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

REVIEW

Termination and beyond: acetylcholinesterase as 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 neurotransmitter hydrolysis is a substantial characteristic of cholinergic synapses. This unique termination mechanism makes acetylcholinesterase (AChE), the enzyme in charge of executing acetylcholine breakdown, a key component of cholinergic signaling. AChE is now known to exist not as a single entity, but rather as a combinatorial complex of protein products. The diverse AChE molecular forms are generated by a single gene that produces over ten different transcripts by alternative splicing and alternative promoter choices. These transcripts are translated into six different protein subunits. Mature AChE proteins are found as soluble monomers, amphipatic dimers, or tetramers of these subunits and become associated to the cellular membrane by specialized anchoring molecules or members of other heteromeric structural components. A substantial increasing body of research indicates that AChE functions in the central nervous system go far beyond the termination of synaptic transmission. The non-enzymatic neuromodulatory functions of AChE affect neurite outgrowth and synaptogenesis and play a major role in memory formation and stress responses. The structural homology between AChE and cell adhesion proteins, together with the recently discovered protein partners of AChE, predict the future unraveling of the molecular pathways underlying these multileveled functions.

G. Zimmerman : H. Soreq (*) The Institute of Life Sciences and the Interdisciplinary Center for Neural Computation (ICNC), The Hebrew University of Jerusalem, Jerusalem 91904, Israel e-mail: [email protected]

Keywords Acetylcholinesterase . Synaptic transmission . Alternative splicing . Alternate promoters . Synapse adherence

Introduction Most neurotransmitters are removed from the synaptic cleft by reuptake. This is the case for dopaminergic, noradrenergic, 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 first suggested in 1914 (Dale 1914) and was subsequently confirmed experimentally by the application of the AChE inhibitor physostigmine to frog heart muscle, which prolonged the effects of ACh on it (Loewi and Navratil 1926). Later utilization of simple separation methods revealed a broad family of cholinesterases and the existence of several catalytically active AChE isoforms that could be distinguished by velocity sedimentation, gel electrophoresis, and their different solubility in diverse buffers, allowing the individual biochemical characterization of each of the forms. Molecular cloning of the ACHE genes from Torpedo californica (Schumacher et al. 1986, 1988) and humans (Soreq et al. 1990; Ben Aziz-Aloya et al. 1993) preceded the determination of the three-dimensional structure of the enzyme (Sussman et al. 1991) and the later identification of the anchoring molecules linking the diverse AChE multimeric forms to the synaptic membrane (Krejci et al. 1991; Perrier et al. 2002). However, subsequent discoveries of an AChE isoform induced by alternative splicing (Kaufer et al. 1998; Meshorer et al. 2002) and involved in diverse stress responses (Birikh et al. 2003; Nijholt et al. 2004) further

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extend previous reports (Layer 1996; Inestrosa and Alarcon 1998; Day and Greenfield 2002; Soreq and Seidman 2001) asserting that AChE functions go far beyond the termination of synaptic transmission.

AChE structure Human AChE is composed of an invariable core of 534 amino acids and a variable C-terminal peptide of 14, 26 or 40 amino acids (Meshorer and Soreq 2006). Alternative promoter choices allow the extension of each of these variants with a recently found extended N-terminal peptide of 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 Å and according to its structure belongs to the alpha/beta hydrolase superfamily. The catalytic domain of AChE is composed of a serine-histidine-glutamate triad, which basically resembles the catalytic site of other serine proteases, except that in this case the acidic residue is glutamate instead of the usual aspartate. Surprisingly, X-ray crystallography has revealed the catalytic triad to be located at the bottom of a 20-Å-long gorge, which penetrates halfway into the protein from its surface (Sussman et al. 1991). This “active site gorge” is flanked by 14 aromatic residues located at the loops between different beta-sheets. A “peripheral anionic site” (PAS) composed of five residues is clustered at the entrance of the gorge and is in turn surrounded by ten acidic residues called the “annular electrostatic motif” (Felder et al. 1997) (Fig. 1).

ACh hydrolysis by AChE The extraordinary turnover rate of AChE (about 100 μs for one substrate molecule; Lawler 1961) impelled biochemists to hypothesize about and analyze the mechanisms of action of the enzyme. Elucidating the crystal structure of AChE revealed a noticeable bipolar distribution of charges on the enzyme surface, with the anionic pole surrounding the entrance to the gorge, and the cationic pole being located at its bottom (Sussman et al. 1991). This evolutionarily conserved feature was initially hypothesized to attract the positively charged substrate toward the entrance of the gorge; however, neutralization by mutagenesis of seven charged residues around the gorge did not alter the hydrolytic rate of AChE (Shafferman et al. 1994). At the gorge entrance, the substrate transiently binds to the PAS (Szegletes et al. 1999), presumably inducing structural changes (Kitz et al. 1970) that may facilitate ACh passage through the gorge. Once inside the gorge, the aromatic groups located around its border guide the substrate from

Fig. 1 Three dimensional structure of Torpedo californica AChE-S (PDB 1GQS; Bar-On et al. 2002). The folded structure of the molecule is presented with the three residues of the catalytic site in red 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 and the anionic residues at its entrance. The helical region leading to the variable C-terminus is given in green and the N-terminus, which may be extended by the “N” peptide, in orange

the protein surface to the active site, where the anionic subsite binds choline and positions the ester at the acylation site. After the serine displaces choline from the substrate, thereby forming an acetyl-enzyme intermediate, the acetate group is liberated by a hydrolysis step. The mechanism by which the acetyl and choline molecules produced by this reaction 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 unequivocal experimental proof for this hypothesis is still lacking. The breakdown of ACh at the synapse immediacies allows the produced choline to be re-uptaken by the synaptic terminal, where it can be reused for further ACh assembling. Intriguingly, the release of a single ACh vesicle from the presynaptic button suffices to saturate all cholinergic receptors and cholinesterases in a diameter of 0.5 μm from the release site (Bartol et al. 1991). Since the binding of ACh to its receptor sites is much faster than the rate at which it is hydrolyzed by AChE, the amount of ACh molecules that will initially bind to synaptic receptors is determined by the ratio of receptors to esterase: roughly 20% esterase and 80% receptors (Anglister et al. 1994). The rate at which ACh is liberated from the activated receptors is slower than the ACh hydrolysis rate, so that, when ACh molecules are released, most AChE units are free and capable of binding a “new” ACh molecule, maintaining in this way a low ACh concentration in the cleft and virtually excluding the possibility of a single ACh molecule activating another receptor. These dynamics explain why the inhibition of AChE has a more acute effect on the duration of the excitatory post-synaptic potential (EPSP) than on its amplitude (Collier and Katz 1971).

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The enzymatic rate of AChE is lowered under excessive substrate concentration. The binding of a second ACh molecule to the PAS apparently slows the deacetylation step or the formation of the induced fit complex during hydrolysis (Krupka 1963; Rosenberry 1975). Mutations at Asp-74 and Trp-286, which lower PAS ligand affinity, dramatically reduce substrate inhibition (Shafferman et al. 1992), supporting the importance of the PAS in such processes. Environmental conditions have also been shown to contribute to changes that influence substrate inhibition. For example, AChE extracted from mosquito shows no substrate inhibition, but when treated with thiocyanate, which converts the enzyme back to its non-amphiphilic derivatives, substrate inhibition returns (Dary and Wedding 1990).

AChE combinatorial complexity AChE is found in an assorted array of forms attained by alternate promoter usage of its gene, alternative splicing of nascent pre-AChE mRNA transcripts, multimerization of individual AChE subunits, and their association with different anchorage or partner proteins. Alternate promoter usage and alternative splicing together modify the 5′ and 3′ termini of AChE mRNA, allowing the production of six protein subunits with different N- and C-termini. Three different 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 join the two different N-termini to yield variants with the common or the “extended” N-terminus (Meshorer et al. 2004; Fig. 2). Whereas the catalytic domain of all AChE isoforms remains invariable, the characteristics of these different terminal peptides alter several key features of the protein. A prominent characteristic of AChE-S, the most abundant form in the nervous system, is a C-terminal cysteine residue located three amino acids from the end of the protein. This cysteine residue allows disulfide bonding with other AChE-S units, giving rise to amphipathic homodimers and homotetramers. Beyond its different amino-acid sequence, the AChE-R C-terminus differs from that of AChE-S in two obvious characteristics: (1) it is hydrophilic, and (2) since it lacks the C-terminal cysteine residue found in the AChE-S C-terminus, it cannot tetramerize by disulfide bonding with other subunits. This produces a soluble monomeric molecule, instead of the amphipathic tetramers composed of dimers. AChE-S tetramers associate with one of two membraneanchoring molecules, which partially determine the synaptic localization of the protein: collagen Q (ColQ) in neuromuscular junctions, and a proline-rich membrane

Fig. 2 ACHE gene structure and protein products. a Exon-intron structures of mouse and human ACHE genes (cylinders exons, lines introns, lines above genes splicing options). Several alternative transcripts may be obtained by alternative splicing of pre-AChE mRNA; six different protein subunits are derived from them. b Alternative protein products of the ACHE gene. AChE-S units can dimerize, tetramerize, or remain as monomers. AChE-S tetramers can remain soluble or become anchored to the membrane by the molecules ColQ or PRiMA. AChE-R inherently remains as a soluble monomer. AChE-E forms glypiated dimers linked to the red blood cells membrane

anchor (PRiMA) in brain synapses (Massoulie et al. 1999). ColQ units homotrimerize by forming a triple helical structure in a proline-rich domain at their C-terminus (Bon and Greenfield 2003; Bon et al. 2003). Since each ColQ can attach an AChE tetramer, hetero-oligomeric complexes may contain four, eight, or 12 AChE subunits, designated A4, A8, or A12, respectively. The glycoprotein PRiMA, in turn, induces the formation of AChE homotetramers and attaches them by a proline-rich motif, keeping them anchored to the cell membrane by a transmembranal domain (Perrier et al. 2002). PRiMA-anchored forms account for 70%–90% of total AChE activity in the central nervous system (CNS; Grassi et al. 1982), the other 10%– 30% being accounted for by asymmetric forms and, in nonstress conditions, by 1% of AChE-R (Perrier et al. 2005). PRiMA-associated AChE tetramers are traditionally designated as G4 (for globular), and monomers and homodimers not associated to any anchorage protein are designated as G1 and G2, respectively (Fig. 2b); however, it is important to remember that the G classification preceded the discovery of PRiMA by two decades. Moreover, a

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significant part of the nascent AChE molecules may never gain catalytic activity (Rotundo 1988), which implies that the traditional ways of quantifying and characterizing AChE isoforms might not have revealed important information. AChE-R lacks a hydrophobic domain and is incapable of binding 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-terminal signal peptide (Soreq et al. 1990) in the rough endoplasmic reticulum, in which they are co-translationally glycosylated, some then being assembled as dimers or tetramers (Rotundo 1988). The non-multimerized units remain as globular monomers and are rendered inactive by as yet unclear post-translational modifications. A retention signal in their C-terminal peptide retains monomers in the cytoplasm (Velan et al. 1991), in which many of them are rapidly degraded (Rotundo and Fambrough 1980). The oligomeric forms transit to the Golgi apparatus, where they acquire complex sugars (Rotundo 1984) and are later assembled into asymmetric forms with ColQ or PRiMA (Rotundo et al. 2005). More recent data suggest that asymmetric heteromeric assembly takes place in the endoplasmic reticulum (Massoulie et al. 1998). In muscle fibers, the newly assembled ColQ-AChE associates intracellularly with the proteoglycan perlecan and is then externalized to the neuromuscular junction (NMJ), where it colocalizes with other components (Rotundo et al. 2005). The role played by PRiMA in AChE targeting within in the CNS is not yet known. Of note, although PRiMA is ubiquitously expressed, it appears to interact with AChE only at CNS synapses. Non-multimerized globular monomers can be secreted from various cell types (Legay et al. 1999); nevertheless, most of the secreted enzymes are attached to an anchorage protein, and the pool of monomeric subunits that is not recruited by them undergoes degradation. Muscle AChE-S finally appears at the surface of the cells about 2.5 h after its synthesis and has a half-life of about 50 h. This time lapse is highly reproducible in cells from different species or origins (Rotundo et al. 1988). In the brain, however, stress insults induce a rapid increase of secretory AChE-R, with a two-fold difference by 30 min (Kaufer et al. 1999). AChE localization AChE is widely expressed in tissues that receive cholinergic innervation, such as neurons, muscle cells, and cells of

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the autonomic nervous system and of the immune nervous system. Interestingly, AChE expression patterns are not always correlated with that of the more faithful cholinergic marker, choline acetyltransferase (ChAT), the enzyme that synthesizes ACh. AChE activity is also found in brain regions with low or no cholinergic inputs, such as the substantia nigra, cerebellum, globus pallidus, and hypothalamus, and in many non-nervous tissues devoid of any known cholinergic innervation. These include testis (Mor et al. 2001), endothelial cells (Deutsch et al. 2002), hematopoietic (Grisaru et al. 2006) and osteogenic cells (Grisaru et al. 1999), and various tumors (Karpel et al. 1994; Perry et al. 2002). AChE is also broadly expressed in nervous tissue before synaptogenesis (Layer 1990; Dori et al. 2005), i.e., before cholinergic transmission is established. The isoformal composition of AChE is probably determined by the different functional characteristics of the diverse synapses at which the enzyme is present. This can initially be divided into two large, rather heterogeneous groups: the cholinergic synapses 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 and short-term, secretion at cholinergic synapses in the CNS is diffuse and has a long-term modulatory character. Moreover, the different anatomical characteristics of these synapses (the former being much more spacious and containing collagens and a basal lamina) appear to determine the isoform types encountered in them. The ColQ subunit is mainly synthesized in muscle cells and attaches AChE oligomers to the basal lamina of the NMJ, between the pre- and post-synaptic membranes, where it represents most of AChE active forms. Rat fast muscles contain exclusively the A12 form, whereas slow muscles contain a higher proportion of the A8 and A4 forms (Sketelj et al. 1992). The PRiMA-anchored tetrameric form appears to be more suited to the smaller synaptic cleft found in the CNS (Legay 2000), where the lack of a basal lamina would hamper ColQ anchoring. At central synapses, anchored forms account for 70%–90% of AChE total activity, whereas asymmetric forms represent less than 3% of this activity. Anchored tetramers are expressed also in fast muscles, in which their concentration is increased or decreased according to the respective physiological exercise (Jasmin and Gisiger 1990; Gisiger et al. 1994) and pathology (Rakonczay et al. 1991). The usually rare variant AChE-R has been found in all studied mammals and is coexpressed with AChE-S, albeit in a distinct neural distribution pattern (Sternfeld et al. 2000) contributing about 1% of total AChE activity under various physiological states (Perrier et al. 2005). During embryogenesis AChE is uniformly distributed in muscle 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 at the endoplasmic reticulum, probably as a precursor pool of heteroligomeric forms (Massoulie 2002). However, they can also be secreted without being attached to any membrane anchor protein.

AChE polymorphisms The marked identity between the human and mouse genomic ACHE sequences and the almost identical crystal structure suggest the importance of virtually each single domain of this enzyme for its proper functioning. Nucleotide polymorphisms in the ACHE gene were considered rare (Soreq and Seidman 2001) until a recent comprehensive study that found AChE polymorphic prevalence at several sites (Hasin et al. 2005). Three identified AChE polymorphisms have been shown to have biological implications and clinical relevance: a polymorphism in the distal promoter of AChE affecting interaction with a glucocorticoid response element (GRE; Shapira et al. 2000), a 4-bp deletion in a hepatocyte nuclear factor 3 (HNF3)-binding site also located at the promoter region (Shapira et al. 2000), and a single nucleotide polymorphism (SNP) producing the His322 Asn substitution, which is responsible for the YT-2 blood group phenotype (Bartels et al. 1993; Ehrlich et al. 1994). Specific but rare polymorphisms at the ACHE/paraoxonase (PON1) locus have further been found to be correlated with hypersensitivity to AChE inhibitors (Shapira et al. 2000), insecticide-induced Parkinson’s disease (PD; Benmoyal-Segal et al. 2005), and trait anxiety (Sklan et al. 2004). Individuals carrying the rare GRE and the HNF3-binding-site polymorphisms show increased constitutive AChE expression and hypersensitivity to AChE inhibitors (Shapira et al. 2000). A later study has indicated a more complex interaction between the HNF3-binding-site polymorphism and PON1 activity as determinants of AChE serum activities (Bryk et al. 2005). Similarly to the carriers of these polymorphisms, TgS transgenic mice overexpressing AChE-S exhibit hypersensitivity to AChE inhibitors (Shapira et al. 2000), suggesting that high basal AChE levels impede further transcriptional activation, which is necessary to overcome exposure to such compounds. The enzyme PON1 degrades organophosphate compounds, which are potent inhibitors of AChE and are notably involved in insecticide-induced PD (Gorell et al. 1998; Kondo and Yamamoto 1998). A recent study aimed at assessing the relationship between polymorphisms in the ACHE/PON1 locus and insecticide-induced PD found a non-significant association between the HNF3-binding-site

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promoter deletion and the pathology. However, when the ACHE and PON1 gene polymorphisms were jointly considered, the debilitated alleles were found to be overrepresented in insecticide-induced PD patients, who showed lower levels of AChE and PON1 activities (Benmoyal-Segal et al. 2005). A synonymous SNP at position P446 of the ACHE gene coding region, present in about 12% of the population (Bartels et al. 1993), has been found to be more frequent in individuals with high levels of trait anxiety compared with those presenting low levels of trait anxiety (Sklan et al. 2004). This SNP is not associated with altered AChE activity in serum (Sklan et al. 2004) and therefore probably reflects an associated change in an as yet undefined adjacent gene(s). A recent study aimed at identifying SNPs in four different populations has found a total of five coding nonsynonymous, three coding synonymous, and five noncoding ACHE SNPs among 96 studied individuals. The five coding SNPs found affect only evolutionarily non-conserved amino acids, which are either located far from the active site or not present in the mature protein (Hasin et al. 2005). No physiological implications have yet been assigned to the new SNPs found in this study.

AChE regulation Several studies indicate that the AChE isoform pattern is regulated by the degree and character of synaptic activity and by some other stimuli with no known relationship to cholinergic transmission. Denervation of mammalian skeletal muscle and the blocking of spontaneous contraction by tetrodotoxin (TTX) have been shown to decrease AChE activity and the appearance of asymmetric forms at the cell surface (Lomo and Slater 1980; Cangiano et al. 1980; Michel et al. 1994; Rossi et al. 2000). This is accompanied by a transient increase in G4 (Gregory et al. 1989), but without altering the G1 and G2 isoforms (Fernandez and Hodges-Savola 1992). The lack of membrane depolarization also negatively affects ColQ expression (Massoulie 2002), which in turn downregulates the rate of heterotetramer assembly and release, thus enlarging the intracellular G4 pool. The conservation of G1 and G2 levels indicates that their expression is controlled by spontaneous ACh release (Fernandez and Hodges-Savola 1992), which is not blocked by TTX. Denervation downregulatory effects can intriguingly be reversed by ectopic reinnervation, the application of neurogenic proteinaceous substances (Fernandez and Hodges-Savola 1992), direct electrical stimulation (Lomo and Slater 1980), treatment with Na+ channel agonists (Rossi et al. 2000), and increase in cytoplasmic 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 distinct AChE compositions may be altered by cross-innervating these muscles or by altering the mode of stimulation imposed upon them (Lomo et al. 1985). Treadmill exercises produce a marked increase in G4 forms in fast, but not low, twitch muscles (Fernandez and Donoso 1988). This may reflect the finding that fast muscles are more readily affected by training than slow ones. A transient increase in AChE has also been detected in the various layers of primary sensorial cortices (Robertson et al. 1985; Robertson 1987) reaching an expression peak at around 2 weeks after birth and attaining adult expression patterns 1 week later. Although enucleation and TTX intraocular injection abolish AChE expression in the primary visual cortex (Robertson et al. 1989), it does not alter ChAT staining in this cortex, suggesting that AChE is not directly related to cholinergic efferents and that it might have roles other than terminating cholinergic transmission during development. Experimental induction of autoimmune myasthenia gravis (MG) in rats stimulates the overexpression of AChE-R, but not of AChE-S, in muscles (Brenner et al. 2003). Destruction of the AChE-R mRNA transcripts by antisense oligonucleotides facilitates muscle action potentials, physiological stamina, and weight gain in experimentally ill rats (Brenner et al. 2003). In antisense-treated monkeys, AChE-R mRNA levels are also selectively decreased in spinal cord neurons, but conferred no movement changes (Evron et al. 2005). Thus, excess, but not normal low, levels of AChE-R change neuromuscular transmission. The composition of AChE in the CNS is subject to dynamic changes under various cellular and physiological stimuli. A gradual AChE increase accompanied by an isoformal shift from G1 to the adult G4 form during embryogenesis has been broadly reported in many organisms (Inestrosa et al. 1994; Anselmet et al. 1994). Reciprocally, the loss of cholinergic circuits characteristic of Alzheimer’s disease (AD) is reflected in reduced AChE activity accompanied by increased monomer fractions in the cerebrospinal fluid (CSF) and in various brain nuclei. These changes are correlated with the clinical severity of the disease, suggesting physiological relevance (Arendt et al. 1992; Darreh-Shori et al. 2004). Interestingly, several AD brain areas show a selective loss of tetrameric AChE (Schegg et al. 1992; Fishman et al. 1986), but the asymmetric and G1 forms are upregulated (Younkin et al. 1986), greatly reducing the ratio between G4 and G1 in various brain regions (Arendt et al. 1992). In AD patients treated with anti-AChEs, an increase in the G1 isoform has recently been shown to represent AChE-R upregulation (Darreh-Shori et al. 2004).

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In healthy mammals, AChE-R is markedly upregulated in the cortex, hippocampus, striatum, and cerebellum of animals 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 the splicing factor SC35 occurring during stress, an increase sustained as long as several weeks after the stress event (Meshorer et al. 2005). A similar AChE-R increase is also produced by corticosterone treatment, organophosphate poisoning, and AChE inhibition by the carbamate antiAChEs 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 did not correlate with the presence of ACh (Greenfield 1991; Appleyard and Jahnsen 1992; Layer 1983) initiated the hypothesis that AChE possessed non-catalytic properties. AChE was found to be released from nigral dopaminergic neurons independently of cholinergic activation (Llinas and Greenfield 1987), in addition to following electrical stimulation (Greenfield and Smith 1979) and intracellular Ca2+ release (Greenfield et al. 1983). Later experiments suggested that released AChE played neuromodulatory functions independent of its catalytic activity and was capable of affecting complex behavioral patterns. Exogenous administration of AChE-S to the striatum of freely behaving rats induced an increased availability of extracellular dopamine, as indicated by overactivation of the nigrostriatal pathway and circling behavior (Hawkins and Greenfield 1992). In vitro studies in various brain areas were aimed at characterizing in more detail the electrophysiological characteristics of AChE. Application of AChE to midbrain slices resulted in marked membrane hyperpolarization and decreased input resistance, even when co-administered with its irreversible catalytic inhibitor soman (Greenfield et al. 1988). This attributed the effect to non-enzymatic mediation. An opposite result was obtained by applying AChE to cerebellar slices, in which the enzyme enhanced the response of Purkinje cells to glutamate and aspartate, facilitating climbing fiber stimulation (Appleyard and Jahnsen 1992). AChE-S application to guinea pig hippocampal slices induced a long-term potentiation (LTP)-like effect in CA1 neurons, as indicated by increased EPSPs and increased population spikes evoked by stimulation of Schaffer collateral fibers (Appleyard 1995). The effects persisted after treatment with the cholinergic antagonists atropine and mecamylamine, suggesting 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 different levels and modify its isoformal composition (EDL extensor digitorum longus muscle, SOL soleus muscle, TTX tetrodotoxin) Manipulation

Outcome

Reference

Denervation of vertebrate skeletal muscles

Upregulation of AChE transcripts after 10 days in avian fast skeletal muscle Downregulation of AChE transcripts after 10 days in rodent fast and slow skeletal muscle Decrease in AChE in spinal cord Preservation of the different AChE isoformal patterns in fast and slow muscles Transient increase in G4 form (24–60 h) in fast muscles, and decrease in all other forms Reduction in AChE activity in fast and slow muscles (14–42 days) and in NMJ Replacement of each muscle characteristic isoformal pattern to that of the other muscle

Rimer and Randall 1999

Cross innervation of fast EDL muscle with slow SOL nerve and vice versa Fast and slow electrical stimulation

Electrical stimulation, incubation with nicotine, but not with histamine or angiotensin II Muscle paralysis by TTX or botulinum toxin

Ca2+ ionophore treatment in TTX paralyzed muscles L-type Ca2+ channel blockers ryanodine and nifedipine, but not the N-type channel blocker ω-conotoxin L-type Ca2+ channel knockout mice High level of K+ in medium Injection of calcitonin gene-related peptide

Treatment with nerve growth factor

Treatment with ciliary neurotrophic factor

Induction of AChE pattern reminiscent of that of EDL in denervated SOL after fast stimulation. Preservation of SOL characteristic AChE pattern in denervated SOL after slow stimulation. No changes in denervated EDL after fast or slow stimulation Two-fold increase in AChE secretion from adrenal gland

Immediate decrease in AChE transcripts lasting for 10 days in rodent fast and slow skeletal muscles Decrease in AChE activity in rodent muscle culture Decrease in AChE activity, mainly in asymmetric forms in quail myotubes culture Induction of slow muscle isoformal pattern in both fast and slow rodent skeletal muscles Increase in AChE activity in cultured avian muscle cells Preservation of AChE activity and increase in its secretion in cultured avian muscle cells Increase in asymmetric AChE concentration Decrease in activity of all forms after 3 h in primary avian pectoral muscle tissue Inhibition of myotube-differentiation induced stabilization of AChE mRNA Decrease in AChE mRNA and activity in skeletal muscle Increase in AChE release from N18TG2 and 108CC15 cell lines Abolition of denervation-caused G4 increase. Preservation of other forms of denervation-caused decrease Reduction in all isoforms in normally innervated rodent skeletal muscle Reduction in AChE transcription in cultured muscle cells Increase in AChE transcription rate in PC12 cells Increase in G4 release to medium from PC12 and chick optic lobe cells Decrease in AChE in denervated, but not intact, adult rat skeletal muscle

Michel et al. 1994 Tsim et al. 1997 Sketelj et al. 1991 Gregory et al. 1989; HodgesSavola and Fernandez 1991 Lomo et al. 1985 Dolenc et al. 1994

Lomo and Slater 1980; Lomo et al. 1985

Small et al. 1993

Michel et al. 1994; Cresnar et al. 1994 Rubin 1985 Fernandez-Valle and Rotundo 1989 Boudreau-Lariviere et al. 1997 Walker and Wilson 1976 Vallette and Massoulie 1991 Rubin 1985 Decker and Berman 1990 Luo et al. 1994 Luo et al. 1996 Biagioni et al. 1995 Hodges-Savola and Fernandez 1995 Fernandez et al. 1999 Rossi et al. 2003 Greene and Rukenstein 1981; Deschenes-Furry et al. 2003 Lucas and Kreutzberg 1985 Boudreau-Lariviere et al. 1996

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Table 1 (continued) Manipulation

Outcome

Reference

RNA-binding protein HuD overexpression Myogenic transcription factors

Stabilization of AChE mRNA in PC12 cells

Deschenes-Furry et al. 2003

Preservation of AChE transcription rate and mRNA stability in cultured fibroblasts Reduction in AChE transcription in 10T1/2 and 10TFL2–3 cells Increase in AChE mRNA in C2C12 myotubes

Mutero et al. 1995

Increase in AChE expression in chicken cultured myotubes Reduction in AChE expression in cultured myotubes Increase in AChE expression in cultured chicken myotubes

Choi et al. 2001a,b

Increase in AChE protein in differentiated myotubes Increase in levels of AChE mRNA, protein, and activity in P19 cells Increase in AChE activity and neurite outgrowth induction in neuroblastoma cell line Inhibition of G4 release to the medium from PC12 and from chick optic lobe cells Inhibition of G4 release to the medium from PC12 and from chick optic lobe cells Reduction in AChE by 70% and preservation of 4S and 6S forms synthesis

Tung et al. 2004 Coleman and Taylor 1996 Sidell et al. 1984

Egr-1 treatment Ephrin A1 application, which activates Stat3 cAMP application P2Y1 receptor activation by ATP, ADP, 2-MeSADP, and 2-MeSATP P2Y2 receptor activation by UTP Retinoic acid treatment, which commits P19 cells to neurons and glia Microtubule inhibitors application (colchicine, taxol) Protein glycosylation inhibitor application (tunicamycin) Reduction of Ca2+ in medium, preventing myoblast fusion in muscle cell line Beta-endorphin application Muscle-preconditioned medium Administration of aluminium and citrate, but not aluminium alone Neostigmine intraventicular injection Cercal nerve ablation Granule cell lesion Combined hypoxia and hypoglycemia (simulated ischemia) Beta-amyloid protein application Donepezil and galantamine administration Infection with nematode parasite Nippostrongylus brasiliensis Experimental induction of myasthenia gravis Repeated session of treadmill exercise

Rapid eye movement (REM) sleep deprivation Confined swim sessions Immobilization stress Perlecan knockout mice Mu-opioid receptor knockout mice Ephrin receptor ephA4 knockout mice

Mutero et al. 1995 Lai et al. 2004

Choi et al. 2001a,b

Lucas and Kreutzberg 1985 Lucas and Kreutzberg 1985 Inestrosa et al. 1983

Decrease in 16S form and increase in 6S and 4S forms in myotube cultures Decrease in AChE activity in rodent sympathetic neurons Increase in AChE in various mouse brain areas

Swerts et al. 1984 Kaizer et al. 2005

Upregulation of AChE-R transcripts in rodent brain

Meshorer et al. 2002

Loss of AChE activity in cercal nerves and terminal ganglion in cockroach AChE increase in molecular layer of rodent dentate gyrus AChE increase in rodent hippocampal slice cultures

Sekhar et al. 1991 McKeon et al. 1989 Saez-Valero et al. 2003

Increase in AChE in rodent primary cortical neurons Increase in AChE in CSF of AD patients

Fodero et al. 2004 Davidsson et al. 2001

Development of discrete foci of intense AChE activity in basal membrane of jejunal mucosa Increase in AChE-R expression in rodent muscle and serum

Russell et al. 2000

Increase in G4 in fast muscles having a dynamic role during exercise. Decrease in G4 in fast muscles having tonic activity during training Small decrease in G4 in slow muscles Increase in AChE activity in several rodent brain areas

Jasmin and Gisiger 1990

Increase in AChE-R expression in diverse rodent brain areas

Kaufer et al. 1998; Meshorer et al. 2002 Nijholt et al. 2004 Arikawa-Hirasawa et al. 2002 Tien et al. 2004 Lai et al. 2004

Increase in AChE-R expression in diverse rodent brain areas Preservation of AChE expression, but without localization to NMJ Increase in AChE activity in striatum, but not in cortex or hippocampus Decrease in AChE expression in skeletal muscle

Haynes et al. 1984

Brenner et al. 2003

Thakkar and Mallick 1991

Cell Tissue Res (2006) 326:655–669

ACh. In order to investigate these phenomena in a simpler scenario, membrane homogenates from rat cortex were incubated with AChE, and the affinity of different a-amino3-hydroxy-5-methylisoxazolepropionate (AMPA) receptor agonists was measured. A dose-dependent increase in the binding of (S)-[3H]5-fluorowillardiine and [3H]AMPA, but not of [3H]kainate, was found after treatment (Olivera et al. 1999). Treatment of neurons with the PAS blocker BW284c51 greatly reduced currents evoked by glutamate, AMPA and N-methyl-d-aspartic acid (NMDA), but not by GABA. PAS blockade increased AChE levels, and impeding this effect by AChE antisense oligonucleotides significantly decreased the induced suppression of glutamate currents (Dong et al. 2004). The interaction of AChE-S and AChE-R with their diverse protein partners may indirectly affect membrane electrophysiological properties, possibly explaining some of the above-mentioned phenomena. Interestingly, splice shift to AChE-R may, under certain conditions, lead to the relocalization and alteration of function(s) of the protein partners of both AChE-S and AChE-R, thus modifying diverse cellular properties. AChE-R, for example, forms complexes with the scaffold protein RACK1 and, through it, with PKC-βII (Birikh et al. 2003; Sklan et al. 2004), which is implicated in synaptic plasticity and memory (Weeber et al. 2000). Transgenic TgR mice overexpressing human AChER exhibit facilitated LTP induction (Nijholt et al. 2004), suggesting profound biological implications for AChE-R/ RACK1/PKC-βII complexes. Like TgR mice, FVB/N strain-matched non-transgenic mice exposed to an acute stress episode show both AChE-R upregulation (Kaufer et al. 1998) and LTP facilitation (Blank et al. 2002), which can be abolished by pretreating the animals with an AChE-R antisense oligonucleotide (Nijholt et al. 2004). Two different short sequences derived from AChE are suggested to be bioactive: a 14-amino-acid peptide located at the AChE-S C-terminus and the 26 most-distal amino acids from the AChE-R C-terminus (Nijholt et al. 2004). The former peptide has been recently shown to modulate NMDA receptor Ca2+ currents (Bon and Greenfield 2003) and α7 nicotinic receptor responses (Greenfield et al. 2004); however, the in vivo existence of this peptide as an 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 the initial observations that a transient peak in AChE activity in postmitotic cells precedes neurite formation by several hours (Weikert et al. 1990; Geula et al. 1995; Dupree and Bigbee 1994) have led researchers to study whether AChE is causally involved in neurite outgrowth. Intriguingly, the

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high amounts of AChE found in the immature embryonic CNS are not accompanied by the appearance of ChAT and precede the establishment of cholinergic transmission, hinting at a non-canonical role for AChE in this scenario. Treatment of different cell cultures with the PAS inhibitors BW254c51, propidium, and fasciculin, but not with the active-site inhibitors echothiophate and galanthamine, induces decreases both in neurite numbers and in their branching (Layer et al. 1993; Dupree and Bigbee 1994; Olivera et al. 2003; Day and Greenfield 2002). This is compatible with the casual involvement of AChE in such processes, indicates a non-catalytic role for AChE, and suggests the PAS as a possible mediator of this process. Diverse primary cultured cells (Bigbee et al. 2000) and cell lines (Sternfeld et al. 1998; Karpel et al. 1996) genetically manipulated to overexpress either AChE-S or a catalytically inactivated form of it (AChE-Sin) show a significant increase in neurite growth, whereas antisense suppression of AChE impairs it (Grifman et al. 1998). AChE application to the medium induces the extension of neuronal processes, synapse formation, and AMPA receptor surface expression in hippocampal cell cultures (Olivera et al. 2003). Some reports claim a more potent role for the embryonic AChE-S monomers than for the adult tetramers (Holmes et al. 1997; Day and Greenfield 2002); however, others contest these observations (De Jaco et al. 2002a,b). Importantly, Xenopus tadpoles overexpressing human AChE-R do not show more extensive neurite outgrowth than control tadpoles, suggesting that AChE membrane attachment is essential to influence such processes. Moreover, ACh modulates neurite growth (Owen and Bird 1995; De Jaco et al. 2002a,b; Zheng et al. 1994), and AChE could exert a trophic role solely by hydrolyzing it. Nevertheless, most studies on neurite outgrowth involve the use of cholinergic antagonists or cell lines incapable of synthesizing ACh, supporting the notion that the observed effects stem from the non-catalytic properties of AChE. Recent investigations indicate that AChE interacts with the basement-membrane protein laminin-1 β in vivo (Paraoanu and Layer 2004; Paraoanu and Layer 2005) and with collagen IV (Johnson and Moore 2003). This interaction is negatively affected by the ionic strength of the medium and by the addition of a monoclonal antibody directed against the PAS (Johnson and Moore 2003). Laminin-1 β is known to interact with integrins, which play a major role in neuronal migration and in CNS development (Benson et al. 2000). Cholinesterase-like adhesion proteins A family of cell adhesion molecules possessing an extracellular domain with notable sequence homology to AChE has been cloned and characterized over the past

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years; their study may generate novel explanations of the non-enzymatic functions of AChE. Five different proteins have been identified comprising the cholinesterase-like adhesion molecules (CLAMs) family: thyroglobulin, glutactin, neurotactin, gliotactin, and neuroligin. The main structural properties shared by CLAMs and AChE are summarized as follows: (1) a 25%–32% amino acid identity between the extracellular domain of CLAMs and the AChE sequence, comprising much of the AChE sequence, (2) a similar hydropathic profile, suggesting a similar threedimensional structure and consequent common functional properties, (3) an unusual surface charge distribution consisting of a negatively charged annular motif around the entrance to the catalytic gorge or around its homologous domain (Botti et al. 1998), (4) an EF-hand motif that apparently binds Ca2+ and has a role in heterologous cell association (Tsigelny et al. 2000), and (5) dimerization and tetramerization of individual subunits, which is mediated by the four-helix bundle of the AChE catalytic domain conserved in all CLAMs (Morel et al. 2001). Of all CLAMs, the physiological roles of neurotactin, gliotactin, and neuroligin are better understood and appear to be closer to the presumed non-canonical functions of AChE (Scholl and Scheiffele 2003). Neurotactin interacts with the secreted protein amalgam through its cholinesterase-like domain (Fremion et al. 2000), promoting heterophilic cell aggregation (Barthalay et al. 1990). Chimeric molecules in which the neurotactin extracellular region is replaced with the homologous AChE domain retain the adhesive properties of the intact molecule (Darboux et al. 1996). An interesting hypothesis is that, under conditions in which AChE is overproduced, it might compete with neurotactin ligands, blocking its growth-inducing functions. This may be especially relevant to the soluble variant AChE-R, which is subject to less limitation on its localization than AChE-S and therefore represents a firmer competitor to neurotactin partners. Gliotactin is expressed in peripheral glia and contributes to the formation of the blood-nerve barrier. Drosophila embryos with mutant gliotactin are nearly paralyzed as a consequence of the incomplete isolation of their motor axons (Auld et al. 1995). Neuroligins constitute a multigenic family of proteins that interact with neurexin transmembrane receptors (Dean et al. 2003). Members of the neuroligin family participate in synapse formation (Scheiffele et al. 2000) through their interaction with neurexins and control the balance between excitatory and inhibitory synapses (Chih et al. 2005), supporting the concept of a neuromodulary function for their extracellular esterasic domain. Moreover, CLAMs might compete with cholinesterases (and viceversa) in interactions with extracellular partners, suggesting putative interrelationships between these two types of proteins in their neuromodulatory activities.

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Concluding remarks AChE is much more than a straightforward terminator of synaptic transmission. Multileveled evidence suggests that it substantially contributes to synaptic transmission, not only in cholinergic synapses, but also in other types (e.g., dopaminergic, glutamatergic). It apparently does this by interacting with signaling cascades, by the complex variety of isoforms that it possesses, and by their modified localization under various conditions. Termination of cholinergic neurotransmission by AChE is thus merely the end of the beginning of multiple subsequent events.

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