(Received for publication, July 3, 1995; and in revised form, November 2, 1995)
From the
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.
Acetylcholinesterase (AChE) ()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
10
M 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.
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.
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.
Run-on
transcription assays were performed essentially as described by
Greenberg and Bender(1987). [-
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
[
-
P]UTP was 3-6
10
cpm/10
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 -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
SSC buffer, incubated with 10
µg/ml RNase A for 30 min at 37 °C, and rewashed with 2
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.
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.
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 100; C-F,
magnification, 630
.)
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.
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 -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.
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
-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).
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 10
M 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.