peptides detected
in cell culture and body fluids are generated by this protease, and therefore, BACE1 is regarded as the authentic
␤-secretase. Upon BACE1 overexpression, an additional
cleavage at the ␤⬘-site, Tyr-10-Glu-11 in the A␤ sequence,
is detected (Fig. 1C). This cleavage removes the aminoterminal part of the A␤ peptide and, when antibodies are
used that only recognize this part of the peptide, less A␤
appears to be generated (49, 319). Interestingly, this alternative cleavage generates an A␤ peptide variant with an
amino-terminal glutamate that can be converted to pyroglutamate by glutamate cyclase as discussed above.
PROTEASES AND PROTEOLYSIS IN ALZHEIMER DISEASE
cerebral energy metabolism is a cardinal feature in AD, as
exemplified by positron emission tomography (PET) imaging studies (207). Energy deprivation induces phosphorylation of the translation initiation factor elF2␣ which may
overcome the 5⬘-UTR-mediated repression of BACE1
translation and thereby increase BACE1 expression and
possibly A␤ generation (221).
The long 3⬘-UTR of BACE contains several microRNA
binding sites, and levels of protein expression are regulated by miRNA expression (29, 110, 331). miRNA is derived from noncoding RNAs that are encoded and transcribed like classical mRNA in the cell nucleus. Precursor
miRNAs (pri-miRs) are sequentially cleaved by two RNase
enzymes, Drosha and Dicer, into small functional ⬃22 nt
oligonucleotides. The miRNAs are finally incorporated
into a “RNA-induced silencing complex” (RISC), which
suppresses translation and/or degrades target mRNA
(157). miRNAs are abundantly expressed in the brain, and
their absence results in severe neurodegeneration (109).
Moreover, loss of miRNA 29a/b-1 correlates with increased BACE1 expression in a subgroup of sporadic AD
patients (110).
Additional noncoding RNA is also involved in positive BACE1 regulation. A short (⬃2 kb) BACE1-antisense
RNA is encoded by a gene located on the opposite strand
of the BACE1 locus on chromosome 11q 23.3 (75). This
antisense BACE1 stabilizes the BACE1 mRNA, and knockdown of the antisense RNA reduces BACE1 expression
and A␤ generation. Increased expression of the antisense
BACE1 RNA was observed in AD patients.
Finally, at the posttranslational (protein) level, BACE1
is regulated by recycling and degradation mechanisms
and by its subcellular localization as discussed in the
previous section. Tesco and co-workers (153, 304) found
that the GGA proteins that control BACE1 localization in
the endosomes are cleaved during apoptosis, resulting in
increased BACE1 expression and A␤ generation. The authors suggest that GGA, in addition to their well-established role in protein trafficking between TGN and the
endosomal compartment, are also involved in the transport of BACE1 to the lysosomes where BACE1 is degraded (153, 304).
D. BACE1 Physiological Functions
FIG. 3. Regulation of expression of BACE1. The promoter of BACE1
is highly regulated by positive (3) and negative (inverted T) feedback.
The BACE1 mRNA transcript is also highly regulated: the 5⬘-untranslated region (UTR) contains several ATG and is alternatively spliced
yielding high and low expressing mRNA variants. The 3⬘-UTR is regulated by miRNA. An antisense BACE1 mRNA is generated from the
opposite DNA strand and stabilizes BACE1 mRNA. See text for further
discussion.
Physiol Rev • VOL
Interestingly, BACE1 is only found in vertebrates but
not in Caenorhabditis elegans and Drosophila melanogaster. Therefore, the functions of BACE1 have been
investigated in vertebrate-specific systems, e.g., the elaborate immune system and the brain. Studies that have
attempted to identify the natural substrates of BACE1
have yielded the best clues to physiological function of
BACE1. In addition to APP, the APP-like proteins 1 and 2
(176, 229), the sialyltransferase ST6Gal I (292), the P-
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derstand the potential contribution of environmental factors and aging to the development of sporadic AD.
BACE1-transcription is under complex regulation
(Fig. 3) (249). Hypoxia induces several transcription factors, including hypoxia-inducible factor 1␣, which binds
to the hypoxia responsive elements (HRE) in the promoter region of BACE1 (294, 360). Hypoxia or other
stresses might also activate cdk5/p25 which, acting via
STAT3 activation, could induce BACE1 expression (338).
Inflammation and anti-inflammatory drugs modulate
BACE1 expression via NF␬〉 (33) and peroxisome proliferator activating protein-␥ (258). Further exploration of
both pathways could yield viable drug targets for AD, not
only to modulate BACE1 expression in a beneficial way,
but also because of the potential neuroprotective and
anti-inflammatory effects of this type of compounds (105,
258).
The BACE1 transcript contains long 5⬘- and 3⬘-untranslated regions (UTR). Alternative splicing generates
BACE1 variants with low or no enzymatic activity (208).
The long GC rich 5⬘-UTR of BACE1 mRNA acts as a
translational repressor (53, 167). It displays extensive
secondary structure and contains three upstream open
reading frames (203, 247, 364). Interestingly, these properties are shared with other 5⬘-UTRs of transcripts that
are translationally controlled by cellular stress. Impaired
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E. BACE1 as a Drug Target for AD
Knockout of Bace1 in mice drastically reduces A␤
production (36, 185, 244) and reduces amyloid plaque
load and AD-related symptoms in AD mouse models (198,
222). A theoretical advantage of blocking BACE instead of
␥-secretase is that this does not result in abnormal accumulation of the APP-CTF␤ (Fig. 1A). The relative mild
phenotypes in mice deficient for BACE1 (discussed
above) compare quite favorably with the severe Notchrelated phenotypes caused by broad spectrum ␥-secretase
inhibition (gastrointestinal bleedings, autoimmune phenoPhysiol Rev • VOL
types, etc.). Moreover, it is not unlikely that part of the
neuregulin-associated phenotypes, including the myelination and behavioral deficits, are developmental in nature
(i.e., will not cause important side effects in adulthood)
(257). Taking all of this into consideration, BACE1 is a
primary drug target for AD. The catalytic site of BACE,
although typical aspartyl protease like, is exceptionally
long, and it has been very difficult to develop small compounds targeting BACE1 in an efficient way. The compounds should be small because they need to cross the
blood brain barrier, and they should have a reasonable
half-life to reduce the number of times older patients have
to take the medication. A flurry of papers has described
BACE inhibitors, but orally available highly efficient
BACE1 inhibitors have only recently become available
(256).
VI. ␥-SECRETASES
A. The Different ␥-Secretase Complexes
In 1993, ␥-secretase was proposed as the generic
name for the proteolytic activity that mediates cleavage of
APP in the transmembrane domain, releasing the A␤ peptide (95). The proof that presenilin 1 (PS1) is a major
constituent of this proteolytic activity was provided in
1998 in a study that showed that loss of PS1 in neurons
leads to an almost complete loss of A␤ peptide generation
(58). The remainder of the ␥-secretase activity is mediated
by presenilin 2 (PS2), a close homolog of PS1 (117, 361).
Missense mutations in both the PS1 and PS2 genes are a
major cause of familial AD (175, 246, 277). Interestingly,
fluorescent life time microscopy studies indicate that these
mutations affect the conformation of the protein (23).
Presenilin is an aspartyl protease (344) but requires
the association of three additional proteins (subunits),
nicastrin, aph-1, and pen-2 to become proteolytically active (70, 152, 299; reviewed in Ref. 54) (Fig. 4A). The
stoichiometry derived from experiments using tagged
overexpressed versions of the individual subunits is likely
1:1:1:1 (224, 261). Technical difficulties in purifying sufficient material make it difficult to analyze the structurefunction of native complexes, and therefore, it remains
uncertain whether larger multimers or different conformations of ␥-secretase exist in situ. Several additional
interacting proteins have been reported, and recently, an
interactome of presenilin was published based on tandem-affinity purification of native complexes (327). These
additional proteins are not in a stoichiometric relation to
the four core proteins and are also not present in ␥-secretase complexes purified using a biotinylated transitionstate inhibitor (343), suggesting that they are not necessary for the activity of the protease per se. Some of these
interacting proteins, like the tetraspanins, might however
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selectin glycoprotein ligand-1 PSGL-1(179), ␤-subunits of
voltage-gated sodium channels (346), and the low-density
lipoprotein receptor-related protein (323) have been identified. The type I membrane protein PSGL-1 contributes to
the rolling of leukocytes along the vessel wall before they
transmigrate through the endothelium. The type II membrane protein ST6Gal I is involved in B-cell expansion and
is required for the correct glycosylation of CD22 ligands.
It is unclear whether BACE1 processing interferes with
these functions.
BACE1 knockout animals display a subtle and complex phenotype that has been ignored for quite a while
(62, 100). A variable number of mice die in the first weeks
after birth, and this lethality is increased by combination
with BACE2 deficiency. The surviving mice are smaller
and present a hyperactive behavior. They display subtle
alterations in the inactivation of the voltage-gated sodium
channels. More recent work has demonstrated transient
changes in the myelination process of peripheral and
central nervous system neurons (130, 341). The changes
manifest as thinner myelin sheets during the first few
weeks after birth in mice when BACE1 expression is
particular high in brain, but the defect is apparently restored later in development by unknown compensation
mechanisms. Nevertheless, remyelination after a crush of
the sciatic nerve appears to be disturbed in adult
Bace1⫺/⫺ mice, indicating that BACE1 might also play a
role in repair of peripheral nerve damage in adulthood
(129). The responsible substrate is apparently neuregulin
I. The BACE1 cleavage site was identified in neuregulin I
and is conserved in neuregulin III as well (129). Both
neuregulin I and neuregulin III accumulate in Bace1-deficient mice (129, 130, 341). Interestingly, Bace1-deficient
mice also display behavioral alterations such as prepulse
inhibition, disturbed operational memory, and other
symptoms that are associated with the schizophrenic phenotype in humans, and which could be restored with the
antipsychotic drug clozapine (262). These symptoms are
probably related to disturbed neuregulin/ErbB4 signaling.
Interestingly, the Aph1b-␥-secretase knockout mice (see
below) display a similar phenotype (59).
PROTEASES AND PROTEOLYSIS IN ALZHEIMER DISEASE
475
exist. If alternative splicing of PS and Aph1 is considered,
additional variants may have to be taken into account as
well (Fig. 4A) (54, 111, 281). It should be noted that PS1
and PS2 differ ⬃33%, and Aph1a and Aph1b even ⬃43% at
the primary amino acid level. These different ␥-secretase
complexes coexist in cell lines in vitro (111), although they
might display different tissue distribution patterns in vivo
(59). Mouse knockout experiments have already demonstrated that the different complexes have divergent biological functions (see below) (59, 63, 116, 186, 271, 272).
FIG. 4. ␥-Secretase complexes and notch signaling. A: ␥-secretase
consists of four subunits: presenilin, nicastrin, Aph1, and Pen2. Two
different presenilin genes and two different Aph1 genes exist. Alternative splicing results in several small variants of presenilin 1 and two
major variants of Aph1a. B: the major role of the presenilin1-Aph1Anicastrin-Pen2 ␥-secretase variant is notch signaling. This ␥-secretase is
responsible for the proteolytic cleavage of notch after ligand binding and
ectodomain shedding by ADAM proteases. The notch fragment remaining after this sheding cleavage event (S2-cleavage) is schematically
represented. The resulting notch intracellular fragment is translocated
to the nucleus and is involved in gene transcription. The analogy with
APP processing is obvious. One of the major problems with ␥-secretase
inhibitors is that they not only block A␤ generation but also notch
cleavage. This could theoretically be circumvented if specific inhibitors
for Aph1b-␥-secretase complexes could be generated, as those are not
involved in notch signaling (see text).
be involved in the regulation or maturation of the activity
of the complex (327).
In the human genome, two PS genes and two Aph1
genes (Aph1a and Aph1b) are present. With the use of
specific antibodies for the different subunits, it was
shown that either PS1 or PS2 and either Aph1a or Aph1b
are incorporated into the mature ␥-secretase complex.
Thus a minimum of four different ␥-secretase complexes
Physiol Rev • VOL
The different tetrameric ␥-secretase complexes are
highly hydrophobic structures with 19 transmembrane
domains. When the complex is purified from cell lines that
overexpress the PS1/Aph1a/Pen2/Nicastrin variant, the estimated mass is ⬃230 kDa (224). A structure at 12-Å
resolution was obtained by single-particle reconstitution
and cryoelectron microscopy (224), revealing a globular
structure with a smooth cytosolic side and a larger irregular extracellular surface. The structure displays three
solvent accessible cavities, one accessible to the cytosolic
side and two accessible to the extracellular side. It should
be noted that, at this level of resolution, it would not be
possible to definitively resolve the issue of whether these
low-density cavities are really pores or merely unresolved
loop structures. However, meticulous cysteine scanning
studies of PS have indicated that the central core of the
complex, consisting of the catalytic site and probably part
of the substrate binding domain, is indeed water accessible (259, 260, 307, 309). Thus it appears that the ␥-secretase, similar to other intramembrane cleaving proteases,
sequesters its active site from the hydrophobic environment of the membrane in a structure that allows entrance
of catalytic water molecules that are necessary for hydrolysis of its substrates. For further discussion and comparison with other intramembrane cleaving proteases, refer
to a recent review (308).
The reason(s) why PS requires three additional proteins for its activity remains unclear, especially in light of
the fact that other intramembrane cleaving proteases,
including the related signal peptide peptidases (SPP), are
apparently active as monomers or homodimers (220, 336).
Nevertheless, the individual ␥-secretase subunits strongly
influence the stability of each other, and correct assembly
is a prerequisite for transit to distinct subcellular compartments (cell surface, endosomes) where the ␥-secretase is active. It has been proposed that the single transmembrane domain protein nicastrin is a “gate-keeper,”
restricting access of substrates to the catalytic site. Nicastrin contains an aminopeptidase-like domain. The carboxylate side chain of a conserved glutamate residue (E333)
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B. Molecular and Cellular Biology of
the ␥-Secretase
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experiments confirmed that the complex proteins are not
active in this early compartment (150). Interestingly, exit
of ␥-secretase from these early compartments is tightly
regulated. “Retrieval to ER protein 1 (RER1p)” recognizes
specific motifs both in Pen2 and nicastrin and continuously recycles these proteins from the ER-Golgi intermediate compartment (ERGIC) back to the ER provided that
these proteins have not been incorporated into the complex (144, 287). In an elegant study, Annaert and colleagues (287) demonstrated that RER1p and Aph1 compete for binding to nicastrin, and only when the RER1p
binding motif in nicastrin is covered by Aph1 can the
assembled subcomplex leave the ER definitively. This
quality control might be a way to control the total levels
of active ␥-secretases in the cell (287). Finally, not all
assembled ␥-secretase complexes in the cell are in active
conformations as transition-state analog inhibitors apparently only bind a small subset of them in situ (18, 162). It
is not unlikely that lipids play an important role in this
regard (225) as in cell free assays, the lipid composition
can affect activity severalfold, and in cells the complex
appears to be associated at least partially with detergentinsoluble patches in the membrane (322, 327). The functional consequences of these associations remain to be
fully explored.
C. ␥-Secretases Proteolytic Functions
To date, most studies have considered the ␥-secretase to be a homogeneous activity. In fibroblasts and
other cell lines, this is probably a valid approach given
that the Aph1a/PS1/Pen2/nicastrin combination is most
abundant. In vivo, however, it appears that the different
complexes have divergent tissue expression patterns and
biological functions. The Aph1a/PS1 combination is crucial in notch signaling, both during embryogenesis and in
adulthood as demonstrated by various knockout mouse
studies (102, 174, 178, 186, 272, 276, 310, 345, 347; reviewed in Ref. 93). In contrast, PS2 knockout animals
(116) only display mild signs of increased apoptosis in
lung tissue. The Aph1b/PS1 (and PS2)/Pen2/nicastrin
combinations display a more restricted expression profile. Deficiency of the Aph1b subunit results in a subtle
behavioral phenotype with disturbed prepulse inhibition,
defects in operational memory, and increased sensitivity
to amphetamine, which are symptoms that have also been
observed in other mouse models of neurodevelopmental
disorders including schizophrenia (59). Interestingly, similar symptoms are observed in BACE1 knockout mice
(262), and the symptoms in both animal models can be
reversed with antipsychotic drugs. Evidence that BACE1
and the Aph1b-␥-secretase complex are both involved in
proteolytic processing of (and likely signaling by) neuregulin provides a molecular basis for the understanding
of this common phenotype.
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in this domain has been suggested to be crucial for the
nicastrin-substrate interaction (274). However, a recent
study has shown that this E333 substitution interferes
with assembly of the ␥-secretase complex, and the small
amount of complexes that are still generated appear to be
as active as the wild-type complexes on a per mole basis,
which contradicts the “gate-keeper” hypothesis (44).
Less is known about the Aph1 and Pen2 subunits.
Aph1 probably has a seven transmembrane domain structure with the carboxy terminus located in the cytoplasm
(82). Aph1 is the most stable component of the complex
and might serve as the initial scaffold for assembly. Aph1
forms a subcomplex with nicastrin before PS and finally
Pen2 are incorporated. The two different Aph1 proteins
alter the conformation of the catalytic subunit PS in the
complex as assessed by fluorescent lifetime imaging microscopy (272), suggesting that they have a structural
effect on the catalytic site of the ␥-secretase complex.
Pen2 is a small hairpinlike protein (50). Its incorporation
into the complex is associated with a major conformational change of the complex (280), resulting in the (likely
auto-) proteolytic cleavage of PS into amino- and carboxyterminal fragments (299) and rendering ␥-secretase a fully
operational proteolytic machine.
An important clue to a true understanding of the
diverse functions of the ␥-secretase complex and cleavage of its many substrates (see below) could potentially
be found in a more precise study of the exact subcellular
localization of the different complexes relative to the
appropriate substrates. This type of analysis has been
very instructive for the signal peptide peptidases (SPP),
which are PS-related multitransmembrane containing aspartyl proteases, and has shown the specific association
of SPP/SPPL1 with the endoplasmic reticulum (ER) and
SPPL2 with endosomes (158). The ␥-secretases provide
obviously a more complex situation: each enzyme consists of four different proteins, and PSs have additional
functions beyond their role in the complex (328). An early
study summarized this problem as “the spatial paradox.”
Indeed, PS, believed at that time to act as an independent
protease, appeared to be mainly localized to the ER,
whereas the ␥-secretase is active in the late compartments of the secretory and endocytic pathways (5, 46, 51,
302). This spatial paradox has been resolved by current
studies that indicate that PSs are present in the ER in a
catalytically inactive conformation. This PS holoprotein
or uncleaved PS forms Ca2⫹ channels that facilitate passive Ca2⫹ leakage across the ER membrane (213, 315).
Apart from more precise morphological localization
studies, dynamic cell-free transport assays are necessary
to investigate how the complex is progressively assembled and matures along the biosynthetic pathway. One of
the first reports describing such results has made use of a
cell-free ER budding assay and analyzed the presence of
the ␥-secretase subunits in COPII transport vesicles. The
PROTEASES AND PROTEOLYSIS IN ALZHEIMER DISEASE
D. Role of ␥-Secretase in the Generation of
Carboxy-Terminal Heterogeneity of A␤
The ␥-secretase is responsible for endoproteolysis of
APP and generation of different A␤ peptides (58). The
major soluble A␤ peptides end at residue Val-40 and at
Ala-42, and mutations of PS systematically increase the
relative ratio of the “long” versus the “short” A␤ peptide
species (A␤42/A␤40 ratio) (32, 263). The two cleavages
occur at the “␥”-sites (Fig. 1C). Concomitantly, the intracellular cleavage peptides (APP intracellular domain or
AICD) are released, which start at Val-51 or Met-52. These
cleavages occur at the “␧”-sites (Fig. 1C). Similar dual
cleavages have been identified for other ␥-secretase substrates such as Notch (223) and CD44 (166). One model
suggests that the ␥-secretase initially cleaves APP at the
␧-site and then progressively truncates the protein by
about three carboxy-terminal acid residues (one ␣-helical
turn) until the ␥-sites are reached (145, 298). Although
further cleavage is possible (generating A␤38 and possibly
even shorter peptides), the remaining peptide becomes
increasingly hydrophilic and therefore is quite readily
liberated from the interior of the ␥-secretase complex
(Fig. 5). In this model, A␤49 is the precursor of A␤46
Physiol Rev • VOL
FIG. 5. Progressive cleavage model for ␥-secretase. ␥-Secretase is
represented as a large hydrophobic complex with a central hydrophilic
core. The different substrates of ␥-secretase likely bind laterally to the
complex and are then translocated into the interior to become cleaved.
The first cleavage occurs close to the cytoplasmic leaflet of the membrane and releases the intracellular domain of the substrates. Progressive removal of hydrophobic amino acids destabilizes the extracellular
part of the substrates, which results finally in the release of small
peptides such as A␤ or similar peptides from other substrates.
(␨-site), A␤43 and A␤40 (␥-site) (84, 145), while A␤48 would
give rise to A␤45 and A␤42 (362). The model remains
descriptive and does not provide an explanation with
regard to how substrates are progressively cleaved within
the same catalytic site. It is possible that the putative
␣-helices formed by the transmembrane domains unwind
after the initial ␧-cleavage, which, in turn, advances the
next peptide bond toward the catalytic site for further
cleavage. The consecutive cleavage model of APP provides an explanation for the paradox that clinical mutations in PS decrease total A␤ generation but increase the
amount of long A␤ peptide (22). Indeed, if these mutations render ␥-secretase less efficient (i.e., cause a partial
loss of function of PS), then a less complete digestion of
A␤ will result in longer and more amyloidogenic A␤ (55).
More than 30 different substrates have been identified for the different ␥-secretase complexes (Fig. 5) (for
an exhaustive list, see Ref. 328). The ␥-secretase cleaves
the transmembrane domains of many proteins with type I
topology (amino terminus oriented to the extracellular
side of the membrane) and short (⬍50 amino acid)
ectodomains with a remarkably relaxed sequence specificity (180, 290). Bulky ectodomains of 200 and more
amino acid residues prevent ␥-secretase cleavage and are
usually removed by membrane-bound (metallo)proteases
at the cell surface (see above for a discussion of the
ADAM proteases and APP shedding). Additional sites in
the substrates might contribute to cleavage efficiency and
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Currently, it is not completely clear what determines
the specificity of the phenotypes in the ␥-secretase knockout mice. Few in vitro experiments have been published
that address the question of the biochemical properties
and substrate specificities of the different complexes.
These studies suggest that the different subunit combinations of the ␥-secretase complex do not considerably
affect the Km and Vmax relative to synthetic substrates
(272) or secretion of A␤ peptides in cell lines (272, 282).
However, these experiments revealed differences in the
conformation of the PS1 catalytic subunit depending on
the presence of the Aph1a or Aph1b subunit, and also
differences in the spectrum of A␤ peptides generated in
various in vitro assays (272). Earlier experiments in cell
cultures suggested that PS2 is a much less efficient protease than PS1 with regard to APP cleavage and A␤
peptide generation (22, 162, 196). At the moment, the
available in vitro assays are too rudimentary to permit
reliable and conclusive analysis of the substrate preferences and critical kinetic parameters of the different
␥-secretase complexes, but it is certainly important to
pursue this avenue of research. Nevertheless, in vitro
experiments, although crucial for further structure-function analysis and the development of specific inhibitors,
have to be supplemented with in vivo experiments if one
really wants to understand the physiological function of
these particular proteases. Indeed, it is crucial to determine whether protease and substrate also meet in real
life.
477
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BART DE STROOPER
specificity (113, 241, 303). Dimerization of APP (and
maybe other substrates?) through the GxxxG motif in its
transmembrane domain might also potentially regulate
processing by ␥-secretase (211). Further work is clearly
needed to more precisely delineate the substrate binding
site in the ␥-secretase and the constraints on this site that
regulate access of substrates to the catalytic site buried in
the interior of the protease.
E. ␥-Secretase as a Drug Target for AD
Physiol Rev • VOL
VII. NEUROFIBRILLAR TANGLES AND TAU
The microtubule-binding protein tau precipitates as
tau tangles in AD and in a whole spectrum of neurodegenerative tauopathies including Pick’s disease, progressive supranuclear palsy, corticobasal degeneration, and
others. Interestingly, the dementia symptoms in AD correlate to a greater extent with the gradual appearance and
spread of tangles throughout the brain than with the
deposition of A␤ in senile plaques (7, 91). The causal
relationship between tau dysfunction and neurodegeneration has been unequivocally established by the identification of more than 40 mutations in the tau gene (http://
www.molgen.ua.ac.be/ADMutations/) that cause familial
frontotemporal dementia with Parkinsonism linked to
chromosome 17 (FTDP-17) (134, 235, 289). However, the
mechanism by which tau causes neurodegeneration remains unclear. Both gain and loss of function mechanisms
have been proposed (12). Mutations and posttranslational
modifications decrease the affinity of tau for microtubules
which will destabilize these structures and will lead to
axonal transport problems. Gain of toxicity could be due
to the aggregates themselves, which mechanistically
block axonal transport or other neuronal functions and
provide a trap for functionally important proteins, including tau itself. A third emerging concept is that small,
soluble oligomeric aggregates of hyperphosphorylated
tau could form highly toxic intermediates that interact
with biological membranes or cause other disturbances
and thereby affect the health of the neuron. This concept
is not unlike the toxic A␤ oligomer concept discussed
above (96).
A. Tangles and the Importance
of Nuclei Formation
The longest version of the tau protein contains 441
amino acid residues. Alternative splicing of exons 2, 3,
and 10 generates six major isoforms in the adult brain
(Fig. 6). Exon 10 encodes one of the four repeat (R)
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Two observations, the essential role of PS in A␤
generation (58) and its role in notch signaling (56, 174,
291), have been a major part of the research agenda in the
drug development for AD for at least a decade (Fig. 4B).
Blocking Notch signaling in the crypts of the intestinal
epithelium induces differentiation into goblet cells and
interferes with the normal replacement of the epithelium,
explaining the gastrointestinal toxicity of ␥-secretase inhibitors (270, 318, 345). In addition, immune suppression
or autoimmune disorders might be caused by notch deficiency in adulthood (97, 310). Several approaches to increase the therapeutic window, i.e., to find compounds
that efficiently block A␤ generation without affecting
notch signaling, have been explored. A rather unexpected
turn in this drug development story is that ␥-secretase
inhibitors are now considered a viable therapeutic strategy for certain cancers in which notch signaling is activated, such as T-cell acute lymphoblastic leukemia (339),
lung cancer (155), and precancerous adenoma (318).
Classical transition state compounds that target the
catalytic site of the ␥-secretase will block cleavage in the
absence of substrate discrimination. A series of non-transition-state ␥-secretase inhibitors have been described
such as peptide-based inhibitors (DAPT), sulfonamides,
and benzodiazepines (compound E). When these inhibitors are used at low concentrations, the ␨-site cleavage
(Fig. 1C) is not affected, but the ␥-site cleavages are
potently inhibited and A␤40 and A␤42 levels decrease
(349). Another class of inhibitors that has attracted much
attention in the last years are the ␥-secretase modulators
(GSM). A subset of non-steroidal anti-inflammatory drugs
(NSAIDs), such as ibuprofen and sulindac sulfide, as well
as enantiomers of flurbiprofen, are apparently able to
shift the cleavage specificity of the ␥-secretase towards
the production of shorter A␤38 peptides, without affecting
A␤40, AICD or, most importantly, notch cleavage (71, 297,
334, 335). The mechanism of action is not fully understood, but they could affect the conformation of ␥-secretase (17), the dimerization of APP (211), or even directly
bind to APP (159). The clinical usefulness of these different approaches needs to be further explored.
More recently, refined strategies that block APP processing and do not affect notch signaling have been pro-
posed. G protein-coupled receptors (GPCR) have been
implicated in the trafficking of the ␥-secretase (214, 305)
and appear to specifically regulate cleavage of APP versus
notch, possibly by redistributing ␥-secretase(s) to subcellular compartments where APP is present, but not notch.
Indeed, downregulating GPR3 leads to a reduction in A␤
production without any notch side effect (305).
A second opportunity might be found in the heterogeneity of the ␥-secretases. The Aph1b complex is expressed in the brain areas affected by AD and is not
involved in notch signaling. Specific inhibition of this
complex might certainly be a viable approach to block A␤
generation in a safe and effective way (272).
PROTEASES AND PROTEOLYSIS IN ALZHEIMER DISEASE
FIG. 6. Schematic representation of tau. Tau is a hydrophilic protein
located in the cytoplasm and binding microtubules by its repeat regions
(R1–R4). Alternative splicing of exons 2, 3, and 10 results in 6 different
isoforms. Several proteolytic cleavage sites as discussed in the text are
indicated. Two amino acid sequences flanking exon 10 are involved in
the aggregation of tau.
B. Hyperphosphorylation of Tau
Tau in tangles is characterized by a high degree of
phosphorylation on its 45 serines, 35 threonine, and 5
tyrosine residues (189). Some of these sites are typically
Physiol Rev • VOL
phosphorylated during embryogenesis, or under specific
physiological conditions (hibernation in certain species),
which might be related to synaptic remodeling, while
other sites are highly pathological. Nevertheless, there is
an ongoing debate with regard to the extent to which
phosphorylation and which specific phosphorylation sites
are crucial for tau toxicity or tangle formation. Phosphorylation affects tau’s affinity for the microtubule to variable
extents, but unexpectedly, phosphorylation at sites that
strongly interfere with microtubule binding (e.g., by the
kinases Mark or protein kinase A) also strongly interfere
with tangle formation (267). Soluble forms of abnormally
phosphorylated tau have been found in mouse models
overexpressing tau containing FTDP-17 mutations, which
might suggest that they appear before tangle formation (2,
60). However, a causal relationship between abnormal
phosphorylation and tangle formation has not been firmly
demonstrated. The best evidence that increased phosphorylation might lead to tangle formation comes from
crosses of mice that overexpress mutated tau with mice
that express the activated kinase CDK5. These mice display increased phosphorylation of tau at multiple sites
and a significant increase in tangle formation (218). Taking all of the evidence into consideration, the debate on
the importance of tau phosphorylation in the pathogenesis of tauopathies, including AD, has not yet been resolved.
FIG. 7. Nucleation-elongation pathway in Tangle formation. Tau
bound to microtubuli (TauMT) can be redistributed to cytoplasmic pools
of Tau (Taucyt) by various factors. Taucyt is highly soluble, and additional
factors, including proteolysis, are necessary to convert this Tau into
nucleating forms of Tau, which induce pretangles, paired helical filaments, and neurofibrillar tangles.
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domains of tau which are involved in microtubule binding. Thus alternative splicing yields either three or four
repeat motifs containing tau. The ratio of four repeats
containing tau (4R) to 3R is ⬃1:1 in adult brain, and
changes in this ratio are characteristic for the different
tauopathies (34, 88, 189). Tau is extensively posttranslationally modified by phosphorylation, glycosylation
and glycation, ubiquitination, and proteolytic processing (92).
Most researchers will agree that the pathway converting soluble tau to filamentous insoluble tau is probably central to the pathogenesis of the tauopathies,
including AD (12). Tau aggregation is a multistep process initiated by a rate-limiting nucleation step, which
is followed by the progressive addition of tau proteins
in an elongation process (Fig. 7). A first step seems to
include the redistribution of microtubule-bound tau to
the soluble cytoplasmic pool of tau as a consequence of
hyperphosphorylation (218), mutations (103, 122), or
other tau modifications. However, the mechanism by
which increased cytoplasmic tau, which is highly soluble and intrinsically unfolded, clusters to generate the
aggregation nuclei required for the further formation of
tau fibrils remains unclear. Mutations (89, 212), posttranslational modifications like proteolysis, or additional factors like the presence of polyanionic structures (86, 90, 147) probably play a crucial rate-limiting
role in this conversion and are therefore therapeutic
targets. The canonical view on the tau cascade is centered on the abnormal phosphorylation status of tau,
but as we will see, interesting evidence is emerging that
abnormal proteolysis of tau plays a role in the transformation of tau to pathological species.
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BART DE STROOPER
C. Proteolysis of Tau and Relevance for AD
FIG. 8. Paper clip conformation of tau. The amino-terminal and
carboxy-terminal domain of tau fold back on the central domain as
indicated in the figure. Proteolytic cleavage of tau may result in truncated tau versions that are more prone to aggregation and tangle formation.
Physiol Rev • VOL
D. Tau as a Drug Target in AD
As mentioned above, it is possible that tangle pathology, once induced in AD, evolves independently of the A␤
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The concept that tau proteolysis contributes to tangle formation in vivo has already been known for a long
time. An important fraction of tau in fibrils is truncated at
positions Glu-391 and Asp-421 (numbering for the longest
splice form of tau) (15, 85, 201, 219). Removal of the
carboxy-terminal part of tau enhances the nucleationelongation reaction in vitro (24, 356), which might indicate that in sporadic AD truncated tau species play an
important (maybe initiating) role. Interestingly, truncated
tau might also be involved in neuronal toxicity or be
responsible for the build up of intermediary toxic oligomeric species. Truncated tau is, when overexpressed in a
rat transgenic model, indeed able to drive neurodegeneration and neurofibrillar pathology (127, 365).
Both amino- and carboxy-terminal parts of tau appear to have an inhibitory effect on the aggregation potential of tau. They fold back in a “hairpin” conformation
(142) on the central domain which contains the microtubule binding repeats (R) (Fig. 8). In repeats R2 and R3,
two hexapeptide motifs are present with a high propensity to form ␤-sheet structures (210, 325). A synthetic tau
4R construct truncated at both the amino and carboxy
terminus has a high tendency to form intracellular tau
aggregates (further enhanced by introducing the deltaK280
FTDP17 mutation) (333). Toxicity is clearly associated with
the aggregation propensity of this construct in vitro (6, 149)
and in vivo (206).
The protease generating the Glu-391 cleavage is unknown, but caspases are potentially involved in the Asp421 cleavage. They are also involved in the removal of
parts of the amino terminus (Fig. 6) by potentially generating a highly aggregation-prone tau fragment. Interestingly, exposure to A␤ can trigger caspase-mediated cleav-
age of tau in neurons in vitro. The truncated tau is readily
phosphorylated at different sites, and an antibody specific
for the Asp-421 site decorated the fibrillar pathology in the
AD brain (85, 243). The link between apoptosis induced
by A␤, abnormal proteolysis of tau, and tau aggregation is,
if confirmed, potentially very interesting given that it provides a link between the two major pathologies of AD and
also suggests new therapeutic targets for the disease (48).
Alternative mechanisms, e.g., activation of calpain by A␤
and generation of an amino-terminal neurotoxic proteolytic 17 kDa tau fragment (AA43-229), have been proposed
to mediate tau-dependent A␤ toxicity as well (Fig. 6)
(228). However, the importance of caspase activation and
apoptosis in the progression of AD has still not been
established (61).
One approach to clarify the role of (limited) proteolysis of tau in disease progression would obviously be to
utilize in vivo modeling in various mouse models. However, the obtained results appear somewhat contradictory
to date. While the rat model (365) supports the hypothesis
that truncated tau can drive neurodegeneration, it is unclear whether truncation of tau is also necessary in the
pathogenesis of AD in humans. For instance, mouse models overexpressing the tau-Pro-301 mutation generate
abundant tangles and display early tau phosphorylation,
whereas truncation at Asp-421 is only a minor occurrence
and probably a late event (60, 359). It should be noted that
rodent models come with their own experimental biases
and assumptions concerning the pathogenic mechanisms
and that any extrapolation to the human situation needs
to be done with caution. For example, truncated tau is
much more abundant in human brains than in these
mouse models (359). Thus it should be taken into consideration that strong overexpression of pathological genes
in mouse models strongly biases the models towards
assumptions that were made when the model was generated.
Several caspase fragments of tau have been reported
but have been insufficiently evaluated in further studies to
draw conclusions with regard to their importance for AD.
Horowitz and co-workers reported a caspase-6 cleavage
at Asp-13 of tau. Amino-truncated tau has been detected
in patients (126), and this amino-terminal domain of tau
interacts with the carboxy-terminal domain to keep tau in
its soluble state (125) (discussed in detail above). Corsetti
et al. (47) report on an amino-terminal tau fragment
(amino acids 26-230) that is generated during apoptosis
and exerts NMDA-mediated neurotoxicity. Removal of the
amino terminus has also been attributed to cleavage by
aminopeptidases (148).
481
PROTEASES AND PROTEOLYSIS IN ALZHEIMER DISEASE
VIII. PROTEOLYTIC DEGRADATION OF THE
AMYLOID A␤ PEPTIDE
A. Clearance of A␤ From the Brain
If abnormal or increased production of A␤ peptide by
genetic mutations causes familial AD, then any biochemical cause of increased A␤, for instance deficient removal
of A␤ peptide, has to be considered as a potential cause of
sporadic AD (252). The normal A␤ fractional clearance
rate is estimated to be ⬃8% per hour (16), and small
chronic defects are theoretically sufficient to cause AD if
the analogy with familial AD is maintained. Physiological
parameters, like blood and cerebrospinal fluid flux in the
brain, together with a series of clearance receptors, including LRP1 and VLDLR, are implicated in the removal of
A␤. A␤ can be taken up by smooth muscle cells delineating the arterioles or by endothelial cells that participate in
TABLE
1.
the blood-brain barrier (19, 20, 64, 104, 366). A portion of
A␤ might also be transcytosed and secreted into the
bloodstream (187). Disturbances in these clearance processes appear particularly relevant for the accumulation
of A␤ peptides in the blood vessel walls, causing the
vascular component or cerebral amyloid angiopathy
(CAA) of AD.
The proteolytic machinery in the brain also certainly
contributes to the degradation of the A␤ peptide and the
aggregates and fibrils that accumulate in AD. The diversity of the proteases implicated in this clearance process
is rather perplexing, but in many cases, in vivo validation
has been provided. A knockout experiment in mice or rats
of a specific protease demonstrating increased steadystate levels of A␤ in brain is strong evidence that the
particular protease is physiologically involved in A␤ turnover (summarized in Table 1). Ectopic overexpression
using transgenes or viral vectors and demonstrating that
the protease is able to lower A␤ levels or prevent amyloid
plaque formation in the brain of AD mouse models is
another piece of in vivo evidence, although the physiological relevance then has to be provided from additional
experiments. Evidence of a genetic association with AD
has been published for a few proteases; however, no
consensus exists with regard to the importance or reproducibility of these associations in the general AD population. From a therapeutic point of view, it remains to be
seen whether any of these proteases provides a viable AD
drug target. Recent reports suggest even that some of the
amyloid degrading proteases (insulin degrading enzyme,
neprilysin, and angiotensin converting enzyme 2, see below) are actually upregulated anyhow in sporadic AD
brain (204, 227). One has to consider the possible side
effects of further upregulating the activity of these proteases in the brain for many years, as would be needed for
A␤ degrading proteases
Protease
Type Protease
Neprilysin (NEP)
Insulin degrading enzyme
(IDE)
Angiotensin converting
enzyme (ACE)
Zn2⫹
Zn2⫹
Endothelin converting
enzyme (ECE1, -2)
Matrix metalloproteinases
(MMP9, MMP2)
Plasmin
Cathepsin B
Membrane Bound?
Degradation of A␤
Monomers, A␤
Oligomers, or A␤
Fibrils
Effects of Genetic
Inactivation on A␤ Levels
(In Vivo)
Reference Nos.
136, 137
77
M, O
M
2
2
Zn2⫹
Type II
Cytosolic, membrane bound,
and secreted
Type I
M
(2?)
Zn2⫹
Type II
M
In discussion
2
Zn2⫹, Ca2⫹
Secreted
M, O, F
(2)
Serine protease
Cysteine protease
Secreted
Secreted
M, O, F
M, F
KO has no clear effect
2
367
66, 114
68
356
140, 317
209
Most proteases involved in A␤ turnover are metalloproteases. It is indicated whether they degrade A␤ mononomers (M), oligomers (O), or
fibrils (F). If knock-out animal models have been described demonstrating increased A␤ levels, this is indicated by KO. See main text for further
discussion.
Physiol Rev • VOL
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pathology. The FTDP-17 mutations actually show that
abnormal tau causes neurodegeneration and dementia.
Unfortunately, no consensus exists with regard to the
treatment of the tau pathology. Abnormal phosphorylation has been considered to be a potential drug target by
many pharmaceutical companies (197), but as discussed
above, proof of concept is clearly lacking. Other possibilities are to interfere with the tau aggregation process, to
reduce tau expression (245), or to improve microtubule
functions (268). Given the importance of nucleation in
tangle formation, it might be worthwhile to explore the
role of abnormal tau proteolysis in AD with more vigor in
the future. Although proteases are reasonable drug targets, caspases might be difficult to block without major
side effects.
482
BART DE STROOPER
a treatment of AD. Moreover, all of the implicated proteases have important functions in the turnover of biologically active peptides. Nevertheless, we will see how some
approaches are currently explored to enhance A␤ proteolytic degradation in human brain.
B. Neprilysin
C. Endothelin Converting Enzymes 1 and 2
The endothelin-converting enzyme (ECE)-1 and -2
are related to neprilysin, displaying several similarities in
sequence and domain structure. ECE-1 cleaves A␤ in vitro
(67), and heterozygous knockout animals (68) show increased A␤ accumulation in brain. Interestingly, the comPhysiol Rev • VOL
D. Angiotensin Converting Enzyme
Several antihypertensive drugs target angiotensin
converting enzyme (ACE), although there is some concern that these drugs could also increase A␤ levels in
brain. In this regard, ACE was shown to degrade A␤
in vitro (128), and polymorphisms in the ACE gene have
been associated with increased or decreased genetic risk
for AD. However, several investigations using genetic inactivation of ACE or inhibitors of ACE in vivo suggest that
ACE does not have a major role in A␤ turnover in the
brains of mice (66, 114, 137). In contrast, one study reported a significant effect on A␤ levels in mice treated
with the ACE inhibitor captopril, an antihypertensive drug
(367). Thus the evidence that implicates ACE as an A␤
degrading enzyme is somewhat conflicting.
E. Insulin Degrading Enzyme
Insulin degrading enzyme (IDE) is a 110-kDa zinc
protease that cleaves physiologically active peptides like
insulin, glucagon, atrial natriuretic factor, and others. The
subcellular localization of IDE is somewhat controversial:
it acts certainly in the cytoplasm where it degrades, for
instance, the AICD once it is released by ␥-secretase (see
above) (69). On the other hand, IDE is also found at the
cell surface and secreted into the medium via the “unconventional secretory pathway” (363).
IDE degrades A␤ in the conditioned medium of cell
cultures, including neurons (320). Both a rat and a mouse
model deficient in IDE have been studied (77, 78), and
increased levels of A␤ in neuronal cultures, and in brain
samples, were discovered. Deficiencies in IDE also lead to
increased insulin levels in the blood and might provide a
clue toward a possible link between diabetes and AD,
although this remains to be further investigated. Overexpression of IDE in brain, similar to neprilysin, improves
pathological aspects in an AD mouse model (172).
IDE together with neprilysin and a third protease
implicated in A␤ degradation, the mitochondrial presequence peptidase or PreP (76), all have a characteristic
catalytic chamber that can encapsulate a variety of peptides of 70 amino acid residues or less. The chamber plays
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Neprilysin was identified as an important A␤ degrading enzyme using an unbiased approach that attempted to
answer the fundamental question of A␤ degradation in the
brain in vivo.
Saido and collaborators (137) injected multilabeled
radioactive A␤ peptides into the rat brain and identified
and characterized the proteolytic fragments generated
over time. The profiles of the fragments and the sensitivity
of the degradation pathway to inhibitors like thiorphan
and phosphoramidon strongly supported a role for neprilysin in A␤ turnover in vivo. This was nicely confirmed in
neprilysin knockout mice, which display a genetic dosedependent increase in endogenous A␤ peptide accumulation in the brain (136). The Dutch, Flemish, Italian, and
Arctic familial AD mutations in the APP gene appear to
further hamper the degradation of A␤ by neprilysin
in vitro, which argues that decreased turnover of these
peptides in addition to their increased tendency to form
fibrils could contribute to the pathogenesis of AD and
CAA in patients (314).
Neprilysin, also referred to as enkephalinase or neutral endopeptidase, is a zinc-dependent metalloprotease
of ⬃90 kDa. It is a type II membrane protein that hydrolyzes biologically active peptides, including enkephalin,
cholecystokinin, substance P, neuropeptide Y, and others.
Overexpression of neprilysin in the brain of AD mouse
models using transgene expression from a calmodulin
kinase II promoter (172) or viral vectors (194, 288) resulted in the clearance of a great deal of the amyloidassociated pathology, but as discussed, it remains unclear
whether such an approach can be translated into a feasible therapy for humans. An alternate idea, to increase
neprilysin levels using agonists of the somatostatin
GPCRs, was elegantly proposed some years ago (255).
Somatostatin levels decline during aging and in AD, and
somatostatin deficiency in mice results in decreased neprilysin activity and increased A␤ levels, which might be
reversed by such a treatment.
bination of neprilysin and ECE-1 knockout further enhanced A␤ levels in brain, confirming that different proteases work together to turn over A␤ (66). ECE-1 exists as
four isoforms that are localized to different regions in the
cell and at the cell membrane. Conversely, ECE-2 displays
a predominantly intracellular localization. Both are type II
membrane-bound zinc metalloproteases that are primarily responsible for processing the vasoconstricting endothelins.
PROTEASES AND PROTEOLYSIS IN ALZHEIMER DISEASE
a key role in the selectivity of these proteases for their
substrates, including A␤ (188).
F. Matrix Metalloproteases
G. Plasmin
Plasmin is generated from plasminogen by the urokinase-type or tissue-type plasminogen activator (uPA and
tPA, respectively). Plasmin is involved in degradation of
fibrin aggregates in the blood. In the brain, plasminogen is
synthesized in neurons, and uPA and tPA can be induced
during ischemia and excitotoxicity. The subsequent plasmin generation and degradation of extracellular matrix
have been implicated in neuronal loss, and tPA-deficient
mice are resistant to this type of insult (312, 313).
The three proteases are serine proteases, but only
plasmin has been directly implicated in A␤ degradation
(169, 316). Interestingly, fibrillar A␤, similar to fibrin, can
stimulate tPA-mediated plasmin activation, and plasmin is
able to degrade fibrillar A␤ (316). In the brain of AD
patients, plasmin activity was found to be decreased (73,
169).
Steady-state A␤ in the brain or plasma does not appear to be affected by plasminogen deficiency, suggesting
that under normal steady-state conditions plasmin contributes little to A␤ turnover (317). However, when radioactively labeled A␤ was injected into the brain of plasminogen-deficient mice, a slower turnover was observed
(200), indicating that under certain experimental conditions plasmin becomes sufficiently activated to significantly contribute to A␤ turnover. As mentioned, fibrillar
A␤ might activate tPA-mediated plasminogen activation
(171, 316). Recently, inhibition of plasmin by neuroserpin
(73) was implicated in AD, while the reverse, activation of
plasminogen activator inhibitor-1 (PAI-1) (140), was proposed as a potential approach to increase plasmin-mediPhysiol Rev • VOL
ated A␤ degradation in AD patients. An orally active,
brain-penetrable inhibitor of PAI-1 lowered A␤ levels in
both plasma and in the brain of an AD transgenic mouse
model and improved memory deficits in these animals.
The possibility that such treatments would lead to increased bleeding has to be balanced against the potential
benefits. However, partial PAI-1 inhibition might be possible without major side effects, as discussed by Jacobsen
et al. (140).
H. Cathepsin B and Cystatin C
Cathepsin B (CatB) is a cysteine protease located in
the endolysosomal system where it degrades peptides.
CatB is also secreted through exocytosis and is involved
in matrix degradation in cancer and arthritis. CatB has
been implicated in APP and A␤ processing for a long time
(40, 41) and has been proposed as an alternative ␤-secretase, see also above (124). However, good experimental
evidence supports a contrasting hypothesis that suggests
that CatB is involved in A␤-monomer and, importantly,
A␤-fibril degradation. Genetic ablation of the gene aggravated, while overexpression of the gene using lentiviral
transfer improved the pathology in an AD mouse model
(209). CatB truncates A␤ peptides at the carboxy terminus, reducing the relative amount of the longer A␤42
peptide. No evidence exists as to whether genetic ablation
of CatB also affects A␤ levels at the endogenous level of
APP expression, which would further strengthen the hypothesis that this protease is indeed part of the natural
degradation machinery of A␤. The contribution of CatB
toward A␤ turnover may strongly depend on its expression levels, which are induced by inflammation and other
factors.
This is also probably the only way to understand the
discrepancies in the results that have been obtained with
cystatin C, a cysteine protease inhibitor of CatB (293).
Genetic ablation of cystatin C resulted in decreased A␤
peptide accumulation in mice and primary neurons and
improved cognition. The beneficial effects of cystatin C
reduction were not observed on a CatB-null background,
providing strong genetic evidence for a cystatin C/CatBregulated pathway for A␤ degradation (293). In contrast,
several studies suggest that cystatin C binds directly to A␤
or A␤ fibrils and is responsible for the clearance of the
peptide. Genetic inactivation of cystatin C did not affect
A␤ load in another AD mouse model (143), while overexpression of cystatin C in the mouse brain of several AD
models actually reduced plaque load (143, 202). Moreover, the clinical reports are conflicting: both increases
and decreases in cystatin C activity have been associated
with AD (42, 295). The point might be that cystatin C has
dual roles, i.e., a proamyloidogenic effect based on its
inhibition of CatB, and an antiamyloidogenic effect by
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Matrix metalloproteases (MMPs) are secreted zincand calcium-dependent endopeptidases. They are expressed at low levels in the brain, but their expression can
be induced in astrocytes by stress, e.g., by the presence of
A␤ peptide itself. MMP-9 can cleave A␤, but unlike most
other A␤ degrading enzymes, it can also degrade fibrillar
A␤ (350). Knockout of MMP2 results in increased steadystate levels of A␤40 and A␤42 in the soluble fraction of
hippocampus and cortex, while infusion of a broad-spectrum metalloprotease inhibitor in the brain also increased
A␤ levels (357), suggesting the potential in vivo relevance
of these (and other metalloproteases) in A␤ turnover. It
remains unclear whether the expression of MMPs is really
altered in the AD brain (10). However, MMP9 is an important factor in ischemic brain insult, and genetic inactivation is protective (8, 332).
483
484
BART DE STROOPER
binding the A␤ peptide in fibrils and clearing it from the
brain. The net result in a given experimental paradigm
will depend on the levels of CatB expression in the given
conditions and on where in the brain cystatin C is expressed.
IX. CONCLUSION
ACKNOWLEDGMENTS
I thank Dr. A. Thathiah for proofreading the manuscript.
Address for reprint requests and other correspondence: B.
De Strooper, Center for Human Genetics, VIB and K.U.Leuven,
O&N1 Herestraat 49, Box 602, 3000 Leuven, Belgium (e-mail:
[email protected]).
GRANTS
Work in my laboratory was supported by a Pioneer Award
from the Alzheimer’s Association; the Fund for Scientific Research Flanders; KULeuven (GOA); Federal Office for Scientific
Affairs, Belgium; a Methusalem grant of the Flemish Government; and an Excellence Award of the Stichting Alzheimer
Onderzoek (SAO).
DISCLOSURES
I am a paid consultant for several pharmaceutical and
biotech companies.
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