Báo cáo khoa học: Amyloid–cholinesterase interactions Implications for Alzheimer’s disease pot

MINIREVIEW
Amyloid–cholinesterase interactions
Implications for Alzheimer’s disease
Nibaldo C. Inestrosa, Margarita C. Dinamarca and Alejandra Alvarez
CRCP Biomedical Center, Pontificia Universidad Cato
´
lica de Chile, Santiago, Chile
Alzheimer’s disease is a progressive and irreversible
neurodegenerative disorder that has emerged as the
most prevalent form of late-life mental failure in
humans. Although a small percentage of Alzheimer’s
disease cases involve mutations in some known genes,
and are referred as familial Alzheimer disease, the
large majority of Alzheimer’s disease cases occur spo-
radically with unknown etiology [1]. There is therefore
a need to search for the mechanisms responsible for
the progressive cognitive decline observed in these
cases. Despite the disparity in age-of-onset, both forms
share common neuropathological features, the amy-
loid-b peptide (Ab) deposition in diffuse and senile
(neuritic) plaques being one of the most relevant.
Ab accumulation and deposition has been causally
implicated in the neuronal dysfunction and loss that
underlies the clinical manifestations [2,3].
One of the several proteins associated with amyloid
plaque deposits is the enzyme acetylcholinesterase,
which is associated predominantly with the amyloid
core of mature senile plaques, pre-amyloid diffuse
deposits and cerebral blood vessels in Alzheimer’s dis-
ease brain [4]. Acetylcholinesterase has been described
in cholinergic and non-cholinergic processes in both

the central and peripheral nervous system [5,6]. The
enzyme is secreted and becomes associated with extra-
cellular structures, namely the synaptic basal lamina at
the neuromuscular junction and, as mentioned above,
the amyloid plaques of Alzheimer’s disease brain [7,8].
Most of the acetylcholinesterase in the central nervous
system is found in a tetrameric form bound to neuro-
nal membranes [9]. Histochemical studies have shown
that the enzyme associated with senile plaques differs
enzymatically in several respects from that associated
Keywords
acetylcholinesterase; acetylcholinesterase–
Ab complexes; Alzheimer’s disease; amyloid
formation; amyloid oligomers; Ab-amyloid
fibrils; butyrylcholinesterase; molecular
chaperone; neurotoxicity; peripherical
anionic site
Correspondence
N. C. Inestrosa, CRCP Biomedical Center,
Pontificia Universidad Cato
´
lica de Chile,
Alameda 340, Santiago, Chile
Fax: +56 2 686 2959
Tel: +56 2 686 2724
E-mail:
(Received 12 October 2007, accepted 12
December 2007)
doi:10.1111/j.1742-4658.2007.06238.x
Acetylcholinesterase is an enzyme associated with senile plaques. Biochemi-

cal studies have indicated that acetylcholinesterase induces amyloid fibril
formation by interaction throughout the peripherical anionic site of the
enzyme forming highly toxic acetylcholinesterase–amyloid-b peptide (Ab)
complexes. The pro-aggregating acetylcholinesterase effect is associated
with the intrinsic amyloidogenic properties of the corresponding Ab pep-
tide. The neurotoxicity induced by acetylcholinesterase–Ab complexes is
higher than the that induced by the Ab peptide alone, both in vitro and
in vivo. The fact that acetylcholinesterase accelerates amyloid formation
and the effect is sensitive to peripherical anionic site blockers of the
enzyme, suggests that specific and new acetylcholinesterase inhibitors may
well provide an attractive possibility for treating Alzheimer’s disease.
Recent studies also indicate that acetylcholinesterase induces the aggrega-
tion of prion protein with a similar dependence on the peripherical anionic
site.
Abbreviations
Ab, amyloid-b peptide; hAChE, human recombinant acetylcholinesterase; PrP
c
, prion protein; PrP
Sc
, scrapie prion protein; Tg, transgenic.
FEBS Journal 275 (2008) 625–632 ª 2008 The Authors Journal compilation ª 2008 FEBS 625
with normal nerve fibrils and neurons [10]. We have
also shown that acetylcholinesterase promotes the
assembly of Ab into amyloid fibrils [11], and that a
mAb directed against the peripheral acidic binding site
(peripherical anionic site) of acetylcholinesterase inhib-
its the effect of the enzyme upon amyloid formation
[12].
Amyloid deposition and the role
of acetylcholinesterase as a

neuropathological chaperone
It is currently thought that the amyloidogenic process
that converts soluble Ab into amyloid fibrils is a nucle-
ation-dependent process [13] associated with structural
transitions of Ab [14]. Although the molecular factors
underlying this transition in vivo remain unknown, the
possible role of additional plaque constituents has been
proposed [4]. We decided several years ago to repro-
duce in vivo the co-localization observed under in vitro
conditions. The first analysis was the binding of acetyl-
cholinesterase to Ab. Using an ELISA it was
determined that the binding of acetylcholinesterase to
Ab-coated wells was five- to sixfold higher than that
observed with bovine serum albumin-coated wells. In
addition, when Ab peptide was loaded onto an acri-
dine–Sepharose column containing bound acetylcholin-
esterase, most of the Ab was recovered during the
loading period, but a significant fraction was recovered
when the bound acetylcholinesterase was eluted with
decamethonium (esterase inhibitor) [15]. In vivo studies
indicated that acetylcholinesterase has the ability to
enhance Ab aggregation and amyloid fibril formation.
In fact, when acetylcholinesterase was infused stereo-
taxically into the CA1 region of the rat hippocampus
novel plaque-like structures were formed [16]. More
recently, independent studies support our initial obser-
vation, indicating that acetylcholinesterase accelerates
Ab deposition: a double-transgenic mouse overexpress-
ing both human APP containing the Swedish mutation
and human acetylcholinesterase has been developed.

Such double-transgenic mice start to form amyloid pla-
ques around three months earlier than mice expressing
only the APP transgene. Moreover, the double acetyl-
cholinesterase–APP transgenic mouse presents more
and larger plaques than control animals, and some
behavioral deterioration, as shown by the working
memory test [17]. By contrast, when two acetylcholin-
esterase inhibitors: physostigmine and donepezil were
subcutaneously administered to the transgenic (Tg)
mouse model of Alzheimer’s disease that overexpresses
a mutant form of human APP (Tg2576), the memory-
related behavioral deficits of the Tg mice were
improved [18]. Both physostigmine and donepezil have
been reported to inhibit acetylcholinesterase-induced
Ab polymerization [19].
Enzymatic properties of the
acetylcholinesterase present in
acetylcholinesterase–Ab complexes
Histochemical studies have demonstrated that the ace-
tylcholinesterase associated with senile plaques differs
from the enzyme found in normal fibers and neurons
with respect to optimal pH, inhibition by excess
substrate and protease inhibitor sensitivity [10,20,21].
Furthermore, biochemical studies have indicated that
senile-plaque-associated acetylcholinesterase is only
partially extracted using collagenase digestion [22],
heparin extractions [23] or high-salt buffers plus deter-
gent [24]. Interestingly, the acetylcholinesterase present
in the acetylcholinesterase–Ab complexes reported by
us showed properties similar to those of senile-plaque-

associated acetylcholinesterase. In fact, the enzyme
associated with the Ab peptide (a) presented K
m
and
V
max
values higher than those observed by the free
enzyme and was more resistant to: (b) incubation
under low pH conditions, (c) inhibition by anti-cholin-
esterase agents and (d) inhibition by excess of substrate
acetylthiocholine [25,26].
Structural motifs of acetylcholinesterase
involved in amyloid formation
In 1996, we found that acetylcholinesterase was able to
accelerate Ab fibril formation [11], forming a high
molecular complex with Ab fibrils that was resistant to
the action of detergent and high ionic strength condi-
tions [26,27]. Several anti-cholinesterase drugs were
able to decrease the effect of acetylcholinesterase on
amyloid formation. No effect was observed when
active-site inhibitors, like tacrin or edrophonium, were
used, however, propidium and fasciculin, anti-cholines-
terase agents that inhibit the peripherical anionic site
of the enzyme, were able to block amyloid formation
[25,28]. These results are entirely consistent with
studies carried out using a mAb directed against the
peripherical anionic site of acetylcholinesterase [12,28].
To identify the acetylcholinesterase motif that pro-
motes Ab fibril formation, we used molecular dynamic
techniques to model the docking of Ab onto the cata-

lytic subunit of acetylcholinesterase. Using this
approach four potential sites were identified, one of
which (site I) spans a major hydrophobic sequence
exposed on the surface of acetylcholinesterase, corre-
sponding to a polypeptide of 3.5 kDa called H peptide
Amyloid–cholinesterase interactions N. C. Inestrosa et al.
626 FEBS Journal 275 (2008) 625–632 ª 2008 The Authors Journal compilation ª 2008 FEBS
(amino acids 274–308 in Torpedo acetylcholinesterase).
This corresponds to a hydrophobic acetylcholinesterase
sequence (L281–M315) previously identified by its
capacity to interact with membranes [29]. These experi-
ments indicated that the acetylcholinesterase motif that
promotes Ab fibril formation is located in a small
hydrophobic peptide that contains a conserved trypto-
phan (W279) which belongs to the peripherical anionic
site of the catalytic subunit of acetylcholinesterase. We
used this hydrophobic peptide to show that it was able
to mimic the capacity to accelerate the Ab aggregation
by intact acetylcholinesterase [30]. A goal of our dis-
coveries was to open a new rational approach to
develop new categories of acetylcholinesterase inhibi-
tors. Such leads might have dual specificity, being
directed to both the active and ‘peripheral’ sites. After
our initial observations, several researcher groups have
been looking for these new properties of ‘classic’ ace-
tylcholinesterase inhibitors and ⁄ or trying to develop
new molecules that can exhibit at least two more phar-
macological properties simultaneously, i.e. the
enhancement of cholinergic transmission and the inhi-
bition of Ab aggregation. In this context, Bartolini

et al. [19] studied the capability of the human recombi-
nant acetylcholinesterase (hAChE) to induce Ab aggre-
gation. Using thioflavine-T fluorescence they
demonstrated that hAChE accelerates amyloid poly-
merization and using CD they showed that hAChE
increases the b-conformation content in Ab prior to
fibril formation. Also, studies with several acetylcholin-
esterase inhibitors effects were performed. The peri-
pherical anionic site inhibitor propidium inhibited
hAChE-induced aggregation, whereas the competitive
acetylcholinesterase inhibitor edrophonium had no
effect [19]. In addition, other molecules with dual ace-
tylcholinesterase inhibitor activity have been developed
from tacrine. The acetylcholinesterase and butyryl-
cholinesterase inhibitory activity, together with the
inhibition of the Ab pro-aggregating effect of the ace-
tylcholinesterase were evaluated. Four indole–tacrine
heterodimer molecules showed a selective inhibition of
the acetylcholinesterase activity and inhibited the
acetylcholinesterase-induced Ab polymerization with
lower IC
50
values than propidium [31,32]. Also, riv-
astigmine analogues [33], xanthostigmine derivatives
[34] and pirimidine derivatives [35] have been synthe-
sized with the same aim: dual inhibitory strength
against acetylcholinesterase and Ab aggregation.
Moreover, the known structure of the peripherical
anionic site could help to design new structure-based
drugs [36,37]. New acetylcholinesterases were designed

following a computational approach based on docking
simulations carried out on the structure of human
acetylcholinesterase. The selected molecules were tested
on the isolated enzyme, following its ability to inhibit
both the catalytic and the Ab pro-aggregating effect
and one molecule (AP2238) had positive effects, show-
ing similar properties to donepezil [38]. These results
suggest that it is possible to obtain compounds that
could have a really therapeutic potential in this area.
Acetylcholinesterase’s ability to
increase the amyloid aggregation
depends of the amyloidogenic
properties of the Ab peptide
A number of studies with synthetic Ab in vitro have
shown that this peptide aggregates and forms amyloid
fibrils similar to the filaments found in the brains of
Alzheimer’s disease patients [39]. For example, the sin-
gle mutation Val18 fi Ala induces a significant incre-
ment of the a-helical content of Ab, and dramatically
diminishes fibrillogenesis [16]. However, the substitu-
tion of Glu22 fi Gln found in hereditary cerebral
hemorrhage with amyloidosis of the Dutch type, yields
a peptide with increased ability to form amyloid fibrils
[40]. In fact, acetylcholinesterase had little effect on the
aggregation of the highly amyloidogenic Dutch variant
[13]. However, when the Ab
Val18 fi Ala
was incubated
with acetylcholinesterase a ninefold increase in the
amyloid amount formed measured by thioflavine-T flu-

orescence was found (Fig. 1). Using SDS ⁄ PAGE we
found that both the wild-type Ab
1–40
, as well as the
mutant Ab
Val18 fi Ala
were able to bind acetylcholines-
terase, while the Dutch variant Ab
Glu22 fi Gln
was not
[13]. Consistent with previous observations is the fact
that the presence of different types of Ab peptide dif-
ferentially affects acetylcholinesterase activity, as indi-
cated in Table 1. In almost all cases, a higher
concentration of the inhibitor was required to block
the acetylcholinesterase–Ab complex than that needed
to block the free acetylcholinesterase. The complex
with Ab
Val18 fi Ala
showed the highest difference com-
pared with the free enzyme, suggesting that this
peptide has the greatest degree of interaction with ace-
tylcholinesterase. Consistent with this, the enzyme
interacting with Ab
Glu22 fi Gln
required a lower concen-
tration of the peripherical anionic site inhibitor to
show a clear inhibition (see propidium and fasciculin
in Table 1). These results are consistent with the idea
that acetylcholinesterase–Ab complex formation alters

the enzymatic properties, and enhancement of amyloid
formation induced by acetylcholinesterase is propor-
tional to the lower amyloidogenic property of the
Ab peptides (Ab
1–40
and Ab
Val18 fi Ala
) in comparison
with the most highly amyloidogenic variants such as
N. C. Inestrosa et al. Amyloid–cholinesterase interactions
FEBS Journal 275 (2008) 625–632 ª 2008 The Authors Journal compilation ª 2008 FEBS 627
Ab
Glu22 fi Gln
, as well as the Ab
1–42
. Recent studies
indicate that the Ab oligomers instead of the amyloid
fibrils ad the real culprit of Alzheimer’s disease. In this
context preliminary data from our laboratory indicates
that acetylcholinesterase increases the Ab
1–42
oligo-
meric formation, the incubation of acetylcholinesterase
with Ab
1–42
for 4 h increases the protofibril and amylo-
spheroids Ab assemblies (Fig. 2B), in comparison
with Ab
1–42
alone (Fig. 2A).

Acetylcholinesterase and prion protein
The prion protein (PrP
c
) is a transmembrane protein
of unknown function [41]. PrP
c
suffers a conforma-
tional change with a decrease in the a-helical and an
increase in its b-sheet secondary structure content. This
altered conformation is known as the scrapie prion
protein (PrP
Sc
). PrP
Sc
is believed to infect and propa-
gate by this refolding abnormally into a structure that
is able to convert normal molecules of the protein into
the abnormally structured form. However, the term in
itself does not preclude other mechanisms of transmis-
sion. All known PrP
Sc
s induce the formation of an
amyloid fold, in which the protein polymerizes into a
fiber, accumulates and it is deposited in the central
nervous system, producing the transmissible spongi-
form encephalopathies. This altered structure changes
the physicochemical properties of the protein, includ-
ing an increased resistance to denaturation by chemical
and physical agents, although infectivity can be
reduced by these treatments, making disposal and con-

tainment of these particles very difficult. Structurally,
the amyloid prion apparently shares the properties
with the Ab aggregates. In some Alzheimer’s disease
patients the Ab and prion pathology coexist and the
both kind of amyloid plaques formed have similar
characteristics [42]. In 2006, the effect of acetylcholin-
esterase was studied on prion peptide aggregation
[43]. The authors used a short fragment of PrP
(PrP 106–126), which corresponds to a specific segment
involved in the conversion reaction and pathogenic
properties of abnormal PrP [44]. Moreover, this pep-
tide forms stable b-sheet structures and assemble in
amyloid fibrils. This specific peptide in the presence of
Table 1. Effect of different inhibitors on acetylcholinesterase in its free state and complexed with Ab analogs containing different substitu-
tions. The IC
50
values were calculated from inhibition curves using the GRAPHPAD PRISM 2.0 program (GraphPad Software, San Diego, CA,
USA). The values correspond to mean ± SD. The P-value obtained for no paired Student’s t-analysis correspond to: *P<0.05, **P<0.01,
***P < 0.001 and ns, not significant.
Inhibitors AChE Ab
1–40
+ AChE Ab
val18-Ala
+ AChE Ab
Glu22-Gln
+ AChE Ab
1–42
+ AChE
Active site IC
50

Tacrine(10
)9
M) 445.0 ± 17.6 1074.8 ± 73.3*** 1422.6 ± 204.9*** 625.8 ± 28.2*** 918.5 ± 0.30***
Edrophonium(10
)6
M) 5.36 ± 0.48 17.6 ± 4.6* 26.9 ± 0.2*** 8.70 ± 0.92* 11.7 ± 0.60***
BW284c51 (10
)9
M) 57.1 ± 1.3 116.9 ± 2.7*** 193.2 ± 22.0*** 91.5 ± 11.3** 123.0 ± 4.2***
Peripheral site IC
50
Propidium (1Q
)6
M) 34.6 ± 1.2 72.0 ± 4.8*** 257.1 ± 24.0*** 47.2 ± 2.1** 51.5 ± 0.4***
Gallamine (1Q
)3
M) 8.76 ± 0.46 13.3 ± 0.45*** 27.0 ± 2.5*** 8.77 ± 0.97
ns
12.2 ± 0.2***
(+)-Tubocuranine (10
)6
M) 883.2 ± 44.5 1060.6 ± 36.2* 201 5.4 ± 275.3** 1008.2 ± 149.O
ns
1266.5 ± 43.2**
Fasiculin (10
)11
M) 24.9 ± 1.5 274.6 ± 28.0*** 1562.7 ± 31.3*** 200.1 ± 10.1*** 324.1 ± 31.0***
Fig. 1. Effect of acetylcholinesterase on amyloid formation by ana-
logs containing different substitutions. Emission fluorescence of
thioflavine-T bound to amyloid formed in the presence of each pep-

tide with and without acetylcholinesterase at the final point of the
aggregation. The presence of acetylcholinesterase increases the
amyloid formation in peptides with reduced amyloidogenic power
(Ab
1–40
and AbV18A), however, the AbE22Q has highly amyloido-
genic properties and the aggregating effect of the acetylcholinester-
ase is less effective.
Amyloid–cholinesterase interactions N. C. Inestrosa et al.
628 FEBS Journal 275 (2008) 625–632 ª 2008 The Authors Journal compilation ª 2008 FEBS
acetylcholinesterase showed an accelerated aggregation.
In addition, the size of the amyloid aggregate increases
with increasing acetylcholinesterase concentrations.
More interesting, the effect of acetylcholinesterase
on Ab aggregation was sensitive to the presence of
peripherical anionic site inhibitors. In fact, propidium
and huperzine X, Y and Z blocked the effect of
acetylcholinesterase on the PrP peptide polymerization
[43,45]. These results suggest that the peripherical anio-
nic site of the acetylcholinesterase is involved both in
the Ab and PrP pro-aggregating effect.
Effect of butyrylcholinesterase over
the amyloidogenic process
At present, the biological function of butyrylcholinest-
erase is unclear. Its activity, however, is known to
increase with age, as well as in patients with Alzhei-
mer’s disease [46]. The normal cerebral cortex contains
low amounts of butyrylcholinesterase, most of which is
located in deep cortical neurons and neuroglia [47].
Histochemically reactive butyrylcholinesterase is asso-

ciated with amyloid plaques where it co-localizes with
the Ab peptide [48]. Butyrylcholinesterase is also pres-
ent in neurofibrillary tangles. In Alzheimer’s disease
the expression of butyrylcholinesterase increases sub-
stantially in Alzheimer’s disease patient’s brain [47].
Butyrylcholinesterase shares many structural and phys-
icochemical properties with acetylcholinesterase [49].
Therefore, butyrylcholinesterase was evaluated as a
possible molecular chaperone for amyloid formation
in vitro, by a thioflavine-T fluorescence assay. The
Ab
1–40
incubated with butyrylcholinesterase, showed
nonsignificant differences in the amyloid formation in
comparison with the assay in the absence of the
enzyme after 24 h. It is well known that butyrylcholin-
esterase lacks Tyr72, Tyr124 and Trp286, residues that
form the peripherical anionic site of acetylcholinester-
ase [49,50], therefore it is possible that the absence of
such amino acids may be involved in the lack of butyr-
ylcholinesterase effect on the amyloid fibrils formation.
Butyrylcholinesterase, in contrast to acetylcholinester-
ase, was found to be present exclusively in the soluble
fraction of an aggregating assay of Ab in a fibrillogen-
esis process [51]. Moreover, using a probe that binds
to low-molecular-mass isoforms of Ab it was observed
that butyrylcholinesterase bound to the soluble
Ab assemblies and slowed down its aggregation.
Butyrylcholinesterase was able to extend the nucleation
phase of Ab polymerization and reduces the rate of

amyloid fibrils formation. Also, it was determined that
the aromatic Trp-8 residue is the responsible for the
butyrylcholinesterase–Ab interaction. These results
indicate that butyrylcholinesterase acts as a molecular
chaperone which suppresses the Ab fibril formation by
stabilization of soluble Ab assemblies. However, it is
not clear whether butyrylcholinesterase would also
affect Ab oligomers formation.
Concluding remarks
Acetylcholinesterase is able to accelerate amyloid
formation at least with two different molecules: the
Ab peptide and the prion protein. In addition, the
AB
Fig. 2. Acetylcholinesterase induces the
formation of Ab oligomers. Ab
1–42
(5 lM)in
the absence (A) or presence (B) of 50 n
M
acetylcholinesterase (human recombinant
enzyme) was aggregated at 37 °C without
stirring. A 5 lL aliquot was obtained
at 4 h incubation, stained with 2% uranyl
acetate and photographed with an electron
microscope. More Ab oligomers were
formed in the presence of acetylcholin-
esterase, which correspond to protofibrils
(black arrow), or amylospheroids (white
arrow).
N. C. Inestrosa et al. Amyloid–cholinesterase interactions

FEBS Journal 275 (2008) 625–632 ª 2008 The Authors Journal compilation ª 2008 FEBS 629
pro-aggregating effect of the enzyme dependents on
the intrinsic amyloidogenic properties of the peptide
used. The acetylcholinesterase effect was sensitive to
drugs that block the peripherical anionic site of the
enzyme, suggesting that new and specific acetylcholin-
esterase inhibitors may well provide an attractive
future therapeutic possibility for Alzheimer’s disease
treatment.
Acknowledgements
We thank Dr Lorena Varela-Nallar for her help with
the manuscript. This work was supported by the FON-
DAP and the Millennium Institute (MIFAB). MD is a
predoctoral fellow from Conicyt.
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