Acetylcholinesterase and apoptosis

A novel perspective for an old enzyme


X.-J. Zhang, Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 YueYang Road, Shanghai 200031, China
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Acetylcholinesterase is indispensable for terminating acetylcholine-mediated neurotransmission at cholinergic synapses. In addition, there is evidence to suggest that acetylcholinesterase contributes to various physiological processes through its involvement in the regulation of cell proliferation, differentiation and survival. The effects of acetylcholinesterase depend on the cell type and cell-differentiation state, the modulation of expression levels, cellular distribution and binding with its protein partners. This minireview highlights recent progress that has advanced our understanding of the role of acetylcholinesterase in the process of cell proliferation and apoptosis.


acetylcholinesterase R-isoform


acetylcholinesterase S-isoform




CCAAT binding factor


c-Jun N-terminal kinase


acetylcholinesterase R-isoform with extended N-terminus


protein kinase C

Acetylcholinesterase is recognized as being actively involved in cholinergic neurotransmission. In addition, levels of this enzyme vary gradually during differentiation, apoptosis and the genesis of neurodegenerative diseases [1,2]. These processes are correlated with the control of cell growth, including both cell proliferation and cell death. In mammals, acetylcholinesterase is encoded by a single gene, ACHE, but, because of alternative splicing at the C-terminus of acetylcholinesterase mRNA, it has three different isoforms: synaptic (S) or tail (T), erythrocytic (E) and read-through (R) [3]. These acetylcholinesterase variants selectively participate in the processes involved in promoting or attenuating cell death that accompany changes in expression, distribution and balance among the enzyme variants. In this minireview, we discuss recent progress in studying the role of acetylcholinesterase in the control of cell growth and suggest the possible underlying molecular basis from a non-classical view.

Evidence regarding the involvement of acetylcholinesterase in apoptosis

Indirect evidence that acetylcholinesterase is involved in regulating cell proliferation and apoptosis is derived from studies in which certain cell-differentiation models were treated with antisense (AS) oligonuleotides corresponding to the ACHE or BCHE gene. Inhibition of acetylcholinesterase gene expression using this method increased the cell count and enhanced cell proliferation in mouse bone marrow primary cultures. Furthermore, AS-acetylcholinesterase suppressed apoptosis-associated DNA fragmentation in progeny cells originating from the differentiation of hematopoietic stem cells [4]. The functions of cholinesterase during tissue differentiation were investigated by cloning a fragment of the BCHE gene in the AS orientation and transfecting it into retinal cells isolated from chick embryos. Although this did not identify the apoptotic functions and isoform types of acetylcholinesterase, increased apoptosis was observed, accompanied by suppressed butyrylcholinesterase expression and enhanced acetylcholinesterase expression in AS-butyrylcholinesterase-treated cells [5]. More direct evidence that acetylcholinesterase contributed to the loss of retinal function was obtained by assessing acetylcholinesterase in retinal photoreceptors following light-induced damage. Although formation of the inner retina network was completely eliminated in newborn acetylcholinesterase-knockout mice, a novel acetylcholinesterase variant in which the acetylcholinesterase R-isoform (AChE-R) has an extended N-terminus (N-AChE-R) exacerbated photic stress-induced death of adult photoreceptors. The orphan AS agent minimized N-AChE-R expression and facilitated the recovery of retinal functions [6,7]. Thus, proteins encoded by the ACHE gene have a role in modulating tissue formation or cell death.

In addition to reports in in vivo systems, we used cultured cells, which can be regarded as a relatively explicit system, and treated them with variable apoptotic stimuli. We observed that the S variant of acetylcholinesterase (AChE-S) emerged in almost all the apoptotic cells of different tissue origin [8]. In PC12 cells, expression of AChE-S but not AChE-R was enhanced in response to apoptosis initiated by calcium influx [9]. Unlike AChE-S or N-AChE-R, AChE-R was suggested to play a role in the body’s response to acute stress and prevention of further injury. Thus, AChE-R, but not AChE-S, transgenic mice display more resistance to age-dependent neurodegeneration [10]. Furthermore, AChE-R, and the C-terminal peptide cleaved from it, exerted proliferative effects on blood cells [11,12]. Although the molecular cascades underlying the reciprocal effects among different acetylcholinesterase variants have not been fully elucidated, these cumulative, multisite observations demonstrate that acetylcholinesterase plays a complex role in modulating cell growth and death.

Regulation of acetylcholinesterase expression in apoptosis

The expression of acetylcholinesterase variants depends on both stress-induced promoter activation and post-transcriptional modulation, including mRNA stability, alternative mRNA splicing, translational control and protein modification. There is evidence regarding the correlation between the stress-activated protein kinase family and apoptosis-associated acetylcholinesterase expression. Phosphorylation of c-Jun N-terminal kinases (JNK) and their downstream transcription factor, c-Jun, was enhanced during apoptosis induced by the DNA topoisomerase inhibitors etoposide or excisanin A. A corresponding increase in acetylcholinesterase expression in the apoptotic cells was observed. This upregulation in acetylcholinesterase was eliminated by administering a JNK inhibitor, silencing JNK with siRNA or antagonizing c-Jun with a dominant-negative c-Jun mutant [13].

The acetylcholinesterase promoter is also sensitive to signals initiated by alterations in intracellular Ca2+ levels. Mobilizing intracellular Ca2+ enhanced acetylcholinesterase mRNA stability and, thereafter, activation of the acetylcholinesterase promoter; however, the mechanism contributing to this enhanced stability during apoptosis remains unclear. One possible mechanism may involve the calcium-mediated RNA-binding protein [14]. It has been shown that acetylcholinesterase promoter activation targets several sites on the acetylcholinesterase promoter. The CCAAT motif was identified and binds the CCAAT binding factor (CBF/NF-Y); this had a suppressive effect on acetylcholinesterase promoter activity [15]. Increased intracellular Ca2+ levels would enable CBF/NF-Y release from this motif and therefore promoted activation of the promoter [16], but factors modulating CBF/NF-Y and CCAAT binding in response to altered calcium signals are unknown. Because of a redundancy in Ca2+ signal transduction during apoptosis, acetylcholinesterase promoter activation is also mediated by two calcium-dependent proteins, namely calpain and calcineurin. The signal cascade among calpain, calcineurin, and nuclear factor of activated T cells (NFAT) is supposedly involved in the final stages of acetylcholinesterase promoter activation [17]. Further investigation uncovered a more complex GSK-3β involved modulating mechanism in AChE level in apoptotic PC12 cells. The increased intracellular Ca2+ levels induced the GSK-3β activation and a parallel upregulation in mRNA and protein levels of AChE-S and AChE-R variants in apoptotic PC12 cells. Although the relationship between AChE variants and GSK-3β remain to be elucidated, the increase in AChE-S but not AChE-R variant was blocked by GSK-3β inhibitor. Considering the GSK-3β is contributed to apoptosis, thus these results suggested the dissimilar functions of the AChE variants [9].

Little is known about the mechanisms underlying the selective splicing choices between AChE-S and AChE-R. Undoubtedly, the balance between the two acetylcholinesterase variants is critical for functional outcome. A shift from AChE-S to AChE-R was observed during promegakaryotic cell differentiation in response to alterations in the level of intracellular Ca2+. It is hypothesized that the microRNA levels modulate the shift of mRNA variant types from AChE-S to AChE-R through acetylcholinesterase, protein kinase C (PKC), and protein kinase A cascades. This shift enhanced cell differentiation and suppression of cell death [18], and was also evident in the Alzheimer’s disease brain [19]. In the stress-induced shift from the AChE-S to AChE-R mRNA variant, the SC35 splicing factor was involved in a reciprocal reinforcing manner [20].

Alternative acetylcholinesterase distributions and apoptosis

It is still not known whether the cellular distribution pattern of acetylcholinesterase is the primary basis for the apoptotic functions of this enzyme. However, specific acetylcholinesterase variants and distributions have been observed in apoptotic cells. We observed that the expression of AChE-S was markedly increased in the perinuclear and nuclear regions of apoptotic cells. During the late stages of apoptosis, acetylcholinesterase accumulates inside the apoptotic body with condensed chromatin [8,21]. The perinuclear distribution of acetylcholinesterase implies that it is located in the endoplasmic reticulum; an implication that was confirmed by co-localization of acetylcholinesterase and calnexin, an endoplasmic reticulum marker protein (data not shown). This distribution pattern is consistent with that of acetylcholinesterase in non-neuronal cells [22]. During apoptosis, AChE-S is synthesized and probably retained in the endoplasmic reticulum and awaits transportation to nuclei or other subcellular fractions. Little is known about the mechanisms underlying acetylcholinesterase nucleic transportation and the restrictions on its delivery to the cell surface. The C-terminal peptide of acetylcholinesterase, which is critical for the membrane-bound character of acetylcholinesterase, is probably responsible for the distribution by virtue of its binding with the other elements [23,24].

AChE-R is mainly a soluble and secreted form of the enzyme due to lack of the C-terminal domain encoded by exon 5 or 6 of the ACHE gene [25]. Similar to AChE-S, AChE-R displays variations in cellular distribution with regard to the promotion of cell proliferation. AChE-R was found to accumulate in homogenate fractions enriched with membrane of myasthenia gravis thymus, which bears a pathological characteristic trait of thymic hyperplasia, probably the result of an enhanced cell proliferation rate. Alterations in the distribution of AChE-R may be responsible for the change in PKCβII, because a notable enhancement in PKCβII expression was detected in thymuses of AChE-R transgenic mice. In addition, thymocytes of AChE-R transgenic mice were less sensitive to apoptosis than were controls. It is still not known whether the location change of AChE-R is due to its binding with PKC [26]. Under oxidative stress conditions, AChE-R can be secreted from glia cells and probably promotes proliferation of the surrounding neurons [27]. However, the extended AChE-R variant N-AChE-R enhances the cell death induced by light damage to photoreceptors [6]. What remains to be established is whether the effects of N-AChE-R occur via a reduction in AChE-R expression or because of antagonizing effects of AChE-R by direct insertion into the membrane with the N-terminus or signal cascades unrelated to other acetylcholinesterase variants. Thus, acetylcholinesterase effects related to the promotion and inhibition of cell proliferation are cell-type and cell-state dependent. It is also possible that acetylcholinesterase functions are correlated with its binding partners that are located in alternative subcellular positions.

Hydrolytic activity of acetylcholinesterase and apoptosis

The hydrolytic activity of acetylcholinesterase is essential for execution of the classical function of the enzyme, however, it appears to be unnecessary for acetylcholinesterase’s regulation of cell growth or death. Overexpression of non-catalytic AChE-S in NRK cells attenuated the proliferation rate and elevated sensitivity to apoptotic stimuli [28]. ARP, a peptide derived from the C-terminus unique for AChE-R, and a 14-amino acid peptide derived from the C-terminal of AChE-S, do not require catalytic activity to execute the functions related to the promotion of cell proliferation or induction of apoptosis [29]. Furthermore, when using acetylcholinesterase inhibitors in Alzheimer’s disease treatment binding to the peripheral site and not the central catalytic active site is more effective [30]. Together, these studies indicate that sites beyond the active center of acetylcholinesterase are critical in modulating cell growth and death.

How does acetylcholinesterase participate in apoptosis?

Acetylcholinesterase isoforms participate in apoptosis in two ways: by promoting or suppressing cell death. The mechanisms underlying the involvement of acetylcholinesterase in modulating cell growth and death are not fully understood, but several models have been proposed. Enhanced acetylcholinesterase variant expression may influence the expression of other group genes, including those involved in apoptosis [31]. Acetylcholinesterase was suggested to function by binding with specific partners or by influencing other elements in the presence of apoptotic stimuli or under stress. This may explain why overexpression of AChE-S does not initiate but rather enhances the sensitivity to cell death [28]. Acetylcholinesterase also contributes to the formation of the apoptosome during apoptosis. Silencing acetylcholinesterase with siRNA blocks the interaction between apoptotic protease activating factor 1 and cytochrome c [32].

Acetylcholinesterase is involved in the pathogenesis of Alzheimer’s disease, which is characterized by amyloid fibril deposition in body tissues, increased acetylcholinesterase staining in Alzheimer disease plaques and loss of cholinergic neurons. There is still no explicit mechanism for the toxicity of acetylcholinesterase in Alzheimer’s disease. A 14-amino acid peptide derived from AChE-S displays toxicity towards cultured cells and is able to assemble into amyloid fibrils under physiological conditions [29]. One possible explanation is that the conformational switch region in the shorter acetylcholinesterase protein fragments has a higher propensity for conformation conversion from α helix to β sheet. Thus, the converted acetylcholinesterase fragments may serve as amyloid nuclei in amyloid β fibril formation [33]. This peptide may also exert toxic or trophic effects by modulating alpha7 nicotinic receptors [34]. This may explain why acetylcholinesterase accumulates in Alzheimer’s disease plaques [35].

Unlike AChE-S, which promotes cell death, AChE-R exerts the inverse effect by positively regulating cell proliferation. The stress-induced AChE-R promotes proliferation by forming a triple complex with PKCε and RACK1 [36], and ARP enhanced AChE-R levels under stress conditions [18]. Although the relationship between acetylcholinesterase and cell-cycle proteins remains unknown, the cell-cycle arrest effects have been observed in differentiating cells with acetylcholinesterase expression [37].

Summary and prospects

The integrated effects of acetylcholinesterase on cell growth control and apoptosis have been reviewed here. Certain functions of acetylcholinesterase with regard to the modulation of cell proliferation and cell death seem to be specific to particular acetylcholinesterase isoforms. The proportion of variants changes in a manner dependent on the intensity of the cellular response to stress. Thus, understanding the regulation of the balance between AChE-R and AChE-S is an important direction for future investigation. How do specific acetylcholinesterase variants achieve selectivity in modulating cell growth and death? The answer is not clear, but their C-terminal peptides are likely to be involved. AChE-R, as a secreted monomer, is likely to modulate proliferation of the neighboring cells [27] possibly by influencing the ligands on the cell surface [38]. ARP and the synthesized 14-amino acid peptide derived from AChE-S represent possible means by which acetylcholinesterase variants may function after being processed and the latter may be a natural entity. The effects of acetylcholinesterase on cell growth control may further correlate with protein modification. The glycosylation of acetylcholinesterase is necessary for maintaining the stability of this enzyme [39]. In the cerebral spinal fluid of Alzheimer’s disease patients, a different glycosylation pattern of acetylcholinesterase was observed [40]. Chaperone heat shock proteins excessively accumulate with overexpressed AChE-S or downregulation of AChE-R during stress [10,41]. The heat shock protein clusters reflect chronic endoplasmic reticulum stress responses. Therefore, it would be interesting to know whether modification of acetylcholinesterase is the basis of the stress-mediating effects of this enzyme. Considering acetylcholinesterase as a binding target, it would be meaningful and exercisable to develop antibodies or small molecular markers as sensitive in situ indicators to distinguish apoptotic cells or cells in stress [42]. In addition, the location and signal transduction of acetylcholinesterase may be influenced by its partners, which is probably the basis of the pathogenesis of acetylcholinesterase-associated diseases. Using a protein–protein comparison blast tool, cholinesterases including acetylcholinesterase-related proteins were found to be common to both Alzheimer’s disease and diabetes, indicating the possible role of acetylcholinesterase in signal transduction in insulin resistance and lipid metabolism [43]. Furthermore, anomalous acetylcholinesterase expression has been found in tumors [44]. Therefore, elucidating the 3D structures and functions of acetylcholinesterase variants and their partners under normal and pathological conditions will provide insights into how acetylcholinesterase contributes to disease etiology and aid in the search for therapeutic agents targeting acetylcholinesterase.


The authors are grateful to Dr Hermona Soreq (The Hebrew University of Jerusalem, Israel) for her critique of the manuscript. This study was supported in part by grants from the National Natural Science Foundation of China (no. 30570920, 30623003), the Third Phase Creative Program of Chinese Academy of Sciences (no. KSCX1-YW-R-13), the Major State Basic Research Development Program of China (973 Program, no. 2007CB947901), and the Science and Technology Commission of Shanghai Municipality (no. 06JC14076 and 06DZ22032).