MINIREVIEW Acetylcholinesterase in cell adhesion, neurite growth and network formation Laura E. Paraoanu and Paul G. Layer Darmstadt University of Technology, Germany Acetylcholinesterase – a protein with many faces Acetylcholinesterase (EC 3.1.1.7) is an enzyme that has an indispensable role in synaptic transmission by cata- lysing hydrolysis of the neurotransmitter acetylcholine. This classical function of acetylcholinesterase is well established, but its diverse localization in non-choliner- gic and non-neural cells and tissues [1], and the diver- sity of its molecular forms [2] strongly suggest additional, ‘non-classical’ functions for acetylcholines- terase. Many proteins, including enzymes, have been found to have more than one function, e.g. functioning as structural proteins in the lens of the eye, or binding to DNA or RNA to regulate translation or transcrip- tion [3]. However, there is currently no straightforward method by which to identify which protein encoded by a given genomic sequence has multiple functions. The dogma one gene–one protein–one function no longer holds true. For acetylcholinesterase, the idea that this protein can serve several biological functions is now accepted, functions that are primarily related to morpho-regulation, adhesion, stress or pathological sit- uations [1,4–6]. One way of achieving increased functional complexity is via alternative splicing. To make things even more complex, many proteins present unfolded components, the function of which remains elusive. Sussman [7] sug- gested that these unstructured molecular modules may be used for various purposes, each one dependent on, and ‘being structurally induced’ by, a given binding partner. Although this hypothesis requires experimental validation, for acetylcholinesterase it is clear that by using different combinations of exons, a single ache gene can yield several polypeptide products with different C-termini. Three alternatively spliced isoforms of acetyl- cholinesterase have been well established: the tetrameric AChE-S isoform (‘S’, synaptic), the dimeric glycophos- phatidylinositol-anchored AChE-E isoform, expressed in blood (‘E’, erythrocytic) and the ‘read-through’ variant of acetylcholinesterase (AChE-R) [2]. These acetylcholinesterase isoforms have the same enzymatic Keywords acetylcholinesterase; adhesion; co-opting proteins; extracellular matrix; knock-out mouse; neurite outgrowth; non-cholinergic functions; laminin-1; retina; structural interactions Correspondence L. E. Paraoanu, Darmstadt University of Technology, Developmental Biology, Schnittspahnstrasse 3, 64287 Darmstadt, Germany Fax: +49 6151 166548 Tel: +49 6151 166105 E-mail: paraoanu@bio.tu-darmstadt.de (Received 27 September 2007, accepted 22 November 2007) doi:10.1111/j.1742-4658.2007.06237.x The expression of acetylcholinesterase is not restricted to cholinergically innervated tissues and relates to both neurotransmission and multiple bio- logical aspects, including neural development, stress response and neurode- generative diseases. Therefore, the classical function of acetylcholinesterase has to be distinguished from its non-classical, e.g. enzymatic from non- enzymatic, functions. Here, the roles of acetylcholinesterase in cell adhe- sion, promoting neurite outgrowth and neural network formation are reviewed briefly, together with potential mechanisms to support these func- tions. Part of these functions may depend on the structural properties of acetylcholinesterase, for example, protein–protein interactions. Recent find- ings have revealed that laminin-1 is an interaction partner for acetylcholin- esterase. The binding of acetylcholinesterase to this extracellular matrix component may allow cell-to-cell recognition, and also cell signalling via membrane receptors. Studies using monolayer and 3D spheroid retinal cul- tures, as well as the acetylcholinesterase-knockout mouse, have been instru- mental in elaborating the non-classical functions of acetylcholinesterase. 618 FEBS Journal 275 (2008) 618–624 ª 2008 The Authors Journal compilation ª 2008 FEBS activity but differ in their tissue specificity, developmen- tal expression, multimeric assembly, membrane-associa- tion patterns and cellular localization. Although acetylcholinesterase is primarily expressed in the central and peripheral nervous systems, different isoforms are found in haematopoietic cells, osteoblasts, vascular endothelial cells and leukocytes, and are induced in vari- ous cell lines undergoing apoptosis [8–11]. Recently, the existence of an acetylcholinesterase protein localized to the nucleus has been documented [12], possibly indicat- ing roles for acetylcholinesterase in angiogenesis and tumour development. Variations in acetylcholinesterase cellular localization, whether membrane-attached, secreted, nuclear or cytoplasmic, have also led to the detection of a variety of interaction partners for the acetylcholinesterase protein (Table 1). Cytoplasmic proteins like RACK [13], nuclear proteins like the trans- criptional co-repressor C-terminal binding protein [14], or b-amyloid [15] can interact with acetylcholinesterase, and consequently may contribute to its functional diversity. Another level of possible multifunctionality of acetylcholinesterase has been introduced by reports on aryl acylamidase as a developmentally regulated enzy- matic side activity located on the acetylcholinesterase protein [16]. Acetylcholinesterase and adhesion There is good evidence that acetylcholinesterase plays a ‘non-classical’ role as an adhesion protein. A molecu- lar basis for this emerged following the detection of a new class of proteins, cholinesterase-like adhesion molecules [17,18]. This protein family includes the Drosophila glutactin, neurotactin, gliotactin and the mammalian neuroligins. These proteins are catalyti- cally inactive, but their cholinesterase-like domain has high sequence similarity with acetylcholinesterase and also acts as a protein–protein interaction domain. In all family members, these domains are found in the extracellular space and may contribute to the forma- tion of cellular junctions by binding to other extracel- lular ligands. The existence of these proteins provided a convincing reason to assume that acetylcholinesterase itself may engage in protein–protein interactions. It is striking that all cholinesterase-like adhesion molecules display similar electrostatic characteristics [17], presenting a typical ‘annular’ electrostatic motif of negative potential around a zone that is homolo- gous to the active-site gorge of acetylcholinesterase. The biological significance of the electrostatic surface potential of acetylcholinesterase is disputed [19]. Because it is known that electrostatic interactions can facilitate target recognition via protein-complex formation, the fact that all cholinesterase-like adhesion molecules share similar electrostatic characteristics strongly supports the notion that acetylcholinesterase can function as an adhesive protein. In vitro evidence for the adhesive functions of acetyl- cholinesterase was provided by Sharma et al. [20] using a microtitre-plate adhesion assay. By using an electri- cal cell–subs trate impedance-sensing method, they demonstrated that the level of cell–substratum adhe- sion of neuroblastoma cells correlates directly with their level of acetylcholinesterase expression. In neuro- nal cells, and also in astrocytes and fibroblasts, acetyl- cholinesterase may have morpho-regulatory roles. There, acetylcholinesterase may form a complex with amyloid precursor protein and perlecan that seems to be involved in substratum adhesion and the polarized migration of adherent cells [21]. Similarly, acetylcholin- esterase has a role in osteoblast adhesion [10,22]. Blockade of sites relevant to acetylcholinesterase adhe- sive properties caused a concentration-dependent decrease in osteoblastic cell adhesion, suggesting Table 1. Acetylcholinesterase interaction partners. Acetylcholinesterase interaction partner Localization Function of the interaction partner Amyloid beta Senile plaques and in the walls of cerebral blood vessels Alzheimer C-terminal binding protein Nucleus Transcriptional co-repressor in association with sequence specific DNA-binding transcriptional repressors Heparin Extracellular matrix proteoglycan Anticoagulant expressed by mast cells and basophils Laminin-1 Extracellular matrix Component of the basement membrane, neural development, neuronal regeneration Perlecan Extracellular matrix proteoglycan Acetylcholinesterase clustering and anchoring at the neuromuscular synapse RACK1 Cytoplasm Scaffold protein L. E. Paraoanu and P. G. Layer Non-classical roles of acetylcholinesterase FEBS Journal 275 (2008) 618–624 ª 2008 The Authors Journal compilation ª 2008 FEBS 619 that acetylcholinesterase may regulate cell–matrix interactions in bone [22]. Thus, the intrinsic capacity of acetylcholinesterase to function in adhesion may be one of the explanations for the neurite outgrowth stimulating role of the enzyme (see below). Acetylcholinesterase interaction with laminin-1 An adhesive protein is defined by its localization on the cell surface and by the fact that it is involved in binding to other cells or to the extracellular matrix. As outlined above in a very abridged manner, acetylcho- linesterase fulfils the requirements to act as a hetero- philic cell adhesion molecule. An important point was therefore to unravel interacting partner(s) for acetyl- cholinesterase localized outside the cell. Using a yeast 2-hybrid approach, we found laminin-1, among others, as a possible binding partner, specifically the globular domain IV of the b1 chain of laminin [23]. In vitro binding studies similarly indicated binding of acetyl- cholinesterase to laminin-1 [24], which was inhibited by peripheral anionic site inhibitors (fasciculin, propi- dium and gallamine) and an anti-acetylcholinester- ase mAb. Increasing the ionic strength, or decreasing the pH decreased this binding [23,24], indicating a dominant role for electrostatic interactions [17]. Laminin is an extracellular matrix protein with a decisive role in neuronal differentiation and adhesion. In non-neuronal tissues, laminin-1 is a component of the basement membrane, for example, the membrane surrounding blood vessels or the membrane underlying epithelial cells. In the developing nervous system, lami- nin is associated with both neuronal and glial cell somata, and is also found along axon tracts [25]. Remarkably, it is not expressed much in the adult cen- tral nervous system of higher vertebrates [26]. Laminin facilitates the migration of neuronal precursors to the appropriate region of the brain [27,28] and also speci- fies the direction of neurite outgrowth [29]. Therefore, one of its prevalent activities is to elicit and direct neu- rite growth, as shown in vitro for a wide variety of neuronal cell types [30,31]. Noticeably, laminin-1 binds to integrin receptors. They comprise the most abun- dant family of receptors for extracellular matrix com- ponents, thereby providing cells with an avenue for the transfer of information from the extracellular space to the cytoskeleton and intracellular signaling pathways [32]. Laminin is able to regulate neurite outgrowth via integrins and also by binding to other proteins, e.g. by its interaction with the b-amyloid precursor protein [33]. How acetylcholinesterase, as ‘the third man’, can affect such interactions, remains to be elucidated. Acetylcholinesterase and neurite growth If an enzyme like acetylcholinesterase has a secondary function in adhesion, what might this mean for the development of a brain? Cell adhesion is a major mech- anism to direct neuritic growth. Indeed, it was a role of acetylcholinesterase in regulating neuritic growth which was the first non-enzymatic activity of acetylcholines- terase to be clearly distinguished from its catalytic activity, by using a pharmacological cell culture approach [34]. These experiments were initiated by observations demonstrating that the expression of ace- tylcholinesterase is a very early step in postmitotic neu- rons [35], preceding the outgrowth of neuritic processes in brain tissues, cultured spheroids [36,37] and non- cholinergic neurons in the rat thalamus [38]. In cell cul- tures, acetylcholinesterase did promote neurite growth from chick nerve cells. This effect was independent of its enzymatic activity, because active-site inhibitors failed to attenuate it [34]. Moreover, overexpression of acetylcholinesterase showed the neuritogenic activity of the protein in neuroblastoma cells [39], phaeochromo- cytoma (PC12) cells [40] and primary dorsal root gan- glion neurons [41] (see Fig. 1, middle panels). Taken together, the role of acetylcholinesterase in neurite out- growth may due to structural interactions with proteins like laminin-1. If this suggestion is valid, then it explains how a protein like acetylcholinesterase, with no transmembrane and intracellular domain, might reg- ulate neurite outgrowth in a non-enzymatic way. At the same time, remaining an active enzyme, acetylcholines- terase may subserve two independent molecular mecha- nisms at the same time. Because acetylcholine can be released from growing neurite tips and stops their fur- ther growth [42], acetylcholinesterase localized at a dis- tant target cell could, via disinhibition of acetylcholine, first act as a neurite attractive molecule and then, dur- ing contact with that target cell, have adhesive (cell binding) functions (Fig. 2). Acetylcholinesterase in neural network formation Alterations in cholinergic innervation during early postnatal development can change various features of cortical ontogeny [43,44]. A transient expression pat- tern for acetylcholinesterase correlates well with the time of thalamo-cortical axon growth into the cerebral cortex, which indicated a morphogenic role of acetyl- cholinesterase in the network formation of thalamo- cortical connections [45]. A wealth of cell-culture approaches demonstrated a neurite supporting role of Non-classical roles of acetylcholinesterase L. E. Paraoanu and P. G. Layer 620 FEBS Journal 275 (2008) 618–624 ª 2008 The Authors Journal compilation ª 2008 FEBS acetylcholinesterase [34,39–41] (and above). In order to come closer to the normal in vivo situation, reaggregat- ed 3D retinal cell spheroids provide a histotypic tissue environment (Fig. 1), in which all cell types become arranged in almost normal spatial 3D relations. Using these 3D in vitro models, overexpression of acetylcho- linesterase leads to an advanced tissue differentiation, larger neuritic growth in inner plexiform layer-like areas and affect photoreceptor survival [46–48]. Most importantly, the acetylcholinesterase knockout mouse Fig. 1. Acetylcholinesterase as player in neurite growth and neural network formation in vivo and in vitro. (Left) Formation of synaptic sub- layers is disturbed in the inner plexiform layer of a P19 retina of the AChE ) ⁄ ) knockout mouse (lower), compared with regular sublayers in the WT mouse (upper) [51]. (Middle) Cells of the R28 retinal cell line grow only short processes (upper), whereas after acetylcholinesterase overexpression they extend very long processes (lower). (Right) In rosetted spheroids reaggregated from dispersed E5 chicken retina [48], acetylcholinesterase-positive amacrine cells surround inner plexiform layer-like plexiform areas to send their processes inwards to organize into synaptic sublayers (upper; cf. with inner plexiform layer in left-hand pictures; cells stained brown by Karnovsky-Roots). In spheroids with low acetylcholinesterase expression, network formation is almost completely inhibited (lower). Bar = 100 lm. 1. Growth cone secretes ACh which inhibits neurite growth ACh ACh AChE ACh AChE Laminin-1 Growth Stabilization AChE as adhesion partner AChE as ACh degrading enzyme Stop Stop Growth cone Direction of growth Direction of growth 2. AChE degrades inhibiting ACh functioning as target attractor 3. AChE might work as an adhesive protein stabilizing cell-cell contacts Fig. 2. Acetylcholinesterase influences neurite outgrowth by making use of enzymatic and structural properties. Schematic drawing showing acetylcholinesterase interconnecting two independent mechanisms in regulating neurite and axonal growth. Further see text. L. E. Paraoanu and P. G. Layer Non-classical roles of acetylcholinesterase FEBS Journal 275 (2008) 618–624 ª 2008 The Authors Journal compilation ª 2008 FEBS 621 [49] provides a suitable in vivo model for evaluating the developmental functions of acetylcholinesterase. This mouse shows extensive behavioural deficits dem- onstrating that acetylcholinesterase has indispensable roles which cannot completely backed-up for by butyr- ylcholinesterase. Whereas the overall expression of cholinergic brain structures was found to be unaltered [50], a detailed study on the formation and long-term survival of the retina revealed drastic changes [51]. During the first 20 postnatal days, formation of synap- tic sublaminae within the inner plexiform layer of the retina was severely distorted, demonstrating a role for acetylcholinesterase in retinal network formation (Fig. 1). Over a long period of up to 180 days, all pho- toreceptors underwent apoptosis, showing that acetyl- cholinesterase has long-lasting supporting effects on photoreceptor survival. These effects are at least par- tially independent of acetylcholinesterase’s enzymatic activity, because in this mouse a non-specific cholines- terase (butyrylcholinesterase) is still normally expressed. Complementing these observations in the acetylcholinesterase knockout mouse, photoreceptor damage by excessive light in rats led to a strong increase of the R-AChE variant [52], independently pinpointing to a link between photoreceptor survival and acetylcholinesterase expression. Concluding remarks Convincing evidence has mounted that acetylcholines- terase has roles in neural development, stress response and degenerative diseases (the latter two issues are dealt with elsewhere in this minireview series), repre- senting an excellent example of a co-opting protein. Its multitude of molecular forms indicates that acetylcho- linesterase subserves not one or two, but several func- tions. Acetylcholinesterase expression coincides with the onset of neural differentiation and it closely pre- cedes axonal outgrowth, but is not restricted to neural tissues. Acetylcholinesterase is homologous to estab- lished cell adhesion molecules, and it is able to interact with various proteins. 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Neuroscience 110, 627–639. 51 Bytyqi AH, Lockridge O, Duysen E, Wang Y, Wolfrum U & Layer PG (2004) Impaired formation of the inner retina in an AChE knockout mouse results in degeneration of all photoreceptors. Eur J Neurosci 20, 2953–2962. 52 Kehat R, Zemel E, Cuenca N, Evron T, Toiber D, Loewenstein A, Soreq H & Perlman I (2007) A novel isoform of acetylcholinesterase exacerbates photorecep- tors death after photic stress. Invest Ophthalmol Vis Sci 48, 1290–1297. Non-classical roles of acetylcholinesterase L. E. Paraoanu and P. G. Layer 624 FEBS Journal 275 (2008) 618–624 ª 2008 The Authors Journal compilation ª 2008 FEBS . globular domain IV of the b1 chain of laminin [23]. In vitro binding studies similarly indicated binding of acetyl- cholinesterase to laminin-1 [24], which was inhibited by. intracellular signaling pathways [32]. Laminin is able to regulate neurite outgrowth via integrins and also by binding to other proteins, e.g. by its interaction