Journal of Molecular Neuroscience 197 Volume 30, 2006

*Author to whom all correspondence and reprint requests should be addressed. E-mail: soreq@cc.huji.ac.il

Readthrough AcetylcholinesteraseA Multifaceted Inducer of Stress Reactions

Gabriel Zimmerman1 and Hermona Soreq*,1,2

1Interdisciplinary Center of Neural Computation; and 2Department of Biological Chemistry,Hebrew University of Jerusalem, Jerusalem 91904, Israel

IntroductionStress insults induce hyperexcitation of choliner-

gic circuits, both peripherally in the sympatheticpathway (Tracey, 2002) and at the central nervoussystem (CNS) (Sapolsky, 1996). This reaction canserve to ensure survival but might also entail a riskto the hyperactivated neurons. Consequent changesin the expression of a series of proteins related toacetylcholine (ACh) metabolism might protect theorganism from the potentially detrimental effects ofthis increase in ACh. Of particular interest amongthese effects is the induction by alternative splicingof the alternative, usually rare, readthrough variantof acetylcholinesterase (AChE), AChE-R. AChE-R isone of the first proteins conveying the signal that theorganism has entered a state of alert. Viewing AChE-R as a stress signal can therefore serve to answer thequestion “How do we expect a stress signal to oper-ate”? This facilitates the generation of hypothesesregarding the triggering of such signals and theeffects it exerts at the molecular, cellular, and phy-siological levels.

Induction of the AChE-R SignalDuring a stress response, both the sympathetic

preganglionic fibers and the septum-hippocampalsystem are activated, secreting large amounts of AChboth at the peripheral nervous system and at theCNS. This secretion activates fast nicotinic AChreceptors, which allows an influx of Na+and Ca2+ ions

Journal of Molecular NeuroscienceCopyright © 2006 Humana Press Inc.All rights of any nature whatsoever are reserved.ISSN0895-8696/06/30:197–200/$30.00JMN (Online)ISSN 1559-1166DOI 10.1385/JMN/30:1-2:197

ORIGINAL ARTICLE

to the cell. Depolarization caused by this influx acti-vates, among others, voltage-dependent Ca2+ chan-nels. The increase in intracellular Ca2+ acts on cAMPresponse–binding elements (CREB) of early tran-scription genes, such as c-fos (Ghosh et al., 1994). Dif-ferent proteins involved in ACh metabolism includec-fos response sites in their promoters, and their tran-scription was found to be up- or down-regulatedshortly after the initiation of this cascade (Kaufer et al., 1998), supporting the notion that they can coun-terbalance cholinergic hyperexcitation. Prominentamong these expression changes is the selectiveinduction of the AChE isoform AChE-R, with simi-lar catalytic capacity, yet greater mobility, than itscounterpart synaptic AChE (AChE-S). In transfectedcells, stimulation of cholinergic receptors inducesAChE transcription. In hippocampal slices, analtered ratio between tetrameric and monomericAChE forms was observed under such stimulation(Kaufer et al., 1998; Soreq and Seidman, 2001).

Converging findings support the hypothesis ofCa2+ modulation, though not necessarily mediatedby ACh receptors, as a critical component in AChEregulation. Null mice devoid of L-type calcium chan-nels, which increase intracellular Ca2+ followingmembrane depolarization, show decreases in AChEmRNA and AChE activity (Luo et al., 1996); and in-activation of this channel by its antagonist nifedipinecauses AChE decreases in different tissues.

Amonomeric form of AChE, presumably AChE-R,was reported to be released from hyperexcited sub-

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stantia nigra slices following different treatments(Llinas and Greenfield, 1987), apparently as a con-sequence of intracellular Ca2+ modifications. Extra-cellular AChE was reported to enhance Ca2+

potentials in the presence of NMDAantagonists andto inhibit them in the presence of NMDA (Bon andGreenfield, 2003), suggesting that it retrieves homeo-stasis following exposure to destabilizing stressinsults.

NMDA receptor effects on AChE-R might bedirect, through the action of CREB motifs in theACHE promoter, or involve c-fos up-regulation orother activating elements on the promoter, e.g., gluco-corticoid response elements (Grisaru et al., 2001;Meshorer et al., 2002). A single-nucleotide substitu-tion, or 4-bp deletion, within this promoter regioncorrelates with intensified susceptibility to environ-mental AChE inhibitors (AChE-I) (Shapira et al., 2000;Benmoyal-Segal et al., 2005). Both the cholinergichyperexcitation that follows psychological stress andthe parallel outcome of AChE blockade thus recruitthis robust reaction.

At a behavioral level, an increase of AChE-RmRNA is observed, with a corresponding increasein AChE-R staining at hippocampal CA1 neurons,up to 3 h after confined swim (Kaufer et al., 1998),following 1 h immobilization stress (Nijholt et al.,2004) or 45 min after exposure to AChE-I (Meshoreret al., 2002).

Putative Roles of the AChE-R SignalAn early stress signal would predictably induce

cellular and organismal reactions to adapt to the newsituation. Experimental modulation of the AChE-Rsignal, both in vivo and in vitro, served to explorethese corresponding processes, many of which yetremain to be delineated.

First and foremost, defining AChE-R as a stresssignal predicts that it will be a sufficient cause forinducing various stress symptoms and that itsabolishment will prevent the emergence of thesesymptoms under stressful situations. Recent find-ings, achieved by different methods, confirm andextend these predictions. Transgenic mice over-producing human AChE-R show an increasedlatency to descend to an open field following a stress-ful intraperitoneal injection (Birikh et al., 2002), andserum AChE elevation, compared with the predictedlevel based on gender, age, body mass index, andethnic origin predicts the transient level of anxietyreactions measured in tested volunteers (Sklan et al.,

2004). Progressive muscle malfunctioning (Farchi et al., 2003), progressive male infertility (Mor et al.,2001), and locomotion abnormalities (Cohen et al.,2002) represent characteristic stress symptoms inother tissues of AChE-R overexpressors.

One of the distinctive effects of acute stress situ-ations, also characteristic of post-traumatic stressdisorder, is a prolonged state of hyperarousal thataffects, among other cognitive functions, the for-mation of new fear memories. Contextual fear learn-ing facilitated 2 and 3 h after exposure to a stressfulaversive stimulus was abolished in animals injectedwith mEN101, an antisense agent inducing selectiveAChE-R mRNA down-regulation (Nijholt et al.,2004). Hippocampal slices of stressed mice, but notmEN101-treated ones, showed enhanced -burst-induced long-term potentiation (LTP) when com-pared with those of naïve mice. This supports thenotion that the cholinergic system operates as a facil-itator of memory consolidation and that AChE-I-mediated improvement in different task-learningparadigms (Shapira et al., 2001) (e.g., in animal exper-iments or in Alzheimer’s disease patients) involvesAChE-R overproduction induced by this treatment.

AChE Functioning at the Molecular LevelChanges in ACHE gene expression appear to be

casually involved in neuronal processes associatedwith rapid changes in Ca2+ concentrations (Day andGreenfield, 2002). However, the mechanismsthrough which these changes occur are not com-pletely understood. The intuitive appealing expla-nation of increased membrane excitability as aconsequence of altered cholinergic transmission hasbeen found to be insufficient to explain AChE trophicfunctions. Although catalytic site blockers, such asechothiophate and galanthamine, failed to inhibitAChE trophic functions, peripheral blockers, suchas BW284c51, succeeded in doing so (Layer et al.,1993). Moreover, the ACh hydrolyzing enzymebutyrylcholinesterase did not show such trophicimplications.

Different parts of the AChE molecule have shownto emulate the trophic functions of the intact pro-tein; however, a unifying theory explaining thisdiversity of results has not yet been proposed. Ashort-amino-acid sequence encoded by the E6 exon of theAChE-S mRNA transcript, which is positioned faraway from the peripheral active site targeted byBW284c51, has been shown to produce neuritegrowth, apoptosis, and necrosis (Day and Greenfield,

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2002) in cultured hippocampal neurons, dependingon concentration and exposure times.

A second candidate for trophic effects is the HNK-1epitope, common both to the AChE-R and AChE-Svariants and involved in cell adhesion. This sequenceis located close to the AChE peripheral site at therim of the catalytic gorge. HNK-1 epitope effects areinhibited by the peripheral AChE-I BW284c51(Munoz et al., 1999).

A third sequence related to trophic effects is theAChE-R carboxyl terminus (ARP51 and mARPpeptides). The ARP51 and mARPpeptides, composedof the C-terminal 51- and 26-amino-acid residues ofAChE-R, showed trophic functions. Thus, mARPreproduced the effects of acute stress in the enhance-ment of fear conditioning in mice, behavior that isassociated with LTPfacilitation in hippocampal slices(Nijholt et al., 2004). Natural cleavage of the mARPpeptide in brain (Dori et al., 2005) suggests that itmight mediate this facilitation after stress. In two-hybrid yeast screening, ARP51 interacted with blades5 and 6 of the scaffold protein RACK1. Coimmuno-precipitation and double-labeling immunofluores-cence confirmed AChE-R/RACK1 interactions invivo (Birikh et al., 2002). RACK1 has been found tomediate phosphorylation of the NR2B subunit of theNMDA receptor by the protein tyrosine kinase Fyn(Yaka et al., 2002). This cascade determines mem-brane insertion and channel opening of NMDAreceptors (Dong et al., 2004). The AChE-R/RACK1complex might also modulate NMDA receptors byits regulation of protein kinase C (II) (PKC- [II])activity (Dong et al., 2004). PKC- (II) was found tobe involved in fear conditioning and to interact withNMDA receptors, altering Ca2+ currents (Lu et al.,2000). Importantly, RACK1 interacts with numerousother protein partners, some of which might also berelevant to stress-induced reactions. For example,RACK1 interaction with the IP3 receptor wasreported to control intracellular Ca2+ concentrations(Patterson et al., 2004). Competition by AChE-R onRACK1 interactions might thus affect stress reactionsthrough more than one pathway.

In summary, recent research points toward AChE-R as a stress signal. One of the ways throughwhich this signal conveys its message is by modi-fying, probably through indirect interactions withthe NMDA receptor, intracellular Ca2+ concentra-tions. AChE-R-induced Ca2+ modification mightplay a central role in the consolidation of fearmemory formation, as well as in stress-relatedapoptosis.

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