REVIEW

Termination and beyond: acetylcholinesteraseas a modulator of synaptic transmission

Gabriel Zimmerman & Hermona Soreq

Received: 28 March 2006 /Accepted: 5 May 2006 / Published online: 27 June 2006# Springer-Verlag 2006

Abstract Termination of synaptic transmission by neuro-transmitter hydrolysis is a substantial characteristic ofcholinergic synapses. This unique termination mechanismmakes acetylcholinesterase (AChE), the enzyme in chargeof executing acetylcholine breakdown, a key component ofcholinergic signaling. AChE is now known to exist not as asingle entity, but rather as a combinatorial complex ofprotein products. The diverse AChE molecular forms aregenerated by a single gene that produces over ten differenttranscripts by alternative splicing and alternative promoterchoices. These transcripts are translated into six differentprotein subunits. Mature AChE proteins are found assoluble monomers, amphipatic dimers, or tetramers of thesesubunits and become associated to the cellular membraneby specialized anchoring molecules or members of otherheteromeric structural components. A substantial increasingbody of research indicates that AChE functions in thecentral nervous system go far beyond the termination ofsynaptic transmission. The non-enzymatic neuromodulatoryfunctions of AChE affect neurite outgrowth and synapto-genesis and play a major role in memory formation andstress responses. The structural homology between AChEand cell adhesion proteins, together with the recentlydiscovered protein partners of AChE, predict the futureunraveling of the molecular pathways underlying thesemultileveled functions.

Keywords Acetylcholinesterase . Synaptic transmission .

Alternative splicing . Alternate promoters .

Synapse adherence

Introduction

Most neurotransmitters are removed from the synaptic cleftby reuptake. This is the case for dopaminergic, noradren-ergic, glutamatergic, gamma aminobutyric acid (GABA)ergic and serotonergic synapses. Cholinergic transmission,in contradistinction, is mainly terminated by acetylcholine(ACh) hydrolysis by the enzyme acetylcholinesterase(AChE; EC 3.1.1.7). Esterase activity in serum was firstsuggested in 1914 (Dale 1914) and was subsequentlyconfirmed experimentally by the application of the AChEinhibitor physostigmine to frog heart muscle, whichprolonged the effects of ACh on it (Loewi and Navratil1926). Later utilization of simple separation methodsrevealed a broad family of cholinesterases and the existenceof several catalytically active AChE isoforms that could bedistinguished by velocity sedimentation, gel electrophore-sis, and their different solubility in diverse buffers, allowingthe individual biochemical characterization of each of theforms. Molecular cloning of the ACHE genes from Torpedocalifornica (Schumacher et al. 1986, 1988) and humans(Soreq et al. 1990; Ben Aziz-Aloya et al. 1993) precededthe determination of the three-dimensional structure of theenzyme (Sussman et al. 1991) and the later identification ofthe anchoring molecules linking the diverse AChE multi-meric forms to the synaptic membrane (Krejci et al. 1991;Perrier et al. 2002). However, subsequent discoveries of anAChE isoform induced by alternative splicing (Kaufer et al.1998; Meshorer et al. 2002) and involved in diverse stressresponses (Birikh et al. 2003; Nijholt et al. 2004) further

Cell Tissue Res (2006) 326:655–669DOI 10.1007/s00441-006-0239-8

G. Zimmerman :H. Soreq (*)The Institute of Life Sciences and the Interdisciplinary Centerfor Neural Computation (ICNC),The Hebrew University of Jerusalem,Jerusalem 91904, Israele-mail: soreq@cc.huji.ac.il

extend previous reports (Layer 1996; Inestrosa and Alarcon1998; Day and Greenfield 2002; Soreq and Seidman 2001)asserting that AChE functions go far beyond the termina-tion of synaptic transmission.

AChE structure

Human AChE is composed of an invariable core of 534amino acids and a variable C-terminal peptide of 14, 26 or40 amino acids (Meshorer and Soreq 2006). Alternativepromoter choices allow the extension of each of thesevariants with a recently found extended N-terminal peptideof 60/66 amino acids in length (Meshorer et al. 2004),hypothetically giving place to six different AChE subunits.The purified protein has an ellipsoidal form of 45×60×65 Åand according to its structure belongs to the alpha/betahydrolase superfamily. The catalytic domain of AChE iscomposed of a serine-histidine-glutamate triad, whichbasically resembles the catalytic site of other serineproteases, except that in this case the acidic residue isglutamate instead of the usual aspartate. Surprisingly, X-raycrystallography has revealed the catalytic triad to be locatedat the bottom of a 20-Å-long gorge, which penetrateshalfway into the protein from its surface (Sussman et al.1991). This “active site gorge” is flanked by 14 aromaticresidues located at the loops between different beta-sheets.A “peripheral anionic site” (PAS) composed of five residuesis clustered at the entrance of the gorge and is in turnsurrounded by ten acidic residues called the “annularelectrostatic motif” (Felder et al. 1997) (Fig. 1).

ACh hydrolysis by AChE

The extraordinary turnover rate of AChE (about 100 μs forone substrate molecule; Lawler 1961) impelled biochemiststo hypothesize about and analyze the mechanisms of actionof the enzyme. Elucidating the crystal structure of AChErevealed a noticeable bipolar distribution of charges on theenzyme surface, with the anionic pole surrounding theentrance to the gorge, and the cationic pole being located atits bottom (Sussman et al. 1991). This evolutionarilyconserved feature was initially hypothesized to attract thepositively charged substrate toward the entrance of thegorge; however, neutralization by mutagenesis of sevencharged residues around the gorge did not alter thehydrolytic rate of AChE (Shafferman et al. 1994). At thegorge entrance, the substrate transiently binds to the PAS(Szegletes et al. 1999), presumably inducing structuralchanges (Kitz et al. 1970) that may facilitate ACh passagethrough the gorge. Once inside the gorge, the aromaticgroups located around its border guide the substrate from

the protein surface to the active site, where the anionicsubsite binds choline and positions the ester at the acylationsite. After the serine displaces choline from the substrate,thereby forming an acetyl-enzyme intermediate, the acetategroup is liberated by a hydrolysis step. The mechanism bywhich the acetyl and choline molecules produced by thisreaction are cleared out of the gorge is not yet understood.Alternative clearance routes, sometimes called “back door”,have been postulated (Gilson et al. 1994), but unequivocalexperimental proof for this hypothesis is still lacking.The breakdown of ACh at the synapse immediaciesallows the produced choline to be re-uptaken by thesynaptic terminal, where it can be reused for further AChassembling.

Intriguingly, the release of a single ACh vesicle from thepresynaptic button suffices to saturate all cholinergicreceptors and cholinesterases in a diameter of 0.5 μm fromthe release site (Bartol et al. 1991). Since the binding ofACh to its receptor sites is much faster than the rate atwhich it is hydrolyzed by AChE, the amount of AChmolecules that will initially bind to synaptic receptors isdetermined by the ratio of receptors to esterase: roughly20% esterase and 80% receptors (Anglister et al. 1994). Therate at which ACh is liberated from the activated receptorsis slower than the ACh hydrolysis rate, so that, when AChmolecules are released, most AChE units are free andcapable of binding a “new” ACh molecule, maintaining inthis way a low ACh concentration in the cleft and virtuallyexcluding the possibility of a single ACh moleculeactivating another receptor. These dynamics explain whythe inhibition of AChE has a more acute effect on theduration of the excitatory post-synaptic potential (EPSP)than on its amplitude (Collier and Katz 1971).

Fig. 1 Three dimensional structure of Torpedo californica AChE-S(PDB 1GQS; Bar-On et al. 2002). The folded structure of themolecule is presented with the three residues of the catalytic site inred and the five residues of the peripheral anionic site (PAS) in blue.Note the location of the catalytic site at the bottom of the gorge andthe anionic residues at its entrance. The helical region leading to thevariable C-terminus is given in green and the N-terminus, which maybe extended by the “N” peptide, in orange

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The enzymatic rate of AChE is lowered under excessivesubstrate concentration. The binding of a second AChmolecule to the PAS apparently slows the deacetylation stepor the formation of the induced fit complex duringhydrolysis (Krupka 1963; Rosenberry 1975). Mutations atAsp-74 and Trp-286, which lower PAS ligand affinity,dramatically reduce substrate inhibition (Shafferman et al.1992), supporting the importance of the PAS in suchprocesses. Environmental conditions have also been shownto contribute to changes that influence substrate inhibition.For example, AChE extracted from mosquito shows nosubstrate inhibition, but when treated with thiocyanate,which converts the enzyme back to its non-amphiphilicderivatives, substrate inhibition returns (Dary and Wedding1990).

AChE combinatorial complexity

AChE is found in an assorted array of forms attained byalternate promoter usage of its gene, alternative splicing ofnascent pre-AChE mRNA transcripts, multimerization ofindividual AChE subunits, and their association withdifferent anchorage or partner proteins. Alternate promoterusage and alternative splicing together modify the 5′ and 3′termini of AChE mRNA, allowing the production of sixprotein subunits with different N- and C-termini. Threedifferent carboxy termini exist: the “synaptic” or S variant,which is also called “tailed” (Massoulie 2002), the“erythrocytic” or E variant (Li et al. 1991), and the“readthrough” or R variant (Kaufer et al. 1998). These jointhe two different N-termini to yield variants with thecommon or the “extended” N-terminus (Meshorer et al.2004; Fig. 2). Whereas the catalytic domain of all AChEisoforms remains invariable, the characteristics of thesedifferent terminal peptides alter several key features of theprotein. A prominent characteristic of AChE-S, the mostabundant form in the nervous system, is a C-terminalcysteine residue located three amino acids from the end ofthe protein. This cysteine residue allows disulfide bondingwith other AChE-S units, giving rise to amphipathichomodimers and homotetramers. Beyond its differentamino-acid sequence, the AChE-R C-terminus differs fromthat of AChE-S in two obvious characteristics: (1) it ishydrophilic, and (2) since it lacks the C-terminal cysteineresidue found in the AChE-S C-terminus, it cannottetramerize by disulfide bonding with other subunits. Thisproduces a soluble monomeric molecule, instead of theamphipathic tetramers composed of dimers.

AChE-S tetramers associate with one of two membrane-anchoring molecules, which partially determine the synap-tic localization of the protein: collagen Q (ColQ) inneuromuscular junctions, and a proline-rich membrane

anchor (PRiMA) in brain synapses (Massoulie et al.1999). ColQ units homotrimerize by forming a triple helicalstructure in a proline-rich domain at their C-terminus (Bonand Greenfield 2003; Bon et al. 2003). Since each ColQcan attach an AChE tetramer, hetero-oligomeric complexesmay contain four, eight, or 12 AChE subunits, designatedA4, A8, or A12, respectively. The glycoprotein PRiMA, inturn, induces the formation of AChE homotetramers andattaches them by a proline-rich motif, keeping themanchored to the cell membrane by a transmembranaldomain (Perrier et al. 2002). PRiMA-anchored formsaccount for 70%–90% of total AChE activity in the centralnervous system (CNS; Grassi et al. 1982), the other 10%–30% being accounted for by asymmetric forms and, in non-stress conditions, by 1% of AChE-R (Perrier et al. 2005).PRiMA-associated AChE tetramers are traditionally desig-nated as G4 (for globular), and monomers and homodimersnot associated to any anchorage protein are designated asG1 and G2, respectively (Fig. 2b); however, it is importantto remember that the G classification preceded thediscovery of PRiMA by two decades. Moreover, a

Fig. 2 ACHE gene structure and protein products. a Exon-intronstructures of mouse and human ACHE genes (cylinders exons, linesintrons, lines above genes splicing options). Several alternative tran-scripts may be obtained by alternative splicing of pre-AChE mRNA; sixdifferent protein subunits are derived from them. b Alternative proteinproducts of the ACHE gene. AChE-S units can dimerize, tetramerize, orremain as monomers. AChE-S tetramers can remain soluble or becomeanchored to the membrane by the molecules ColQ or PRiMA. AChE-Rinherently remains as a soluble monomer. AChE-E forms glypiateddimers linked to the red blood cells membrane

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significant part of the nascent AChE molecules may nevergain catalytic activity (Rotundo 1988), which impliesthat the traditional ways of quantifying and characterizingAChE isoforms might not have revealed importantinformation.

AChE-R lacks a hydrophobic domain and is incapable ofbinding to ColQ or PRiMA. Therefore, it remains soluble,and its secreted form shows greater mobility than AChE-S(Soreq and Seidman 2001).

AChE biosynthesis

AChE polypeptides are synthesized with an N-terminalsignal peptide (Soreq et al. 1990) in the rough endoplasmicreticulum, in which they are co-translationally glycosylated,some then being assembled as dimers or tetramers(Rotundo 1988). The non-multimerized units remain asglobular monomers and are rendered inactive by as yetunclear post-translational modifications. A retention signalin their C-terminal peptide retains monomers in thecytoplasm (Velan et al. 1991), in which many of them arerapidly degraded (Rotundo and Fambrough 1980). Theoligomeric forms transit to the Golgi apparatus, where theyacquire complex sugars (Rotundo 1984) and are laterassembled into asymmetric forms with ColQ or PRiMA(Rotundo et al. 2005). More recent data suggest thatasymmetric heteromeric assembly takes place in theendoplasmic reticulum (Massoulie et al. 1998). In musclefibers, the newly assembled ColQ-AChE associates intra-cellularly with the proteoglycan perlecan and is thenexternalized to the neuromuscular junction (NMJ), whereit colocalizes with other components (Rotundo et al. 2005).

The role played by PRiMA in AChE targeting within inthe CNS is not yet known. Of note, although PRiMA isubiquitously expressed, it appears to interact with AChEonly at CNS synapses. Non-multimerized globular mono-mers can be secreted from various cell types (Legay et al.1999); nevertheless, most of the secreted enzymes areattached to an anchorage protein, and the pool ofmonomeric subunits that is not recruited by them undergoesdegradation. Muscle AChE-S finally appears at the surfaceof the cells about 2.5 h after its synthesis and has a half-lifeof about 50 h. This time lapse is highly reproducible in cellsfrom different species or origins (Rotundo et al. 1988). Inthe brain, however, stress insults induce a rapid increase ofsecretory AChE-R, with a two-fold difference by 30 min(Kaufer et al. 1999).

AChE localization

AChE is widely expressed in tissues that receive choliner-gic innervation, such as neurons, muscle cells, and cells of

the autonomic nervous system and of the immune nervoussystem. Interestingly, AChE expression patterns are notalways correlated with that of the more faithful cholinergicmarker, choline acetyltransferase (ChAT), the enzyme thatsynthesizes ACh. AChE activity is also found in brainregions with low or no cholinergic inputs, such as thesubstantia nigra, cerebellum, globus pallidus, and hypothal-amus, and in many non-nervous tissues devoid of anyknown cholinergic innervation. These include testis (Mor etal. 2001), endothelial cells (Deutsch et al. 2002), hemato-poietic (Grisaru et al. 2006) and osteogenic cells (Grisaru etal. 1999), and various tumors (Karpel et al. 1994; Perry etal. 2002). AChE is also broadly expressed in nervous tissuebefore synaptogenesis (Layer 1990; Dori et al. 2005), i.e.,before cholinergic transmission is established. The isofor-mal composition of AChE is probably determined by thedifferent functional characteristics of the diverse synapsesat which the enzyme is present. This can initially be dividedinto two large, rather heterogeneous groups: the cholinergicsynapses mediating muscular contraction found at the NMJ,and the modulatory cholinergic synapses found at the CNS.Whereas ACh release at the NMJ is spatially defined andshort-term, secretion at cholinergic synapses in the CNS isdiffuse and has a long-term modulatory character. More-over, the different anatomical characteristics of thesesynapses (the former being much more spacious andcontaining collagens and a basal lamina) appear todetermine the isoform types encountered in them.

The ColQ subunit is mainly synthesized in muscle cellsand attaches AChE oligomers to the basal lamina of theNMJ, between the pre- and post-synaptic membranes,where it represents most of AChE active forms. Rat fastmuscles contain exclusively the A12 form, whereas slowmuscles contain a higher proportion of the A8 and A4forms (Sketelj et al. 1992). The PRiMA-anchored tetramer-ic form appears to be more suited to the smaller synapticcleft found in the CNS (Legay 2000), where the lack of abasal lamina would hamper ColQ anchoring. At centralsynapses, anchored forms account for 70%–90% of AChEtotal activity, whereas asymmetric forms represent less than3% of this activity. Anchored tetramers are expressed alsoin fast muscles, in which their concentration is increased ordecreased according to the respective physiological exercise(Jasmin and Gisiger 1990; Gisiger et al. 1994) andpathology (Rakonczay et al. 1991). The usually rare variantAChE-R has been found in all studied mammals and is co-expressed with AChE-S, albeit in a distinct neural distribu-tion pattern (Sternfeld et al. 2000) contributing about 1% oftotal AChE activity under various physiological states(Perrier et al. 2005).

During embryogenesis AChE is uniformly distributed inmuscle cells accumulating, after nerve contact, at the NMJ(Inestrosa 1984) in which it remains abundantly expressed

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(Legay et al. 1995) allowing sustained high translation.AChE-S dimers and larger homomers are usually located atthe endoplasmic reticulum, probably as a precursor pool ofheteroligomeric forms (Massoulie 2002). However, theycan also be secreted without being attached to anymembrane anchor protein.

AChE polymorphisms

The marked identity between the human and mousegenomic ACHE sequences and the almost identical crystalstructure suggest the importance of virtually each singledomain of this enzyme for its proper functioning. Nucleo-tide polymorphisms in the ACHE gene were considered rare(Soreq and Seidman 2001) until a recent comprehensivestudy that found AChE polymorphic prevalence at severalsites (Hasin et al. 2005). Three identified AChE poly-morphisms have been shown to have biological implica-tions and clinical relevance: a polymorphism in the distalpromoter of AChE affecting interaction with a glucocorti-coid response element (GRE; Shapira et al. 2000), a 4-bpdeletion in a hepatocyte nuclear factor 3 (HNF3)-bindingsite also located at the promoter region (Shapira et al.2000), and a single nucleotide polymorphism (SNP)producing the His322 Asn substitution, which is responsi-ble for the YT-2 blood group phenotype (Bartels et al.1993; Ehrlich et al. 1994). Specific but rare polymorphismsat the ACHE/paraoxonase (PON1) locus have further beenfound to be correlated with hypersensitivity to AChEinhibitors (Shapira et al. 2000), insecticide-inducedParkinson’s disease (PD; Benmoyal-Segal et al. 2005),and trait anxiety (Sklan et al. 2004). Individuals carryingthe rare GRE and the HNF3-binding-site polymorphismsshow increased constitutive AChE expression and hyper-sensitivity to AChE inhibitors (Shapira et al. 2000). A laterstudy has indicated a more complex interaction between theHNF3-binding-site polymorphism and PON1 activity asdeterminants of AChE serum activities (Bryk et al. 2005).Similarly to the carriers of these polymorphisms, TgStransgenic mice overexpressing AChE-S exhibit hypersen-sitivity to AChE inhibitors (Shapira et al. 2000), suggestingthat high basal AChE levels impede further transcriptionalactivation, which is necessary to overcome exposure tosuch compounds.

The enzyme PON1 degrades organophosphate com-pounds, which are potent inhibitors of AChE and arenotably involved in insecticide-induced PD (Gorell et al.1998; Kondo and Yamamoto 1998). A recent study aimedat assessing the relationship between polymorphisms in theACHE/PON1 locus and insecticide-induced PD found anon-significant association between the HNF3-binding-site

promoter deletion and the pathology. However, when theACHE and PON1 gene polymorphisms were jointlyconsidered, the debilitated alleles were found to beoverrepresented in insecticide-induced PD patients, whoshowed lower levels of AChE and PON1 activities(Benmoyal-Segal et al. 2005).

A synonymous SNP at position P446 of the ACHE genecoding region, present in about 12% of the population(Bartels et al. 1993), has been found to be more frequent inindividuals with high levels of trait anxiety compared withthose presenting low levels of trait anxiety (Sklan et al.2004). This SNP is not associated with altered AChEactivity in serum (Sklan et al. 2004) and therefore probablyreflects an associated change in an as yet undefinedadjacent gene(s).

A recent study aimed at identifying SNPs in fourdifferent populations has found a total of five codingnonsynonymous, three coding synonymous, and five non-coding ACHE SNPs among 96 studied individuals. The fivecoding SNPs found affect only evolutionarily non-con-served amino acids, which are either located far from theactive site or not present in the mature protein (Hasin et al.2005). No physiological implications have yet beenassigned to the new SNPs found in this study.

AChE regulation

Several studies indicate that the AChE isoform pattern isregulated by the degree and character of synaptic activityand by some other stimuli with no known relationship tocholinergic transmission. Denervation of mammalian skel-etal muscle and the blocking of spontaneous contraction bytetrodotoxin (TTX) have been shown to decrease AChEactivity and the appearance of asymmetric forms at the cellsurface (Lomo and Slater 1980; Cangiano et al. 1980;Michel et al. 1994; Rossi et al. 2000). This is accompaniedby a transient increase in G4 (Gregory et al. 1989), butwithout altering the G1 and G2 isoforms (Fernandez andHodges-Savola 1992). The lack of membrane depolariza-tion also negatively affects ColQ expression (Massoulie2002), which in turn downregulates the rate of heterote-tramer assembly and release, thus enlarging the intracellularG4 pool. The conservation of G1 and G2 levels indicatesthat their expression is controlled by spontaneous AChrelease (Fernandez and Hodges-Savola 1992), which is notblocked by TTX. Denervation downregulatory effects canintriguingly be reversed by ectopic reinnervation, theapplication of neurogenic proteinaceous substances(Fernandez and Hodges-Savola 1992), direct electricalstimulation (Lomo and Slater 1980), treatment with Na+

channel agonists (Rossi et al. 2000), and increase incytoplasmic Ca2+ (Rubin 1985).

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Fast and slow muscles possess only the A12 and A12,and the A8 and A4 AChE forms, respectively. The distinctAChE compositions may be altered by cross-innervatingthese muscles or by altering the mode of stimulationimposed upon them (Lomo et al. 1985). Treadmill exercisesproduce a marked increase in G4 forms in fast, but not low,twitch muscles (Fernandez and Donoso 1988). This mayreflect the finding that fast muscles are more readilyaffected by training than slow ones.

A transient increase in AChE has also been detected inthe various layers of primary sensorial cortices (Robertsonet al. 1985; Robertson 1987) reaching an expression peak ataround 2 weeks after birth and attaining adult expressionpatterns 1 week later. Although enucleation and TTXintraocular injection abolish AChE expression in theprimary visual cortex (Robertson et al. 1989), it does notalter ChAT staining in this cortex, suggesting that AChE isnot directly related to cholinergic efferents and that it mighthave roles other than terminating cholinergic transmissionduring development.

Experimental induction of autoimmune myastheniagravis (MG) in rats stimulates the overexpression ofAChE-R, but not of AChE-S, in muscles (Brenner et al.2003). Destruction of the AChE-R mRNA transcripts byantisense oligonucleotides facilitates muscle action poten-tials, physiological stamina, and weight gain in experimen-tally ill rats (Brenner et al. 2003). In antisense-treatedmonkeys, AChE-R mRNA levels are also selectivelydecreased in spinal cord neurons, but conferred nomovement changes (Evron et al. 2005). Thus, excess, butnot normal low, levels of AChE-R change neuromusculartransmission.

The composition of AChE in the CNS is subject todynamic changes under various cellular and physiologicalstimuli. A gradual AChE increase accompanied by anisoformal shift from G1 to the adult G4 form duringembryogenesis has been broadly reported in many organ-isms (Inestrosa et al. 1994; Anselmet et al. 1994).Reciprocally, the loss of cholinergic circuits characteristicof Alzheimer’s disease (AD) is reflected in reduced AChEactivity accompanied by increased monomer fractions inthe cerebrospinal fluid (CSF) and in various brain nuclei.These changes are correlated with the clinical severity ofthe disease, suggesting physiological relevance (Arendt etal. 1992; Darreh-Shori et al. 2004). Interestingly, severalAD brain areas show a selective loss of tetrameric AChE(Schegg et al. 1992; Fishman et al. 1986), but theasymmetric and G1 forms are upregulated (Younkin et al.1986), greatly reducing the ratio between G4 and G1 invarious brain regions (Arendt et al. 1992). In AD patientstreated with anti-AChEs, an increase in the G1 isoform hasrecently been shown to represent AChE-R upregulation(Darreh-Shori et al. 2004).

In healthy mammals, AChE-R is markedly upregulatedin the cortex, hippocampus, striatum, and cerebellum ofanimals exposed to various stressors (Kaufer et al. 1998;Meshorer et al. 2002; Nijholt et al. 2004; Perrier et al.2005). This upregulation depends on an increase of thesplicing factor SC35 occurring during stress, an increasesustained as long as several weeks after the stress event(Meshorer et al. 2005). A similar AChE-R increase is alsoproduced by corticosterone treatment, organophosphatepoisoning, and AChE inhibition by the carbamate anti-AChEs pyridostigmine and physostigmine (Kaufer et al.1998; Meshorer et al. 2002; Table 1).

Beyond transmission

AChE neuromodulatory activity

The initial observation that AChE expression patterns didnot correlate with the presence of ACh (Greenfield 1991;Appleyard and Jahnsen 1992; Layer 1983) initiated thehypothesis that AChE possessed non-catalytic properties.AChE was found to be released from nigral dopaminergicneurons independently of cholinergic activation (Llinas andGreenfield 1987), in addition to following electricalstimulation (Greenfield and Smith 1979) and intracellularCa2+ release (Greenfield et al. 1983). Later experimentssuggested that released AChE played neuromodulatoryfunctions independent of its catalytic activity and wascapable of affecting complex behavioral patterns. Exoge-nous administration of AChE-S to the striatum of freelybehaving rats induced an increased availability of extracel-lular dopamine, as indicated by overactivation of thenigrostriatal pathway and circling behavior (Hawkins andGreenfield 1992). In vitro studies in various brain areaswere aimed at characterizing in more detail the electro-physiological characteristics of AChE. Application ofAChE to midbrain slices resulted in marked membranehyperpolarization and decreased input resistance, evenwhen co-administered with its irreversible catalytic inhib-itor soman (Greenfield et al. 1988). This attributed theeffect to non-enzymatic mediation. An opposite result wasobtained by applying AChE to cerebellar slices, in whichthe enzyme enhanced the response of Purkinje cells toglutamate and aspartate, facilitating climbing fiber stimula-tion (Appleyard and Jahnsen 1992). AChE-S application toguinea pig hippocampal slices induced a long-term poten-tiation (LTP)-like effect in CA1 neurons, as indicated byincreased EPSPs and increased population spikes evokedby stimulation of Schaffer collateral fibers (Appleyard1995). The effects persisted after treatment with thecholinergic antagonists atropine and mecamylamine, sug-gesting that these modulatory effects are not mediated by

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Table 1 Regulation of AChE expression. Several electrophysiological, chemical, and behavioral manipulations alter AChE expression at differentlevels and modify its isoformal composition (EDL extensor digitorum longus muscle, SOL soleus muscle, TTX tetrodotoxin)

Manipulation Outcome Reference

Denervation of vertebrateskeletal muscles

Upregulation of AChE transcripts after 10 daysin avian fast skeletal muscle

Rimer and Randall 1999

Downregulation of AChE transcripts after 10 daysin rodent fast and slow skeletal muscle

Michel et al. 1994

Decrease in AChE in spinal cord Tsim et al. 1997Preservation of the different AChE isoformal patternsin fast and slow muscles

Sketelj et al. 1991

Transient increase in G4 form (24–60 h) in fastmuscles, and decrease in all other forms

Gregory et al. 1989; Hodges-Savola and Fernandez 1991

Reduction in AChE activity in fast and slow muscles(14–42 days) and in NMJ

Lomo et al. 1985

Cross innervation of fast EDLmuscle with slow SOL nerveand vice versa

Replacement of each muscle characteristic isoformalpattern to that of the other muscle

Dolenc et al. 1994

Fast and slow electricalstimulation

Induction of AChE pattern reminiscent of thatof EDL in denervated SOL after fast stimulation.Preservation of SOL characteristic AChE pattern indenervated SOL after slow stimulation. No changesin denervated EDL after fast or slow stimulation

Lomo and Slater 1980;Lomo et al. 1985

Electrical stimulation,incubation with nicotine,but not with histamine orangiotensin II

Two-fold increase in AChE secretion from adrenal gland Small et al. 1993

Muscle paralysis by TTX orbotulinum toxin

Immediate decrease in AChE transcripts lastingfor 10 days in rodent fast and slow skeletal muscles

Michel et al. 1994;Cresnar et al. 1994

Decrease in AChE activity in rodent muscle culture Rubin 1985Decrease in AChE activity, mainly in asymmetricforms in quail myotubes culture

Fernandez-Valle andRotundo 1989

Induction of slow muscle isoformal pattern in bothfast and slow rodent skeletal muscles

Boudreau-Lariviere et al. 1997

Increase in AChE activity in cultured avianmuscle cells

Walker and Wilson 1976

Preservation of AChE activity and increase in itssecretion in cultured avian muscle cells

Vallette and Massoulie 1991

Ca2+ ionophore treatmentin TTX paralyzed muscles

Increase in asymmetric AChE concentration Rubin 1985

L-type Ca2+ channel blockersryanodine and nifedipine,but not the N-typechannel blocker ω-conotoxin

Decrease in activity of all forms after 3 h in primaryavian pectoral muscle tissue

Decker and Berman 1990

Inhibition of myotube-differentiation induced stabilizationof AChE mRNA

Luo et al. 1994

L-type Ca2+ channel knockoutmice

Decrease in AChE mRNA and activity in skeletal muscle Luo et al. 1996

High level of K+ in medium Increase in AChE release from N18TG2 and 108CC15cell lines

Biagioni et al. 1995

Injection of calcitoningene-related peptide

Abolition of denervation-caused G4 increase. Preservationof other forms of denervation-caused decrease

Hodges-Savola andFernandez 1995

Reduction in all isoforms in normally innervated rodentskeletal muscle

Fernandez et al. 1999

Reduction in AChE transcription in cultured muscle cells Rossi et al. 2003Treatment with nervegrowth factor

Increase in AChE transcription rate in PC12 cells Greene and Rukenstein 1981;Deschenes-Furry et al. 2003

Increase in G4 release to medium from PC12 and chickoptic lobe cells

Lucas and Kreutzberg 1985

Treatment with ciliaryneurotrophic factor

Decrease in AChE in denervated, but not intact, adultrat skeletal muscle

Boudreau-Lariviere et al. 1996

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Table 1 (continued)

Manipulation Outcome Reference

RNA-binding proteinHuD overexpression

Stabilization of AChE mRNA in PC12 cells Deschenes-Furry et al. 2003

Myogenic transcription factors Preservation of AChE transcription rate and mRNAstability in cultured fibroblasts

Mutero et al. 1995

Egr-1 treatment Reduction in AChE transcription in 10T1/2 and 10TFL2–3 cells Mutero et al. 1995Ephrin A1 application, whichactivates Stat3

Increase in AChE mRNA in C2C12 myotubes Lai et al. 2004

cAMP application Increase in AChE expression in chicken cultured myotubes Choi et al. 2001a,bReduction in AChE expression in cultured myotubes

P2Y1 receptor activation byATP, ADP, 2-MeSADP, and2-MeSATP

Increase in AChE expression in cultured chicken myotubes Choi et al. 2001a,b

P2Y2 receptor activation by UTP Increase in AChE protein in differentiated myotubes Tung et al. 2004Retinoic acid treatment, whichcommits P19 cells to neuronsand glia

Increase in levels of AChE mRNA, protein, and activity in P19 cells Coleman and Taylor 1996Increase in AChE activity and neurite outgrowth inductionin neuroblastoma cell line

Sidell et al. 1984

Microtubule inhibitorsapplication (colchicine, taxol)

Inhibition of G4 release to the medium from PC12 and fromchick optic lobe cells

Lucas and Kreutzberg 1985

Protein glycosylation inhibitorapplication (tunicamycin)

Inhibition of G4 release to the medium from PC12 and fromchick optic lobe cells

Lucas and Kreutzberg 1985

Reduction of Ca2+ in medium,preventing myoblast fusion inmuscle cell line

Reduction in AChE by 70% and preservation of 4S and 6Sforms synthesis

Inestrosa et al. 1983

Beta-endorphin application Decrease in 16S form and increase in 6S and 4S forms inmyotube cultures

Haynes et al. 1984

Muscle-preconditioned medium Decrease in AChE activity in rodent sympathetic neurons Swerts et al. 1984Administration of aluminium andcitrate, but not aluminium alone

Increase in AChE in various mouse brain areas Kaizer et al. 2005

Neostigmine intraventicularinjection

Upregulation of AChE-R transcripts in rodent brain Meshorer et al. 2002

Cercal nerve ablation Loss of AChE activity in cercal nerves and terminal ganglion incockroach

Sekhar et al. 1991

Granule cell lesion AChE increase in molecular layer of rodent dentate gyrus McKeon et al. 1989Combined hypoxia andhypoglycemia(simulated ischemia)

AChE increase in rodent hippocampal slice cultures Saez-Valero et al. 2003

Beta-amyloid protein application Increase in AChE in rodent primary cortical neurons Fodero et al. 2004Donepezil and galantamineadministration

Increase in AChE in CSF of AD patients Davidsson et al. 2001

Infection with nematode parasiteNippostrongylus brasiliensis

Development of discrete foci of intense AChE activity inbasal membrane of jejunal mucosa

Russell et al. 2000

Experimental induction ofmyasthenia gravis

Increase in AChE-R expression in rodent muscle and serum Brenner et al. 2003

Repeated session of treadmillexercise

Increase in G4 in fast muscles having a dynamic role duringexercise. Decrease in G4 in fast muscles having tonicactivity during training

Jasmin and Gisiger 1990

Small decrease in G4 in slow musclesRapid eye movement (REM)sleep deprivation

Increase in AChE activity in several rodent brain areas Thakkar and Mallick 1991

Confined swim sessions Increase in AChE-R expression in diverse rodent brain areas Kaufer et al. 1998;Meshorer et al. 2002

Immobilization stress Increase in AChE-R expression in diverse rodent brain areas Nijholt et al. 2004Perlecan knockout mice Preservation of AChE expression, but without localization to NMJ Arikawa-Hirasawa et al. 2002Mu-opioid receptor knockout mice Increase in AChE activity in striatum, but not in cortex or hippocampus Tien et al. 2004Ephrin receptor ephA4 knockout mice Decrease in AChE expression in skeletal muscle Lai et al. 2004

662 Cell Tissue Res (2006) 326:655–669

ACh. In order to investigate these phenomena in a simplerscenario, membrane homogenates from rat cortex wereincubated with AChE, and the affinity of different a-amino-3-hydroxy-5-methylisoxazolepropionate (AMPA) receptoragonists was measured. A dose-dependent increase in thebinding of (S)-[3H]5-fluorowillardiine and [3H]AMPA, butnot of [3H]kainate, was found after treatment (Olivera et al.1999). Treatment of neurons with the PAS blockerBW284c51 greatly reduced currents evoked by glutamate,AMPA and N-methyl-d-aspartic acid (NMDA), but not byGABA. PAS blockade increased AChE levels, and imped-ing this effect by AChE antisense oligonucleotides signif-icantly decreased the induced suppression of glutamatecurrents (Dong et al. 2004).

The interaction of AChE-S and AChE-R with theirdiverse protein partners may indirectly affect membraneelectrophysiological properties, possibly explaining someof the above-mentioned phenomena. Interestingly, spliceshift to AChE-R may, under certain conditions, lead to therelocalization and alteration of function(s) of the proteinpartners of both AChE-S and AChE-R, thus modifyingdiverse cellular properties. AChE-R, for example, formscomplexes with the scaffold protein RACK1 and, through it,with PKC-βII (Birikh et al. 2003; Sklan et al. 2004), which isimplicated in synaptic plasticity and memory (Weeber et al.2000). Transgenic TgR mice overexpressing human AChE-R exhibit facilitated LTP induction (Nijholt et al. 2004),suggesting profound biological implications for AChE-R/RACK1/PKC-βII complexes. Like TgR mice, FVB/Nstrain-matched non-transgenic mice exposed to an acutestress episode show both AChE-R upregulation (Kaufer etal. 1998) and LTP facilitation (Blank et al. 2002), which canbe abolished by pretreating the animals with an AChE-Rantisense oligonucleotide (Nijholt et al. 2004).

Two different short sequences derived from AChE aresuggested to be bioactive: a 14-amino-acid peptide locatedat the AChE-S C-terminus and the 26 most-distal aminoacids from the AChE-R C-terminus (Nijholt et al. 2004).The former peptide has been recently shown to modulateNMDA receptor Ca2+ currents (Bon and Greenfield 2003)and α7 nicotinic receptor responses (Greenfield et al.2004); however, the in vivo existence of this peptide asan independent entity has not been corroborated.

AChE involvement in neurite outgrowth

The finding that ACh modulates growth cone motility(Owen and Bird 1995; De Jaco et al. 2002a,b) and theinitial observations that a transient peak in AChE activity inpostmitotic cells precedes neurite formation by severalhours (Weikert et al. 1990; Geula et al. 1995; Dupree andBigbee 1994) have led researchers to study whether AChEis causally involved in neurite outgrowth. Intriguingly, the

high amounts of AChE found in the immature embryonicCNS are not accompanied by the appearance of ChAT andprecede the establishment of cholinergic transmission,hinting at a non-canonical role for AChE in this scenario.Treatment of different cell cultures with the PAS inhibitorsBW254c51, propidium, and fasciculin, but not with theactive-site inhibitors echothiophate and galanthamine,induces decreases both in neurite numbers and in theirbranching (Layer et al. 1993; Dupree and Bigbee 1994;Olivera et al. 2003; Day and Greenfield 2002). This iscompatible with the casual involvement of AChE in suchprocesses, indicates a non-catalytic role for AChE, andsuggests the PAS as a possible mediator of this process.Diverse primary cultured cells (Bigbee et al. 2000) and celllines (Sternfeld et al. 1998; Karpel et al. 1996) geneticallymanipulated to overexpress either AChE-S or a catalyticallyinactivated form of it (AChE-Sin) show a significantincrease in neurite growth, whereas antisense suppressionof AChE impairs it (Grifman et al. 1998). AChE applicationto the medium induces the extension of neuronal processes,synapse formation, and AMPA receptor surface expressionin hippocampal cell cultures (Olivera et al. 2003). Somereports claim a more potent role for the embryonic AChE-Smonomers than for the adult tetramers (Holmes et al. 1997;Day and Greenfield 2002); however, others contest theseobservations (De Jaco et al. 2002a,b). Importantly, Xenopustadpoles overexpressing human AChE-R do not show moreextensive neurite outgrowth than control tadpoles, suggest-ing that AChE membrane attachment is essential toinfluence such processes. Moreover, ACh modulates neu-rite growth (Owen and Bird 1995; De Jaco et al. 2002a,b;Zheng et al. 1994), and AChE could exert a trophic rolesolely by hydrolyzing it. Nevertheless, most studies onneurite outgrowth involve the use of cholinergic antagonistsor cell lines incapable of synthesizing ACh, supporting thenotion that the observed effects stem from the non-catalyticproperties of AChE.

Recent investigations indicate that AChE interactswith the basement-membrane protein laminin-1 β in vivo(Paraoanu and Layer 2004; Paraoanu and Layer 2005) andwith collagen IV (Johnson and Moore 2003). Thisinteraction is negatively affected by the ionic strength ofthe medium and by the addition of a monoclonal antibodydirected against the PAS (Johnson and Moore 2003).Laminin-1 β is known to interact with integrins, whichplay a major role in neuronal migration and in CNSdevelopment (Benson et al. 2000).

Cholinesterase-like adhesion proteins

A family of cell adhesion molecules possessing anextracellular domain with notable sequence homology toAChE has been cloned and characterized over the past

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years; their study may generate novel explanations of thenon-enzymatic functions of AChE. Five different proteinshave been identified comprising the cholinesterase-likeadhesion molecules (CLAMs) family: thyroglobulin, glutac-tin, neurotactin, gliotactin, and neuroligin. The mainstructural properties shared by CLAMs and AChE aresummarized as follows: (1) a 25%–32% amino acid identitybetween the extracellular domain of CLAMs and the AChEsequence, comprising much of the AChE sequence, (2) asimilar hydropathic profile, suggesting a similar three-dimensional structure and consequent common functionalproperties, (3) an unusual surface charge distributionconsisting of a negatively charged annular motif aroundthe entrance to the catalytic gorge or around its homologousdomain (Botti et al. 1998), (4) an EF-hand motif thatapparently binds Ca2+ and has a role in heterologous cellassociation (Tsigelny et al. 2000), and (5) dimerization andtetramerization of individual subunits, which is mediated bythe four-helix bundle of the AChE catalytic domainconserved in all CLAMs (Morel et al. 2001).

Of all CLAMs, the physiological roles of neurotactin,gliotactin, and neuroligin are better understood and appearto be closer to the presumed non-canonical functions ofAChE (Scholl and Scheiffele 2003). Neurotactin interactswith the secreted protein amalgam through its cholinester-ase-like domain (Fremion et al. 2000), promoting hetero-philic cell aggregation (Barthalay et al. 1990). Chimericmolecules in which the neurotactin extracellular region isreplaced with the homologous AChE domain retain theadhesive properties of the intact molecule (Darboux et al.1996). An interesting hypothesis is that, under conditions inwhich AChE is overproduced, it might compete withneurotactin ligands, blocking its growth-inducing functions.This may be especially relevant to the soluble variantAChE-R, which is subject to less limitation on itslocalization than AChE-S and therefore represents a firmercompetitor to neurotactin partners. Gliotactin is expressedin peripheral glia and contributes to the formation of theblood-nerve barrier. Drosophila embryos with mutantgliotactin are nearly paralyzed as a consequence of theincomplete isolation of their motor axons (Auld et al.1995). Neuroligins constitute a multigenic family ofproteins that interact with neurexin transmembrane recep-tors (Dean et al. 2003). Members of the neuroligin familyparticipate in synapse formation (Scheiffele et al. 2000)through their interaction with neurexins and control thebalance between excitatory and inhibitory synapses (Chihet al. 2005), supporting the concept of a neuromodularyfunction for their extracellular esterasic domain. Moreover,CLAMs might compete with cholinesterases (and vice-versa) in interactions with extracellular partners, suggestingputative interrelationships between these two types ofproteins in their neuromodulatory activities.

Concluding remarks

AChE is much more than a straightforward terminator ofsynaptic transmission. Multileveled evidence suggests thatit substantially contributes to synaptic transmission, notonly in cholinergic synapses, but also in other types (e.g.,dopaminergic, glutamatergic). It apparently does this byinteracting with signaling cascades, by the complex varietyof isoforms that it possesses, and by their modifiedlocalization under various conditions. Termination ofcholinergic neurotransmission by AChE is thus merely theend of the beginning of multiple subsequent events.

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