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: 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. 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. 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