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

Histochemical studies have shown that the enzyme associated with senile plaques differs enzymatically in several respects from that associated Keywords acetylcholinesterase; acetylcholin

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: ninestrosa@bio.puc.cl

(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.

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 containoverexpress-ing 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 Km and

Vmaxvalues 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

(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 IC50values 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 Val18fi Ala induces a significant incre-ment of the a-helical content of Ab, and dramatically diminishes fibrillogenesis [16] However, the substitu-tion of Glu22fi 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 AbVal18fi 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 Ab1–40, as well as the mutant AbVal18fi Alawere able to bind acetylcholines-terase, while the Dutch variant AbGlu22fi 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 AbVal18fi 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 AbGlu22fi Glnrequired 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 (Ab1–40 and AbVal18fi Ala) in comparison with the most highly amyloidogenic variants such as

AbGlu22fi Gln, as well as the Ab1–42 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 Ab1–42

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 Ab1–42alone (Fig 2A)

Acetylcholinesterase and prion protein

The prion protein (PrPc) is a transmembrane protein

of unknown function [41] PrPcsuffers 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 (PrPSc) PrPScis 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 PrPScs 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.

Active site IC 50

Tacrine(10)9M ) 445.0 ± 17.6 1074.8 ± 73.3*** 1422.6 ± 204.9*** 625.8 ± 28.2*** 918.5 ± 0.30***

Peripheral site IC50

(+)-Tubocuranine (10)6M ) 883.2 ± 44.5 1060.6 ± 36.2* 201 5.4 ± 275.3** 1008.2 ± 149.Ons 1266.5 ± 43.2** Fasiculin (10)11M ) 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.

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

Ab1–40incubated 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

Fig 2 Acetylcholinesterase induces the

formation of Ab oligomers Ab 1–42 (5 l M ) 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).

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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