©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
Regulation of Acetylcholinesterase Expression during Neuronal Differentiation (*)

(Received for publication, July 3, 1995; and in revised form, November 2, 1995)

Barbara A. Coleman Palmer Taylor (§)

From the Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

We have examined the developmental expression of acetylcholinesterase (AChE) during the process of neuronal differentiation from a pluripotent stem cell. P19 embryonic carcinoma cells form embryoid bodies, which, when cultured with retinoic acid, are induced to differentiate into neurons and glia. No AChE activity is present in the undifferentiated stem cells, and mRNA protection analyses do not detect AChE mRNA. Commitment to a neuronal differentiation pathway results in increased levels of AChE mRNA, production of a tetrameric form of the enzyme, and secretion of AChE into the culture medium. Concomitant with subsequent morphological differentiation into neurons, enzyme secretion diminishes and AChE becomes largely tethered to the neuronal cell membranes. The enzyme is attached to the cell surface as a globular tetramer. Its hydrodynamic properties are consistent with association through a noncatalytic hydrophobic subunit rather than anchorage by a glycophospholipid tail. No change in the rate of transcription of the Ache gene was detected during the course of differentiation, suggesting that the gene is actively transcribed at very early stages of development. Results suggest that stabilization of a labile mRNA governs the increase in AChE mRNA and gene product. The studies presented indicate that an early event in neuronal differentiation is the stabilization of the mRNA leading to expression of a secreted form of AChE. A subsequent step associated with neurite outgrowth results in a transition from secretion of the tetrameric enzyme to its localization on the cell membrane.


INTRODUCTION

Acetylcholinesterase (AChE) (^1)catalyzes the hydrolysis of the neurotransmitter, acetylcholine, in the central and autonomic nervous systems and at neuromuscular synapses. In vertebrates, AChE also is expressed in hematopoietic cells. Distinct molecular forms of the enzyme predominate in each cell type due to alternative splicing at the 3`-end of the gene. The carboxyl-terminal sequences encoded by the alternatively spliced exons direct the cellular localization of the enzyme but do not affect its catalytic properties.

AChE exists as asymmetric, collagen-tailed forms or as globular entities. Globular monomers (G1), dimers (G2), or tetramers (G4) are found as soluble or membrane-associated species. Tetramers, linked to a noncatalytic hydrophobic subunit, are most common in mammalian nervous tissue. Indeed, G4 AChE represents 80-90% of the enzyme in mammalian brain (Inestrosa et al., 1994). The appearance of cholinesterase activity on nerve cells often precedes synaptogenesis, and it has been hypothesized that cholinesterase is important in development of the mammalian nervous system (Robertson and Yu, 1993; Layer and Willbold, 1995). Although often used as a marker for terminal differentiation of neurons (Levine et al., 1974; Teresky et al., 1974), developmentally regulated expression of AChE in neuronal cells is not well understood. Neuroblastoma and rat pheochromocytoma cell lines have previously been used to study AChE expression (see references in Denis-Donini and Augusti-Tocco(1980) and Greene and Rukenstein(1981)). However, studies of developmental expression may be limited in such cell lines since they are already committed to a neuronal phenotype and are sufficiently differentiated to express AChE. We sought to investigate the regulation of AChE gene expression throughout the course of neuronal development in order to understand better its expression during the differentiation process.

Murine embryonic carcinoma cells are the pluripotent stem cells of mouse teratocarcinomas. Undifferentiated monolayers of these cells can be maintained in culture, or embryoid bodies (EBs) can be generated in vitro by growing cells in bacteriological Petri dishes in which they spontaneously aggregate without attachment. Little differentiation occurs in EBs themselves; however, when replated onto tissue culture dishes, several cell types develop. The murine P19 cell line can be induced in vitro to differentiate only along the neuroectodermal pathway by culturing EBs with >5 times 10M retinoic acid (Jones-Villeneuve et al., 1982). When replated into tissue culture dishes, the RA-treated aggregates adhere and develop into neurons, glia, and fibroblast-like cells in a manner similar to that observed in vivo (Jones-Villeneuve et al., 1982, 1983). Both aggregation and RA are necessary to induce neuronal differentiation of P19 cells, but the continued presence of the inducer is not required. Cultures containing up to 90% neurons can be obtained by treating differentiating P19 cells with mitotic inhibitors. These neurons express AChE as well as a wide variety of biochemical, morphological, and electrical properties characteristic of mammalian neurons (Jones-Villeneuve et al., 1982, 1983; McBurney et al., 1988; Cheun and Yeh, 1991; Staines et al., 1994). Thus, this cell line provides an appropriate system for examining the developmental regulation of mammalian AChE during the neuronal differentiation process.


EXPERIMENTAL PROCEDURES

Tissue Culture

P19 cells were obtained from American Type Culture Collection (Atlanta, GA) and cultured as undifferentiated monolayers at 10^5 cells/ml in 90% minimal essential medium alpha supplemented with 7.5% newborn calf serum, 2.5% heat-inactivated fetal bovine serum, and antibiotics (100 units/ml penicillin G and 100 µg/ml streptomycin sulfate). Cells were grown at 37 °C in a 5% CO(2) atmosphere. Neuronal differentiation was induced by culturing 10^5 cells/ml in Fisherbrand 100-mm Petri dishes containing normal culture medium plus 5 times 10M RA. Cells did not adhere and instead formed free-floating EBs. Media containing fresh RA was replaced every 48 h. To induce extensive morphological differentiation, EBs were exposed to 0.25% trypsin for 1 min, resuspended in regular culture medium, and plated in tissue culture dishes containing serum-free OptiMEM medium (Life Technologies, Inc.) lacking RA and supplemented with 0.5 µg/ml recombinant human fibronectin (Life Technologies, Inc.) plus antibiotics. Subsequently, cells received fresh OptiMEM media without fibronectin every 48 h. After 2 days of differentiation, 5 µg/ml cytosine arabinoside was included to inhibit proliferation of nonneuronal cells.

To assay for secreted AChE, EBs were cultured with media containing serum in which endogenous esterase activity was inhibited by treatment with DFP. To accomplish this, calf serum and fetal bovine serum were incubated with 0.1 mM DFP overnight at room temperature on a rocking platform. Sera were sterile filtered, held for 48 h at 4 °C, and then assayed for residual DFP and AChE activity. Less than 1% of the original AChE activity remained after treatment, and the serum displayed no inhibitory activity toward exogenously added AChE.

AChE Activity Determination

EBs were allowed to settle for 15 min in conical tubes, washed twice with 15 ml of cold PBS, and collected by centrifugation for 3 min at 500 times g. Monolayers were washed twice with 5 ml of cold PBS and removed from the plates by scraping. Cells generally were solubilized in 1% Triton X-100 in PBS and briefly sonicated. Insoluble material was removed by centrifugation for 10 min at 12,000 times g. Aliquots of supernatants were used for enzyme assays. To inhibit extracellular and cell surface AChE, a final concentration of 100 nM ecothiophate was added to the cells for 10 min prior to extensive washing with cold PBS and harvesting by scraping. Soluble AChE was extracted into a low salt soluble fraction by freezing and thawing cells 3 times in PBS. Cell membranes were sedimented as above and extracted with 1% Triton X-100 in PBS to obtain the detergent-soluble (DS) forms. Media collected for enzyme analysis were spun at 500 times g for 10 min to remove cells and then concentrated using Centriprep 30 concentrators (Amicon, Inc., Beverly, MA) prior to assay. AChE was measured as described by Ellman et al.(1961) in a reaction mixture containing 100 mM sodium phosphate (pH 7.0), 0.1% Triton X-100, 0.75 mM acetylthiocholine iodide, and 0.3 mM 5,5`-dithiobis(nitrobenzoic acid). Samples were assayed with and without 10 µM AChE-specific inhibitor, BW284C51. Protein determinations were made using the Bio-Rad DC protein assay. All chemicals used were obtained from Sigma unless otherwise noted.

Histochemistry and Immunolabeling

Cells were washed 3 times with cold PBS before fixing for 20 min in 0.4% formaldehyde, 0.04% glutaraldehyde in cold PBS. After fixing, cells were again washed 3 times with PBS and stained for activity using the Karnovsky and Roots (1964) method. Fixed cells were incubated with 20 µM iso-OMPA and then stained in the presence of iso-OMPA to inhibit nonspecific cholinesterase. To assess other esterase activity, 10 µM BW284C51 was added instead of iso-OMPA.

For immunohistochemistry studies, dissociated EBs were plated onto polylysine-treated coverslips and allowed to differentiate in serum-free media for 4 days without cytosine arabinoside. Coverslips were fixed, washed, and incubated with 1% normal goat serum in PBS for 20 min to block nonspecific binding. Polyclonal rabbit antisera raised in the laboratory against purified, recombinant DNA-derived mouse AChE was used as the primary antibody. Immunoblots showed that the antiserum detected as little as 20 ng of purified mouse AChE at a 1:500 dilution but had no cross-reactivity with 80 ng of purified mouse butyrylcholinesterase at 1:100 dilution. Coverslips were incubated for 60 min in antiserum diluted with 0.1% goat serum. Cells were subsequently washed 3 times for 5 min in PBS. Immunodetection was performed using a Vectastain anti-rabbit peroxidase kit (Vector Laboratories, Burlingame, CA) and developed with a fresh solution of 0.5 mg/ml 3`,3`-diaminobenzidine, 0.01% hydrogen peroxide, and 0.04% nickel chloride. Neuron-specific labeling was accomplished by incubating 10 µg/ml fluorescein isothiocyanate-labeled tetanus toxin C fragment (List Biological Laboratories, Campbell, CA) for at least 60 min with fixed, neuronally selected cultures grown on coverslips. After washing with PBS, coverslips were mounted with Slow Fade (Molecular Probes, Eugene, OR) and examined using a Zeiss Photomicroscope equipped with epifluorescent optics.

RNA Preparation and RNase Protection Assay

A radioactive probe for the hydrophilic form of AChE was generated from cDNA encoding 460 bases in exons 4 and 6, as described by Luo et al.(1994). A cDNA fragment encoding the glycophospholipid-linked form of AChE (Li et al., 1991) was ligated to the upstream invariant coding region of the gene and subcloned into a pRc/CMV expression vector (Invitrogen), digested with BamHI and transcribed with SP6 to produce a probe to detect splicing of Ache exon 4 to exon 5. A U1 small nuclear RNA probe from human cDNA cloned into pSP65 (Howe and Steitz, 1986) was linearized with HindIII and transcribed by SP6 polymerase at one-tenth the specific radioactivity of other probes. Total cell RNA was prepared as described by Chomczynski and Sacchi(1987). For mRNA protection assays, 40 µg of total RNA was hybridized overnight with 5 times 10^5 cpm of antisense RNA probes at 55 °C. Nonhybridized RNA was digested with 10 µg/ml RNase A and 150 units/ml RNase T1 (Bio 101, Vista, CA) for 1 h at 30 °C. Bands were quantified by densitometric analysis and confirmed using the Ambis(TM) radioanalytical system.

Nuclear Run-on Transcription and mRNA Decay Rates

P19 cells in varying stages of differentiation were washed 3 times in ice-cold PBS, collected in the same buffer using 1 ml/plate of cells, and centrifuged at 500 times g for 5 min at 4 °C. Cells were resuspended in 4 ml of cold lysis buffer (10 mM Tris-HCl (pH 7.4), 10 mM NaCl, 3 mM MgCl(2), 0.5% Nonidet P-40) and swelled on ice for 5 min. After rupturing cells in a Dounce homogenizer, nuclei were sedimented at 500 times g for 5 min, resuspended in another 4 ml of lysis buffer, counted on a hemacytometer and again sedimented. Final resuspension achieved a concentration of 10^7 nuclei/0.2 ml storage buffer (50 mM Tris-HCl (pH 8.3), 40% glycerol, 5 mM MgCl(2), 0.1 mM EDTA). Nuclei were frozen in liquid nitrogen and stored at -70 °C.

Run-on transcription assays were performed essentially as described by Greenberg and Bender(1987). [alpha-P]UTP, (20 mCi/ml, Amersham Corp.) was used to label nascent transcripts for 30 min at 30 °C. After DNase I digestion and protein denaturation, radiolabeled RNA was isolated by precipitation and filtration through Millipore HA filters. RNA incorporation of [alpha-P]UTP was 3-6 times 10^6 cpm/10^7 nuclei.

Radiolabeled RNA was hybridized to slot blots containing 5 µg of plasmid DNA/slot. Single-stranded M13 DNAs containing 2.1 kilobases of either AChE sense or antisense cDNA were used for hybridizations. Nonspecific binding was assessed using single-stranded M13 vector. A 1.4-kilobase mouse alpha-tubulin probe was used to normalize for RNA loading. Probes were hybridized in 2 ml of 10 mM TES buffer (pH 7.4) containing 10 mM EDTA, 0.1% SDS, and 300 mM NaCl. After incubation for 48 h at 60 °C, blots were washed in 2 times SSC buffer, incubated with 10 µg/ml RNase A for 30 min at 37 °C, and rewashed with 2 times SSC. Hybridized blots were exposed to Kodak X-Omat AR film for 4-7 days at -70 °C. Autoradiographs were quantified by densitometric analysis.

To measure AChE mRNA decay rates, EBs were cultured for 5 days in the presence of RA and either plated for neuronal differentiation as described or maintained as EBs without further RA treatment. After an additional 2 days, cells were treated with 20 µg/ml DRB for up to 24 h. RNA was harvested at various times and AChE mRNA levels analyzed by mRNA protection.

Sedimentation Velocity in Sucrose Density Gradients

Soluble or detergent-solubilized AChE in 150 µl was layered over 3-30% (w/v) sucrose gradients containing 0.1 M NaCl, 0.04 M MgCl(2), 0.01 M Tris-HCl (pH 8.0), and 1% (v/v) detergent and sedimented at 200,000 times g for 20 h at 4 °C in an SW41 Ti Beckman rotor. A cushion of 40% (w/v) sucrose was layered at the bottom of the gradients to recover enzyme aggregates. Carbonic anhydrase (3.3 S), alkaline phosphatase (6.1 S), catalase (11.4 S), and beta-galactosidase (16 S) were used as sedimentation markers. Gradients were fractionated into 96-well microtiter plates, and aliquots were assayed spectrophotometrically for enzyme activities using a Ceres UV900 H Di microtiter plate reader (Bio-Tek Instruments) and Kineticalc II software.


RESULTS

Induction of Expression of AChE by Retinoic Acid

Murine P19 cells were induced to differentiate along the neuroectodermal pathway by culturing free-floating EBs in RA. After 6 days, EBs were plated into tissue-culture dishes, whereupon extensive morphological differentiation occurred. Neurons were distinguished as phase-bright rounded cells with extended processes. Glial and other nonneuronal cells were often seen beneath neuron-like cells and appear as flattened, nonphase bright cells. Although nonneuronal cells were abundant in cultures 2 days after plating RA-treated EBs, their numbers steadily declined upon treatment with 5 µg/ml cytosine arabinoside. This treatment was selective for retaining postmitotic neurons and resulted in highly enriched neuronal populations. Despite reports that serum-free media itself is selective for neuronal P19 cells (Rudnicki and McBurney, 1987), cytosine arabinoside was required to inhibit growth of nonneuronal cells in OptiMEM medium.

P19 embryonic carcinoma stem cells exhibited no AChE activity. However, after aggregation with RA and plating for morphological differentiation, neuronally induced cells showed increasing levels AChE activity in cell homogenates (Fig. 1). Increased AChE activity paralleled the appearance of morphologically identifiable neurons and a decrease in number of nonneuronal cell types. EBs cultured in the presence or absence of RA up to 6 days failed to express detectable levels of AChE (Fig. 1). If EBs were maintained in culture, the free floating aggregates also expressed AChE activity but at a much lower level and appearing at later time than in the morphologically differentiating neuronal cells. RA treatment of P19 monolayers without aggregation results only in formation of fibroblast cells (Jones-Villeneuve et al., 1982), which did not express AChE (Fig. 1). Thus, both aggregation and exposure to RA were required for induction of AChE expression, consistent with requirements for neuronal differentiation of P19 cells (Jones-Villeneuve et al., 1982). Replating onto tissue-culture dishes for morphological differentiation was necessary to observe high levels of cell-associated AChE activity in RA-treated, neuronally induced cells.


Figure 1: AChE expression in P19 cells under different culture conditions. After 6 days of culturing EBs, cells were plated (arrow) onto tissue culture dishes for morphological differentiation. Alternatively, EBs were cultured with RA for 6 days and maintained as free-floating aggregates without the drug for an equivalent time period. AChE activities represent means of 3-9 determinations from separate experiments ± S.E.



RA Induced P19 Neurons Express AChE Activity

EBs cultured with RA were replated onto tissue culture dishes, grown for 4 days and stained for AChE activity. As shown in Fig. 2A, AChE activity in the presence of the butyrylcholinesterase inhibitor, iso-OMPA, was localized to the network of neuronal processes and their associated cell bodies. Flattened, background cells did not have appreciable AChE activity unless they were in contact with neurites or neuritic processes. No staining of differentiated cultures occurred when the AChE specific inhibitor, BW284C51, was used in the assay (Fig. 2B). To confirm that AChE activity was associated with neuronal cell populations, immunohistochemistry was done using rabbit antiserum generated against purified mouse AChE. As seen in Fig. 2C, neuron-like cells showed intense immunoreactivity, whereas nonneuronal cell types showed diffuse, background staining. Immunoblots revealed no cross reactivity of our AChE antiserum with butyrylcholinesterase, indicating that the staining was not due to antibodies that recognized serum cholinesterase. Antiserum incubated with purified mouse AChE prior to use in the assay stained far fewer cells (Fig. 2D). Background staining of nonneuronal cells was also reduced, and no staining was observed when normal rabbit serum was substituted for AChE antiserum (Fig. 2F). To further establish that neurons were expressing AChE, immunohistochemically stained cultures were additionally incubated with fluorescein isothiocyanate-conjugated tetanus toxin, which binds specifically to neuronal gangliosides (Dimpfel et al., 1977). Binding of the fluorescent toxin to stained cells (Fig. 2E) confirmed that cells expressing AChE were, in fact, of a differentiated neuronal phenotype. Not every neuronal cell expressed membrane-associated AChE as some cells labeled with fluorescent tetanus toxin but did not react immunohistochemically. Cells that had been aggregated in the absence of RA and replated showed no neuronal differentiation or AChE reactivity. Thus, AChE activity appeared restricted to neuronally differentiated P19 cells.


Figure 2: Histochemical and immunohistochemical detection of AChE activity. P19 cells were cultured as EBs for 6 days in the presence of RA and then allowed to differentiate morphologically for 4 days in tissue culture dishes. A, Karnovsky-Roots histochemical stain for AChE activity in the presence of 20 µM iso-OMPA to inhibit nonspecific cholinesterase activity; B, Karnovsky-Roots stain with 10 µM BW284C51 used to specifically inhibit AChE activity; C, immunohistochemical staining of P19 neuronal cultures using polyclonal rabbit serum generated against purified recombinant-derived mouse AChE; D, immunohistochemical staining of P19 neuronal cultures using anti-mouse AChE polyclonal rabbit serum, which had been pre-adsorbed with purified mouse AChE. E, neuron-specific labeling with fluorescein isothiocyanate-conjugated tetanus toxin of cultures previously stained immunohistochemically for AChE; F, immunohistochemical staining of P19 neuronal cultures using normal rabbit serum. (A and B, magnification 100times; C-F, magnification, 630times.)



Expression of AChE mRNA

The open reading frame of the mouse Ache gene consists of 5 exons of which the first 3 in the open reading frame (exons 2-4) are invariantly spliced and, when transcribed by themselves, generate active enzyme. Exons 5 and 6 are alternatively spliced. The splicing pattern influences the cellular disposition of AChE but not its catalytic constants (Massoulie et al., 1993; Taylor and Radic, 1994). Hydrophilic forms of AChE, generated from splicing of exon 4 to exon 6, are the predominant forms expressed in most mammalian tissues. Soluble dimers and tetramers, as well as forms linked to structural subunits all result from use of exon 6. RNase protection experiments using a probe specific for splicing of exon 4 to exon 6 were conducted with RNA isolated from EBs or neuronally differentiated P19 cultures. As seen in Fig. 3A, mRNA representing the exon 4-6 splicing pattern was observed in long term embryoid bodies and differentiated neurons and accumulated during the differentiation process. RA-treated EBs maintained in culture as free-floating aggregates beyond 6 days expressed significant levels of AChE mRNA, despite lower levels of activity in the cells (compare Fig. 3with Fig. 1). In fact, the amount of mRNA in EBs or neuronally selected cultures of similar age were generally comparable (Fig. 3B).


Figure 3: AChE mRNA increases in RA treated EBs and morphologically differentiating neuronal P19 cultures. A, a representative autoradiograph from a message protection experiment showing protection of labeled probe by AChE mRNA during neuronal differentiation of P19 cells. Probe A, shown below, was used to assess splicing of AChE exon 4 to exon 6 (see ``Results'' for an explanation). The protected U1 band was used to normalize for RNA loading densities. No bands were observed in the tRNA control lane (not shown). B, densitometric analysis of the protected mRNA graphed after normalizing AChE to U1 levels. C, P19 cells were cultured as EBs for 6 days with RA and then either maintained in suspension without drug for an additional 6 days (12 d EBs) or differentiated in tissue culture plates for 6 days (6+6 neurons). tRNA control lane is also shown. Probe B, shown at bottom, was used to examine the level of splicing to exon 5, represented by the band at 445 nucleotides. The band at 257 nucleotides represents splicing to exon 6. Minor bands at 363 and 188 nucleotides likely result from unspliced message. No evidence for polymerase readthrough and retention of the intron separating exons 4 and 5 was obtained from P19 cells.



With Probe A (Fig. 3), either splicing of exon 4 to exon 5 or retention of the intron separating exons 4 and 5 would be indicated by a protected band of 160 nucleotides; this band was not evident. To confirm that minimal splicing to exon 5 occurred in P19 neurons, a riboprobe spanning the splice junction of exons 4 and 5 was also used in message protection experiments (Fig. 3, probe B). Alternative splicing to exon 5 results in amphiphilic dimers with a glycophospholipid tail, which anchors the enzyme to the cell membrane. These forms are common to hematopoietic cells in mammals and certain cell lines (Li et al., 1993; Karpel et al., 1994). Adult mouse brains show AChE mRNA spliced almost exclusively to exon 6 (Li et al., 1991, 1993; Karpel et al., 1994). Only a minor band representing splicing of exon 4 to exon 5 and equal to less than 2% of total AChE mRNA was observed at all stages of P19 cell differentiation. Thus, P19 embryonic carcinoma cells splice AChE mRNA in a manner consistent with the predominant pattern seen in mouse brain throughout the neuronal differentiation process.

Increased Levels of AChE mRNA Are Not Due to Transcriptional Activation

To determine whether accumulation of AChE message and increased enzyme activity result from changes in the rate of Ache gene transcription during differentiation, nuclear run-on experiments were conducted using nuclei isolated from pluripotent P19 cells, EBs, and morphologically differentiated P19 neuronal cultures. RNA elongated in the presence of [alpha-P]UTP was hybridized to 2.1-kb fragments of AChE cloned in the sense and antisense orientations in M13. As seen in Fig. 4, the Ache gene was actively transcribed even in untreated P19 embryonic carcinoma stem cells. Although transcription above background rates appeared in the antisense direction, the majority of AChE message was derived from transcription of the sense strand. This pattern continued during subsequent stages of commitment and differentiation. Transcription in both directions was inhibited by 2 µg/ml of the RNA polymerase II inhibitor, alpha-amanitin, indicating that AChE transcripts did not the result from RNA polymerase I or III activity (results not shown). No change in transcription rate of the AChE gene was observed over the course of commitment and differentiation. This indicated that the enhanced levels of AChE mRNA resulted from post-transcriptional control. Furthermore, we consistently observed a direct correlation between amount of AChE mRNA present and the amount of activity measured, suggesting that AChE protein expression is regulated through stabilization of AChE mRNA.


Figure 4: Analysis of transcription rate and rate of decay for AChE mRNA. A, transcriptional activity assessed by run-on transcription assays using nuclei isolated from undifferentiated P19 stem cells (lane 1), EBs cultured 4 days in the presence of RA (lane 2), EBs cultured 6 days with RA and maintained in suspension for an additional 5 days (lane 3), and P19 neuronal cultures derived from EBs treated for 6 days and subsequently differentiated in tissue culture plates for 5 days. Bands represent hybridization of P19 AChE sense and antisense RNA to the complementary DNA strand in M13. Single-stranded M13 vector was used to assess nonspecific binding. B, quantification of bands by densitometric analysis using hybridization to alpha-tubulin to normalize for RNA loading. Data are means ± S.E. for the number of independent experiments indicated. C, decay of AChE mRNA in EBs and P19 neurons after inhibition of transcription by DRB. Cells were treated with 20 µg/ml DRB for up to 24 h. RNA was harvested at various time points and assayed for levels of AChE mRNA by message-protection experiments. Bands were quantified by densitometry and confirmed using the Ambis(TM) radioanalytical system before normalizing to RNA levels in untreated controls. Each line represents the mean ± S.E. for three independent experiments.



Experiments were done to determine decay rates for AChE mRNA during differentiation. P19 neurons and long-term EBs were treated with DRB to inhibit transcription and the levels of AChE mRNA determined by RNase protection experiments. For both neurons and EBs, the half-life for decay of the AChE mRNA was approximately 12 h (Fig. 4C). In fact, decay profiles for both cell types appear very similar. AChE mRNA levels were not sufficient for a precise analysis of decay rates in P19 stem cells or at early times after treatment with RA. However, turnover at this stage appears to be far more rapid. Nonetheless, experiments indicate that when AChE is expressed by neurons or neuronal precursors, their mRNAs become stabilized and exhibit similar half-lives of about 12 h.

Differences in Expression of AChE Molecular Species During Differentiation

The high levels of AChE mRNA present in EBs led to speculation that the enzyme might be synthesized but not appreciably retained by the cells. Thus, enzyme activity was assayed in both neuronal and EB cells and media (Fig. 5). As also seen earlier, AChE activity associated with the EBs themselves remained quite low even up to 12 days in culture. EBs maintained as free-floating aggregates after treatment with RA, however, were found to synthesize significant amounts of enzyme, of which greater than 90% was secreted (Fig. 5A). By contrast, P19 cells plated to allow extensive neuronal morphological differentiation to occur also secreted AChE, but retained 50% as cell-associated enzyme (Fig. 5B). When normalized to the amount of protein, EBs expressed the enzyme at a cumulative level equivalent to that observed in P19 neurons (Fig. 5, A and B).


Figure 5: Distribution of AChE activity between cell extracts and media. AChE activity was determined from cultures grown as EBs for 6 days with RA and maintained as EBs without drug (A), or plated for neuronal differentiation (B). Measurements are the means of at least four independent experiments expressed ± S.E.



Sedimentation in sucrose density gradients was used to evaluate the molecular forms expressed during differentiation. Globular forms of mouse AChE monomers (G1), dimers (G2), and tetramers (G4) sediment at approximately 3.6 S, 5.5 S, and 10.6 S, respectively, in Triton X-100-containing gradients (Brimijoin, 1983). AChE retained by the EBs consisted mostly of monomers at 3.6 S (Fig. 6A). These forms likely function as precursors to the more complex oligomeric forms of the enzyme (Brimijoin, 1983; Massoulie et al., 1993; Inestrosa et al., 1994). To further assess the amphiphilic character of the AChE molecules, EB cell extracts were also run on sucrose gradients containing Brij-96. The amphiphilic nature of G1 monomers in EBs and neurons was evidenced by a reduced sedimentation coefficient from approximately 3.6 S in Triton X-100-containing gradients to 3.0 S in Brij-96 (Fig. 6, A and B). Neurons, by comparison, contained G1, G2, and a large amount of G4 enzyme associated with the cells (Fig. 6B). At both stages of differentiation, cells secreted G4 into the culture medium (Fig. 6, C and D). Trace amounts of AChE monomers and dimers also were occasionally detected in the culture media. The distinguishing feature in expression of AChE during the differentiation process was that tetrameric AChE was not retained by neuronally committed cells when morphological differentiation was limited by culture conditions.


Figure 6: Sucrose density gradient separation of AChE molecular forms. Comparison of Triton X-100 and Brij-96 sucrose gradient analyses of AChE activity extracted from 11-day-old EBs cultured 6 days in presence of RA and maintained 5 days as free-floating aggregates (A) or P19 neurons morphologically differentiated for 5 days (B). C, molecular forms of AChE in the media of 11-day-old EBs, or D, P19 neurons differentiated for 5 days analyzed on Triton-X sucrose gradients. E, comparison of Triton X-100 and Brij-96 sucrose gradient analyses of DS AChE activity extracted from P19 neurons. F, DS AChE activity from 4-day differentiated control or ecothiophate-treated P19 neurons analyzed on Triton X-100 sucrose gradients. Sedimentation of the markers carbonic anhydrase (3.3 S), alkaline phosphatase (6.1 S), catalase (11.4 S), and beta-galactosidase (16 S) (left to right) are indicated by triangles at top of panels.



Low salt soluble and DS forms of AChE were extracted separately from neuronal P19 cells, and the detergent soluble fraction was analyzed on gradients. When the DS fraction was assayed on gradients containing either Triton X-100 or Brij-96, a shift in the sedimentation of the G4 form could be detected in addition to the shift in G1 (Fig. 6E). In studies where total cellular AChE is analyzed (Fig. 6B), this shift is less evident, a likely consequence of the presence of soluble tetramers destined for secretion, which obscure the shift in membrane-associated G4 AChE. Sucrose gradients using extracts from cells treated with the irreversible, cell impermeant AChE inhibitor, ecothiophate, confirmed that the majority of G1 AChE enzyme was intracellular, while G4 was the form located extracellularly on the neuronal surface (Fig. 6F).


DISCUSSION

Several neural cell lines have been used over the years to study AChE expression (cf. Massoulie et al.(1993)). PC12 cells more fully differentiate into sympathetic neurons when treated with nerve growth factor, but they are not completely differentiated as removal of the hormone results in retraction of neuronal processes and a reduction in AChE production (Greene and Rukenstein, 1981). Neuroblastoma and neuroblastoma hybrid cells commonly express molecular forms of AChE not normally found in mammalian nervous tissue (Lazar and Vigny, 1980; Li et al., 1993), suggesting that they lack important tissue- or developmentally specific regulatory controls. P19 embryonic carcinoma cells have been shown to differentiate into neurons in a manner consistent with that observed in vivo (Jones-Villeneuve et al., 1982, 1983; McBurney et al., 1988). Thus, we examined the regulation of AChE expression during differentiation of pluripotent P19 stem cells into terminally differentiated, postmitotic neurons. Undifferentiated P19 cells did not express AChE activity, nor was there sufficient mRNA for detection by message protection assays. However, P19 EBs cultured in the presence of 5 times 10M RA became committed to differentiating along the neuroectodermal pathway. Indeed, when such EBs were replated into adherent tissue culture dishes, rapid and extensive neuronal differentiation occurred. During this period, cells began to express AChE on their membrane surfaces as shown by histochemical and immunohistochemical staining. In addition to exhibiting morphology characteristic of neurons, the AChE-expressing cells also bound tetanus toxin, confirming their neuronal character. Fluorescent toxin labeling of AChE immunoreactive cells also showed that not all P19 neurons expressed the hydrolytic enzyme on their cell surfaces. This may be due to mixed neuronal phenotypes, which are reportedly present in differentiating P19 cultures (Sharma and Notter, 1988; Staines et al., 1994) or to some neurons continuing to express only secreted forms of AChE.

Experiments to determine transcriptional activity of Ache during development demonstrated that the gene was actively transcribed at the stem cell stage. Furthermore, while the level of AChE mRNA increased dramatically during differentiation, the transcription rate of the Ache gene remained constant in pluripotent, committed, and differentiated cells. This indicates that AChE mRNA levels are regulated post-transcriptionally. Experiments to determine rates of mRNA decay during different stages of differentiation were hampered by our inability to detect AChE mRNA in P19 stem cells or during early differentiation. However, EBs maintained in culture to a point where they expressed appreciable AChE activity showed a similar degree of mRNA stability to early differentiating neurons. The half-lives of AChE mRNA for both EBs and neurons were approximately 12 h. In the C2C12 muscle cell line, AChE mRNA and activity also increase during differentiation due to enhanced mRNA stability (Fuentes and Taylor, 1993). C2C12 myoblasts are already committed to differentiate into muscle, an AChE-expressing cell type. It is noteworthy that the AChE mRNA in neurons appears to turn over even more slowly than the stabilized mRNA in C2C12 myotubes. Results of studies presented here show that the Ache gene is turned on even prior to cell type determination. Certain neurons and muscle and hematopoietic cells express AChE, although each cell type is derived from a distinct developmental lineage. AChE may also be expressed only transiently by certain cells during development (Bear et al., 1985; Robertson et al., 1985). Permanent activation of the Ache gene and regulation of expression through mRNA stabilization may function to ensure rapid availability of the enzyme at any time during differentiation.

Both soluble and membrane-bound forms of neuronal AChE result from splicing of the invariant exons to exon 6. Neuronally induced P19 cells expressed AChE from nearly exclusive use of exon 6. Only minimal splicing to exon 5, which encodes the glycophospholipid-linked subunit, was detected. In contrast to certain neuronal cell lines which often express both amphiphilic and hydrophilic forms of AChE (Li et al., 1993), expression of AChE in differentiating P19 neurons is consistent throughout development with the splicing pattern found in adult mammalian brain (Li et al., 1993, Karpel et al., 1994).

Density gradient sedimentation shows that AChE associated with EBs exists predominantly as amphiphilic monomers. Yet, globular AChE tetramers are being assembled and secreted into the culture medium. AChE sequence homology to cellular adhesion proteins, its transient expression during brain development, and changes in neurite growth seen in the presence of AChE inhibitors combine to suggest that AChE plays a role in neuronal development (for review, see Layer and Willbold (1995)). Our results using differentiating P19 neurons suggest that secretion of AChE prior to neurite outgrowth may reflect the normal developmental process, which is not readily discernible in vivo.

The vast majority of membrane bound AChE in mammalian nervous tissue exists as globular tetramers. Our hydrodynamic analysis indicates P19 neurons anchor G4 through a hydrophobic structural subunit as expressed in rat, human, monkey, and bovine brains (Boschetti et al., 1994; Gennari et al., 1987; Liao, et al., 1993; Inestrosa et al., 1987). Presently, this structural subunit has not been cloned from any species. Indeed, its exact composition has yet to be determined. Thus it is not yet possible to examine whether synthesis of the hydrophobic structural subunit or its linkage to AChE tetramers is directly coupled to neurite outgrowth in P19 cells or is simply a temporally related event.

During rat and mouse brain development, an overall change in the expression of AChE molecular forms is observed in which the proportion of G1 decreases as the relative amounts of cell-associated G4 increase (Rieger and Vigny, 1976; Wade and Timiras, 1980; Inestrosa et al., 1994). Indeed, Layer and Willbold(1995) consider cholinesterase expression to occur in two discrete developmental phases; a morphogenetic period where low molecular weight forms predominate and a synaptogenetic phase during which expression of tetrameric AChE prevails. In light of the results of these studies, it is plausible that in the morphogenetic stages of embryogenesis, neuronal precursor cells assemble G1 AChE into tetramers, which are initially secreted. Subsequently, and concomitant with neurite extension, the G4 forms become localized to the neuronal cell surface. This presents an alternative explanation to simple maturation by assembly for the increase in ratio of G4 to G1 forms seen during brain development. Finally, disparities between AChE mRNA and cellular activity, which have been observed during embryonic development (Anselmet et al., 1994; Hammond et al., 1994), also may be explained, in part, by secretion of the enzyme.


FOOTNOTES

*
This work was supported by United States Public Health Service Grants GM18360 and GM24437, a grant from the Muscular Dystrophy Association, and by American Heart Association Postdoctoral Fellowship 93-58. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed. Tel.: 619-534-1366; Fax: 619-534-8248.

(^1)
The abbreviations used are: AChE, acetylcholinesterase; EB, embryoid body; RA, retinoic acid; DFP, diisopropyl fluorophosphate; PBS, phosphate buffered saline; DS, detergent-soluble; BW284C51, 1,5-bis(4-allyldimethylammoniumphenyl)pentan-3-one dibromide; iso-OMPA, tetraisopropyl pyrophosphoramide; TES, N-tris(hydroxymethyl)methyl2-aminoethanesulfonic acid; DRB, 5,6-dichlorobenzimidzole riboside.


ACKNOWLEDGEMENTS

We thank Shelley Camp for cloning assistance and technical advice, Dr. Mark Ellisman for help with microscopy, Mark Bodman and Claudine Prowse for aid in histo- and immunohistochemical staining, and Dr. Damon Getman for helpful discussions.


REFERENCES

  1. Anselmet, A., Fauquet, M., Chatel, J.-M., Maulet, Y., Massoulie, J., and Vallette, F.-M. (1994) J. Neurochem. 62, 2158-2165 [Medline] [Order article via Infotrieve]
  2. Bear, M. F., Carnes, K. M., and Ebner, F. F. (1985) J. Comp. Neurol. 237, 519-532 [Medline] [Order article via Infotrieve]
  3. Boschetti, N., Lioa, J., and Brodbeck, U. (1994) Neurochem. Res. 19, 359-365 [Medline] [Order article via Infotrieve]
  4. Brimijoin, S. (1983) Prog. Neurobiol. 21, 291-322 [Medline] [Order article via Infotrieve]
  5. Cheun, J. E., and Yeh, H. H. (1991) Int. J. Dev. Neurosci. 9, 391-394 [Medline] [Order article via Infotrieve]
  6. Chomczynski, P., and Sacchi, N. (1987) Anal. Biochem. 162, 156-159 [CrossRef][Medline] [Order article via Infotrieve]
  7. Denis-Donini, S., and Augusti-Tocco, G. (1980) Curr. Top. Dev. Biol. 16, 323-348 [Medline] [Order article via Infotrieve]
  8. Dimpfel, W., Huang, R. T. C., and Habermann, E. (1977) J. Neurochem. 29, 329-334 [Medline] [Order article via Infotrieve]
  9. Ellman, G. L., Courtney, K. D., Andres, V., Jr., and Featherstone, R. M. (1961) Biochem. Pharmacol. 7, 88-95 [CrossRef][Medline] [Order article via Infotrieve]
  10. Fuentes, M. E., and Taylor, P. (1993) Neuron 10, 679-687 [Medline] [Order article via Infotrieve]
  11. Gennari, K., Brunner, J., and Brodbeck, U. (1987) J. Neurochem. 49, 12-18 [Medline] [Order article via Infotrieve]
  12. Greenberg, M. E., and Bender, T. P. (1987) in Current Protocols in Molecular Biology (Ausubel, F. M., Brent, R., Kingston, R. E., Moore, D. D., Seidman, J. G., Smith, J. A., and Struhl, K., eds) pp. 4.10.1-4.10.9, John Wiley and Sons, New York
  13. Greene, L. A., and Rukenstein, A. (1981) J. Biol. Chem. 256, 6363-6367 [Abstract/Free Full Text]
  14. Hammond, P., Rao, R., Koenigsberger, C., and Brimijoin, S. (1994) Proc. Natl. Acad. Sci. U. S. A. 91, 10933-10937 [Abstract/Free Full Text]
  15. Howe, J. G., and Steitz, J. A. (1986) Proc. Natl. Acad. Sci. U. S. A. 86, 9006-9010
  16. Inestrosa, N. C., Roberts, W. L., Marshall, T. L., and Rosenberry, T. (1987) J. Biol. Chem. 262, 4441-4444 [Abstract/Free Full Text]
  17. Inestrosa, N. C., Moreno, R. D., and Fuentes, M.-E. (1994) Neurosci. Lett. 173, 155-158 [Medline] [Order article via Infotrieve]
  18. Jones-Villeneuve, E. M. V., McBurney, M. W., Rogers, K. A., and Kalnins, V. I. (1982) J. Cell Biol. 94, 253-262 [Abstract]
  19. Jones-Villeneuve, E. M. V., Rudnicki, M. A., Harris, J. F., and McBurney, M. W. (1983) Mol. Cell. Biol. 3, 2271-2279 [Medline] [Order article via Infotrieve]
  20. Karnovsky, M. J., and Roots, L. (1964) J. Histochem. Cytochem. 12, 219-221 [Medline] [Order article via Infotrieve]
  21. Karpel, R., Aziz-Aloya, R. B., Sternfeld, M., Ehrlich, G., Ginzberg, D., Tarroni, P., Clementi, F., Zakut, H., and Soreq, H. (1994) Exp. Cell Res. 210, 268-277 [CrossRef][Medline] [Order article via Infotrieve]
  22. Layer, P. G., and Willbold, E. (1995) Prog. Histochem. Cytochem. 29, 1-94 [Medline] [Order article via Infotrieve]
  23. Lazar, M., and Vigny, M. (1980) J. Neurochem. 35, 1067-1079 [Medline] [Order article via Infotrieve]
  24. Levine, A. J., Torosian, M., Sarokhan, A. J., and Teresky, A. K. (1974) J. Cell. Physiol. 84, 311-317 [Medline] [Order article via Infotrieve]
  25. Li, Y., Camp, S., Rachinsky, T. L., Getman, D., and Taylor, P. (1991) J. Biol. Chem. 266, 23083-23090 [Abstract/Free Full Text]
  26. Li, Y., Camp, S., and Taylor, P. (1993) J. Biol. Chem. 268, 5790-5797 [Abstract/Free Full Text]
  27. Liao, J., Norgaard-Petersen, B., and Brodbeck, U. (1993) J. Neurochem. 61, 1127-1134 [Medline] [Order article via Infotrieve]
  28. Luo, Z., Fuentes, M. E., and Taylor, P. (1994) J. Biol. Chem. 269, 27216-27223 [Abstract/Free Full Text]
  29. Massoulie, J., Pezzementi, L., Bon, S., Krejci, E., and Vallete, F.-M. (1993) Prog. Neurobiol. 41, 31-91 [CrossRef][Medline] [Order article via Infotrieve]
  30. McBurney, M. W., Reuhl, K. R., Ally, A. I., Nasipuri, S., Bell, J. C., and Craig, J. (1988) J. Neurosci. 8, 1063-1073 [Abstract]
  31. Rieger, F., and Vigny, M. (1976) J. Neurochem. 27, 121-129 [Medline] [Order article via Infotrieve]
  32. Robertson, R. T., and Yu, J. (1993) News Physiol. Sci. 8, 266-272 [Abstract/Free Full Text]
  33. Robertson, R. T., Tijerina, A. A., and Gallivan, M. E. (1985) Dev. Brain. Res. 21, 203-214
  34. Rudnicki, M. A., and McBurney, M. W. (1987) in Teratocarcinomas and Embryonic Stem Cells: A Practical Approach (Robertson, E. J., ed) pp. 19-50, IRL Press, Washington, D. C.
  35. Sharma, S., and Notter, M. F. D. (1988) Dev. Biol. 125, 246-254 [Medline] [Order article via Infotrieve]
  36. Staines, W. A., Morassutti, D. J., Reuhl, K. R., Ally, A. I., and McBurney, M. W. (1994) Neuroscience 58, 735-751 [CrossRef][Medline] [Order article via Infotrieve]
  37. Taylor, P., and Radic, Z. (1994) Annu. Rev. Pharmacol. Toxicol. 34, 281-320 [CrossRef][Medline] [Order article via Infotrieve]
  38. Teresky, A. K., Marsden, M., Kuff, E. L., and Levine, A. J. (1974) J. Cell. Physiol. 84, 319-332 [Medline] [Order article via Infotrieve]
  39. Wade, P. D., and Timiras, P. S. (1980) Dev. Neurosci. 3, 101-108 [Medline] [Order article via Infotrieve]

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