 |
 Previous Article | Next Article 
The Journal of Neuroscience, February 15, 1998, 18(4):1240-1249
Acetylcholinesterase Enhances Neurite Growth and Synapse
Development through Alternative Contributions of Its Hydrolytic
Capacity, Core Protein, and Variable C Termini
Meira
Sternfeld1,
Guo-li
Ming2,
Hong-jun
Song2,
Keren
Sela1,
Rina
Timberg1,
Mu-ming
Poo2, and
Hermona
Soreq1
1 Department of Biological Chemistry, The Life Sciences
Institute, The Hebrew University of Jerusalem, 91904, Israel, and
2 Department of Biology, University of California at San
Diego, La Jolla, California 92093-0357
 |
ABSTRACT |
Accumulated indirect evidence suggests nerve growth-promoting
activities for acetylcholinesterase (AChE). To determine unequivocally whether such activities exist, whether they are related to the capacities of this enzyme to hydrolyze acetylcholine and enhance synapse development, and whether they are associated with alternative splicing variants of AChEmRNA, we used four recombinant human AChEDNA
vectors. When Xenopus laevis embryos were injected with a vector expressing the synapse-characteristic human AChE-E6, which
contains the exon 6-encoded C terminus, cultured spinal neurons
expressing this enzyme grew threefold faster than co-cultured control
neurons. Similar enhancement occurred in neurons expressing an
insertion-inactivated human AChE-E6-IN protein, containing the same C
terminus, and displaying indistinguishable immunochemical and
electrophoretic migration properties from AChE-E6, but incapable of
hydrolyzing acetylcholine. In contrast, the nonsynaptic secretory human
AChE-I4, which contains the pseudointron 4-derived C terminus, did not
affect neurite growth. Moreover, no growth promotion occurred in
neurons expressing the catalytically active C-terminally truncated human AChE-E4, demonstrating a dominant role for the E6-derived C
terminus in neurite extension. Also, AChE-E6 was the only active enzyme
variant to be associated with Xenopus membranes.
However, postsynaptic length measurements demonstrated that both
AChE-E6 and AChE-E4 enhanced the development of neuromuscular junctions in vivo, unlike the catalytically inert AChE-E6-IN and
the nonsynaptic AChE-I4. These findings demonstrate an evolutionarily
conserved synaptogenic activity for AChE that depends on its hydrolytic capacity but not on its membrane association. Moreover, this
synaptogenic effect differs from the growth-promoting activity of AChE,
which is unrelated to its hydrolytic capacity yet depends on its exon 6-mediated membrane association.
Key words:
acetylcholinesterase; alternative C termini; neurogenesis; neurite extension; noncatalytic function; Xenopus spinal neurons; synaptogenesis; neuromuscular
junctions
| |
INTRODUCTION |
Acetylcholinesterase (AChE)
hydrolyzes the neurotransmitter acetylcholine (ACh) released from nerve
terminals at neuromuscular junctions (NMJs) and brain cholinergic
synapses, thus terminating synaptic transmission (Salpeter, 1967 ).
Potential noncatalytic functions of AChE were implicated by findings
that certain AChE inhibitors decrease chick neurite outgrowth in
culture and that externally added AChE stimulates this process
regardless of the presence of specific inhibitors (Layer et al., 1993;
Jones et al., 1995; Small et al., 1995). Sequence homology between AChE and several adhesion molecules (de La Escalera et al., 1990; Auld et
al., 1995; Ichtchenko et al., 1995) and the early appearance of AChE in
developing embryos before the onset of cholinergic neurotransmission
(Layer and Willbold, 1995) also suggest that AChE may play a
developmental function in cellular development and neuronal growth that
is unrelated to its classic ACh hydrolyzing activity. However,
experiments aimed at the noncatalytic nature of the neurogenic activity
of AChE were all based on the indirect use of inhibitors or involved
external addition of AChE to the culture medium (Jones et al., 1995) or
solid substrate (Layer et al., 1993; Small et al., 1995). This called
for studies in which the activity levels of AChE would be changed
within the tested neurons themselves.
Human pre-AChEmRNA may be alternatively spliced at its 3' end to yield
three mature AChEmRNAs encoding protein products with three distinct C
termini (Ben Aziz-Aloya et al., 1993; Karpel et al., 1994). These
include the brain-abundant exon 6-encoded C-terminal peptide, the
hematopoietic exon 5-encoded C terminus, which enables
glycophospholipid attachment, and the C terminus derived from the open
reading frame of the tumor-abundant pseudointron 4. The brain and
muscle human (h) AChE form (hAChE-E6), expressed in developing
Xenopus laevis embryos, accumulates in and enlarges the
postsynaptic length of neuromuscular junctions (NMJs) (Seidman et al.,
1994; 1995). Transgenic mice expressing hAChE-E6 show NMJ enlargement
and late onset neuromotor deterioration (Andres et al., 1997). In
contrast, DNA encoding the read-through form of hAChEmRNA (ACHE-I4/E5)
causes production and secretion of an enzyme C terminated by the
I4-encoded peptide (hAChE-I4) in ciliated and secretory epidermal cells
of Xenopus embryos. Moreover, hAChE-I4 did not reach NMJs or
affect their length (Seidman et al., 1995). When transfected into
glioma cells, ACHE-I4/E5 caused the appearance of small, processless
round cells, whereas hAChE-E6 transfection induced process extension
(Karpel et al., 1996). To explore the involvement of the catalytic
activity and the alternative C termini of AChE in its neurogenic or
synaptogenic activities, we constructed two novel hAChEDNA vectors. One
of these encodes a truncated form of the enzyme, devoid of any of the
natural C termini; the other encodes an insert-disrupted form of the
enzyme, incapable of hydrolyzing ACh yet recognized by anti-AChE
antibodies. These two constructs and the above hACHE-E6 and hACHE-I4/E5
DNAs were microinjected into Xenopus oocytes and embryos.
The biochemical and hydrodynamic properties of the resultant proteins
were then compared with the effects of each of these AChE variants on
neurite extension from spinal neurons and on in vivo NMJ
development in Xenopus.
| |
MATERIALS AND METHODS |
Construction of vectors. The plasmids referred to
here as ACHE-E6 and ACHE-I4/E5 have been described in detail (Ben
Aziz-Aloya et al., 1993; Seidman et al., 1995). To create a DNA
construct encoding a truncated form of hAChE, lacking either of the
native C termini, we used a two-phase PCR engineering procedure
(Higuchi, 1990) using the recombinant human ACHE cDNA and genomic
clones (Soreq et al., 1990). In the first PCR phase, performed
essentially as described (Karpel et al., 1994), ACHE-E6 served as
template. Two partially overlapping products were produced in which
ACHE exon 4 and the SV40 polyadenylation signal were joined together, using primers containing the overlapping sequence: E3/1522+
5'-CGGCTCTACGCCTACGTCTTTGAACAC CGTGCTTC-3';
E4del4-5'-TAACGTCGACTATCAGGTGGCGCTGAGCAATTTGGGGG-3'; E4del3+
5'-TTGCTCAGCGCCACCTGATAGTCGACGTTAACTTGTTTATTGCAGCTTATAATGG-3'; SV40
PolyA-5'ATGATTTGGACAAACCACAACTAGAATGCAGTG-3'.
Primers were named according to their position in the human ACHE
alternative sequences and vectors. After removal of the primers, the
two products were combined into one longer product by a second PCR
reaction in which they served as templates. External primers E3/1522+
and SV40 polyA were used in the second phase to create a fragment consisting of the 3' end of exon 3, exon 4, and the polyA signal. PCR
reaction was as above except that in the first cycle the denaturing step was at 94°C for 5 min and the annealing step was from 94 to
50°C (slope rate of 1°C per 30 sec). The product and the original ACHE-E6 plasmid were restricted using enzymes NotI and
SalI, and the two products were then ligated.
To construct the disrupted ACHE coding sequence we inserted an in-frame
sequence of 21 nucleotides, six bases downstream of the codon for the
active site serine, located in exon 2. The technique used was the same
two-phase PCR described above. In the first PCR phase we used primers
containing the inserted sequence. Primers used were E2/340+
5'-GCTTTCCTGGGCATCCCCTTTGCGGAGCCA-3';
E2ins2-5'-TCCaccgaattgaggatgtcgccacgcgctCTCCCCAAACAGCGT-3'; E2ins1+
5' - AGCGCGtggcgacatcctcaattcggtggaGCCGCCTCGGCGGGCAT - 3' ;
1212-5'-GAAGTCTCCCGCGTTGATCAGGGCCTCTGG-3'. The inserted sequence is
designated by lower case letters. Primers E2/340+ and E2ins2 were
used to link the inserted sequence to the upstream PCR product, and
primers E2ins1+ and E2/1212 were used to link it to the downstream product. The second PCR phase was performed using external primers E2/340+ and E2/1212. Stage II PCR products and ACHE-E6 were
restricted using enzymes BstEII and SphI and
ligated. First and second phase PCR reactions were as above. After
construction, the accuracy and integrity of both constructs was
validated by DNA sequencing.
Recombinant AChE production and assays of hydrolytic
activity. Xenopus oocytes were microinjected with 10 ng
of DNA of each recombinant AChE plasmid, incubated for 48 hr, and
homogenized in high-salt-detergent buffer as described previously
(Neville et al., 1990). Homogenates were frozen until use. AChE
activity was measured by evaluating acetylthiocholine (ATCh) hydrolysis using 96-well microtiter plates. pH dependence of AChE activity was
assessed using phosphate buffer at the pH range 5.8-8.0, with intervals of 0.2. For Km and substrate
inhibition experiments, we used ATCh in the concentration range of
0.05-60 mM and the GraFit 3.0 program (Erithacus Software
limited, Staines, UK). For enzyme stability studies, oocyte homogenates
were incubated at 19-42°C for 0-5 hr, after which AChE catalytic
activities in each of the homogenates were assessed as above.
Xenopus embryo microinjection and subcellular
fractionation. In vitro fertilization of mature
Xenopus eggs and blastomere microinjection were performed as
described elsewhere (Seidman et al., 1994), except that embryos were
raised in 19-21°C. Subcellular fractionation of 1-, 2-, and 3-d-old
embryos into low-salt (0.01 M Tris-HCl, pH 7.4, 0.05 M MgCl2, 144 mM NaCl),
low-salt-detergent (1% Triton X-100 in 0.01 M sodium
phosphate, pH 7.4), and high-salt (1 M NaCl in 0.01 M sodium phosphate, pH 7.4) buffers was performed as
described previously (Seidman et al., 1994).
Protein blot analyses and immunocytochemistry. Denaturing
SDS-PAGE and blotting were essentially as described elsewhere (Seidman et al., 1994), except that after transfer the blots were washed with
1× PBS (80 mM NaH2PO4, 20 mM Na2HPO4, pH 7.4), 0.5%
Tween-20, 18% glucose, 10% glycerol, 2.5% bovine serum albumin, and
1% skim milk. Immunodetection was performed using a pool of monoclonal antibodies (132-1,2,3; 6 µg/ml each) raised against denatured human
brain AChE, and a 1:2 × 104 dilution of a
horseradish-peroxidase-conjugated sheep anti-mouse IgG (Jackson
Laboratories, Bar Harbor, ME). Chemiluminescent detection was performed
with the ECL kit (Amersham Life Sciences) as instructed. Ten microliter
samples of oocyte homogenate (equivalent to ~50 ng AChE) were loaded
on each lane. For enzyme activity blots, we used nondenaturing gel
electrophoresis followed by incubation in ATCh staining mixture
(Seidman et al., 1995), using similar amounts of oocyte
homogenates.
For immunochemical AChE detection in situ, cells were fixed
with 2% paraformaldehyde in PBS for 30 min at room temperature and
then permeabilized with 0.1% Triton X-100 (20 min). After they were
washed with TBST (10 mM Tris-HCl, pH 7.0, 150 mM NaCl, 0.05% Tween-20), cells were incubated with
anti-human AChE antibodies (mouse monoclonal antibody 132-1; 1:1000
dilution in TBST containing 10% normal goat serum) at 4°C overnight.
After washes with TBST (5 × 30 min), cells were incubated with
goat anti-mouse IgG conjugated to fluorescein (Sigma) (1:100 in TBST
containing 10% normal goat serum) at 22°C for 2 hr. After the same
washing procedure as described above, cells were mounted and observed
under a Nikon Diaphot microscope with a 20×/1.25 objective.
Culture preparation. Xenopus nerve-muscle
cultures were prepared according to previously reported methods
(Spitzer and Lamborghini, 1976; Anderson et al., 1977; Tabti and Poo,
1995). Briefly, neural tubes and the associated myotomal tissue of
1-d-old Xenopus embryos (stage 20-23 according to Nieuwkoop
and Faber, 1967) were dissociated in
Ca2+-Mg2+-free saline
supplemented with EDTA (115 mM NaCl, 2.6 mM
KCl, 10 mM HEPES, 0.4 mM EDTA, pH 7.6) for
15-20 min. The cells were plated on glass coverslips and used for
experiments after 6 hr of incubation at room temperature. The culture
medium consisted (vol/vol) of 49% Leibovitz L-15 medium (Life
Technologies, Gaithersburg, MD), 1% bovine serum (Life Technologies),
and 50% Ringer's solution (115 mM NaCl, 2 mM
CaCl2, 2.6 mM KCl, 10 mM
HEPES, pH 7.6).
Neurite length measurements. Line drawings of isolated
neurons and their neuritic processes were traced from the video monitor display of recorded microscopic images. The tip of the growing neurite
was defined as the distal leading edge of the phase-dark palm of the
growth cone, without considering the filopodial extension. The entire
trajectory of the neurite, including all of its branches, was measured
with a digitizing pad (Houston Instruments), and the total length of
each neurite was calculated by a microcomputer. The rate of extension
(micrometers per hour) was determined by dividing the net increase in
neurite length (in micrometers) by the duration of observation (in
hours).
Electron microscopy and morphometric analyses. Histochemical
staining, transmission electron microscopy, and morphometric analyses
were performed as described previously (Ben Aziz-Aloya et al., 1993;
Seidman et al., 1995).
| |
RESULTS |
Construction and analyses of hAChE variants
To delineate functions and domains of AChE variants that are
involved in neuron growth and synaptogenesis, two novel hAChEDNA vectors were constructed by a two-phase PCR procedure (see Materials and Methods). One of these constructs, ACHE-E4 (Fig.
1A), carries a
truncated coding sequence containing exons 2, 3, and 4 of the human ACHE gene. The other construct, ACHE-E6-IN (Fig.
1A), encodes a protein identical to the
synapse-accumulating AChE-E6, except that it carries an in-frame insert
of seven amino acids near the active site protein sequence, which
should render it inactive. Similar to the previously used ACHE-E6 (Ben
Aziz-Aloya et al., 1993) (Fig. 1A) and ACHE-I4/E5
(Seidman et al., 1995) (Fig. 1A), transcription of
both these constructs was regulated by the cytomegalovirus promoter,
and they both contain the SV40 polyadenylation signal. Together, this
set of four constructs enabled us to explore the biochemical and
morphogenic activities of AChEs with three distinct C termini (encoded
by E6, I4, or E4) and of the synaptic AChE-E6 enzyme with or without
catalytic capacity.
View larger version (44K):
[in this window]
[in a new window]
|
Figure 1.
Biochemical properties of recombinant AChE
variants. A, Analyzed AChE DNAs and the DNA constructs
encoding each of the examined AChE variants. Common exons are
designated by black boxes, exon 6 by a hatched
box, and pseudointron 4 and exon 5 by dotted
boxes. See Materials and Methods for details on the
construction of these vectors. B, Hydrolytic
cholinesterase activities. ATCh hydrolyzing activities of each of the
enzyme forms encoded by the above ACHE constructs were tested in
homogenates of microinjected Xenopus oocytes. Presented
are average results of three experiments for each construct. Endogenous
Xenopus AChE activities were subtracted from activities
found for cDNA-injected oocytes. Note that AChE-E4 activity levels are
comparable with those of AChE-E6 and AChE-I4 and that AChE-E6-IN
displayed no significant catalytic activity. C,
Electrophoretic properties and antibody recognition of the AChE
variants. Homogenates of Xenopus oocytes microinjected
with each of the ACHE cDNA constructs and of control buffer-injected oocytes (C) were subjected to denaturing gel
electrophoresis followed by protein blot and immunodetection
(top) and to nondenaturing gel electrophoresis followed
by AChE activity staining (bottom). Each lane represents
~50 ng AChE. Note that AChE-E6-IN is highly immunoreactive but
displays no catalytic activity and that AChE-E4 migrates faster than
the other variants in the denaturing gel. In the bottom
panel, note that AChE-I4 displays heterogeneous bands and
migrates faster than AChE-E6 and AChE-E4. D, Substrate inhibition. The above oocyte homogenates were assayed for
cholinesterase activity in the presence of 0.05-60 mM ATCh
as substrate. Cholinesterase activity in each substrate concentration
is shown as percentage of the highest activity for each homogenate,
after subtraction of spontaneous ATCh hydrolysis. Shown is one
representative of two experiments. Inset,
Km values of the recombinant hAChE
variants.
|
|
For biochemical characterization of their protein products, all four
plasmids were microinjected into Xenopus oocytes. The catalytic activity of the resultant proteins was then assessed by
measuring the hydrolysis rate of acetylthiocholine (ATCh) in oocyte
homogenates. As predicted, oocytes expressing the disrupted form,
ACHE-E6-IN, displayed exceedingly low activity levels (Fig. 1B), which were similar to those of the two
experimental controls: buffer-injected and uninjected oocytes (not
shown), most probably reflecting the endogenous Xenopus AChE
activity levels. As expected, both natural AChE variants, terminated
with the E6-encoded C terminus or with that encoded by I4, showed high
activity levels (Fig. 1B), confirming previous
results (Schwarz et al., 1995a; Seidman et al., 1995). The novel
truncated form of AChE, encoded by ACHE-E4, displayed activity levels
within the range of the other two variants (Fig. 1B).
This demonstrated that neither of the natural C termini encoded by E6
or I4 is essential for the ACh hydrolytic activity of AChE.
Electrophoretic distinctions and hydrolytic similarities
In consideration of the possibility that the low activity levels
observed for the ACHE-E6-IN homogenates did not reflect inactivation but were caused by impaired production of this protein in oocytes, we
subjected the various recombinant hAChEs to denaturing gel electrophoresis followed by immunoblotting. Selective immunodetection of hAChE bands (Fig. 1C, top panel)
demonstrated that the Xenopus system is capable of producing
AChE-E6-IN in size and amounts comparable to those of the other AChE
variants. The electrophoretic migration distance of all variants except
AChE-E4 matched the expected molecular weight of 66 kDa, whereas
AChE-E4 migrated somewhat faster, consistent with its truncated C
terminus. Minor amounts of an immunopositive protein, with a migration
distance similar to that of AChE-E4, could also be seen in the lanes
loaded with the protein products of ACHE-E6 and ACHE-E6-IN (Fig.
1C, top panel). Catalytic activity and
intact C termini are therefore not obligatory requirements for
production and stability of this protein in the Xenopus
milieu.
When subjected to nondenaturing gel electrophoresis followed by AChE
activity staining, the enzyme produced by ACHE-E6-IN showed no
detectable catalytic activity, as opposed to the other variants, which
were all highly active (Fig. 1C, bottom
panel). The migration of AChE-E6 was considerably slower
and the band was much sharper than those of AChE-I4, which displayed
faster migrating heterogeneous bands. AChE-E4 showed an intermediate band. The four tested hAChE variants thus differed in their Stokes radius and charge and active site conformation, whereas they maintained similar primary folding and production efficiency.
Having shown that ACHE-E6, ACHE-I4/E5, and ACHE-E4 all encode for
active enzymes, we wished to examine whether changing the C terminus of
AChE did not affect its catalytic properties in a more subtle manner.
Interestingly, the Xenopus enzyme retained its full
catalytic activity after 5 hr at 42°C, whereas all hAChE variants
lost 50% of their activity (not shown). However, all three hAChE
variants were found to have Km values within the
same range of the previously reported value (0.3 mM for
AChE-E6 in Xenopus oocytes) (Seidman et al., 1994) and were
similarly inhibited by high substrate concentrations (Fig.
1D). Also, substrate hydrolysis by all AChE forms was
similarly enhanced within the pH range of 5.8-8.0 (data not shown), in
agreement with reports of others (for review, see Schwarz et al.,
1995b). There was therefore no indication whatsoever for involvement of
the variable C termini of AChE in its catalytic properties, consistent
with previous reports in which enzymatic cleavage of the C terminus was
used to obtain a homogeneous catalytically active AChE preparation for
x-ray diffraction analysis (Sussman et al., 1991).
Expression of human AChE in Xenopus spinal neurons
Expression of hAChE in Xenopus embryonic neurons was
examined after injection of each of the above hAChEDNA constructs into one of the blastomeres of two-cell stage Xenopus embryos. To
facilitate identification of living neurons expressing hAChE during
neurite growth assays, fluorescent dextran was co-injected with the
DNA. The progeny neurons of the injected blastomere could then be
identified by the presence of fluorescent dextran. Confirmation of AChE
expression in individual spinal neurons was then obtained by
immunocytochemical staining of the dissociated neurons from 1-d-old
embryos, using monoclonal antibodies specific for hAChE (Seidman et
al., 1995). The reliability of fluorescent dextran as a marker for
neurons overexpressing AChE was examined in the following experiment. Nerve-muscle cultures were prepared from embryos injected with ACHE-E6
cDNA and rhodamine-dextran. Dextran-positive neurons in 1-d-old
cultures were identified and recorded. The same cultures were then
processed for immunocytochemical staining with antibodies against AChE.
In control cultures prepared from embryos not injected with AChE-E6
cDNA, there was a negligible level of AChE staining. In cultures
prepared from AChE-E6 cDNA and dextran-injected embryos, we found that
92% (36 of 39 cells) of dextran-positive neurons exhibited AChE
staining, whereas 95% (55 of 58 cells) of dextran-negative neurons
exhibited undetectable AChE staining. Figure
2 depicts examples of (AChE+) and
(AChE) neurons, together with their bright-field images at 7, 8, and
9 hr in culture, before the staining of AChE. In this culture,
dextran-positive muscle cells also showed, as expected, elevated
staining with AChE. Thus, immunostaining for hAChE shows that hAChE-E6
is expressed in these spinal neurons and that dextran fluorescence is a
reliable marker for AChE expression.
View larger version (99K):
[in this window]
[in a new window]
|
Figure 2.
Neurons expressing human AChE-E6 (+) and control
neurons () in Xenopus cultures. Xenopus
embryos were co-injected with AChE-E6 DNA and rhodamine-dextran
complexes, and their spinal neurons were dissociated into culture
1 d later. Bright-field images were taken at 7, 8, and 9 hr after
cell plating. Both the total neurite length and the rate of neurite
growth were measured during this period. Fluorescence micrographs on
the right of the 9 hr photographs depict the rhodamine fluorescence of
dextran complexes, which were co-injected with the cDNA. Indirect
fluorescein immunofluorescence staining of AChE observed at the end of
the experiment is shown on the last right panel. Note
the correlation between dextran fluorescence and AChE staining.
Staining and imaging conditions were identical for all four cells,
which were from the same culture. Fluorescently labeled cells (+) were
positive with both red and green filters, whereas negative () cells
remained invisible in both. Scale bar, 20 µm.
|
|
Effects of expressing human AChE on neurite growth
Six hours after plating of dissociated Xenopus neural
tube cells in culture, many spinal neurons exhibit active neurite
outgrowth. Only isolated neurons not in contact with any other cell
were used in this study, on which two types of neurite growth assays were made. First, the total neurite length of each neuron in the culture was measured. Second, extension of individual neurites was
measured for a 3-4 hr period at 1 hr intervals (from 6 to 10 hr after
plating). The hAChE-expressing (AChE+) neurons were identified by the
presence of fluorescent dextran in the cell. To reduce
culture-to-culture variation, similar numbers of (AChE+) and control
neurons in the same culture or cultures from the same batch of embryos
were examined. This analysis revealed that the average neurite length
(the entire trajectory of the neurite, including all its branches) was
124.0 ± 11.7 µm (SEM; n = 32) in
(AChE-E6+)-expressing neurons, which is significantly longer (p < 0.001; two-tailed t test) than
that observed for noninjected neurons (88.2 ± 10.3 µm; SEM;
n = 33) or AChE-I4-expressing neurons (77.5 ± 8.2 µm; SEM; n = 27). To illustrate the overall
difference in neurite growth for a large number of neurons, composite
drawings were made by superimposing tracings of the video images of
randomly chosen neurons, 15 from each of the noninjected control,
(AChE-E6+), and (AChE-I4+) groups, respectively (Fig.
3). It is clear from this figure that the
net neurite extension over the first 9 hr in culture was substantially
longer in (AChE-E6+) neurons.
View larger version (22K):
[in this window]
[in a new window]
|
Figure 3.
Effects of expressing human AChE on the growth of
Xenopus spinal neurons. Composite concentric line
drawings were made from video images of 12 isolated spinal neurons at
6, 7, 8, and 9 hr after cell plating. The center of the neuronal soma
(deleted from this image) was placed in the center of each drawing.
Note consistent overall neurite length promotion in neurons that
expressed AChE-E6 but not AChE-I4. Neurons derived from uninjected
blastomeres served as controls. Scale bar, 20 µm.
|
|
The higher total neurite length at a particular time in culture may
reflect a higher growth rate of each neurite, an earlier onset of
neurite outgrowth from the soma after cell plating, or both. Direct
measurements of the growth rate of individual neurites during the 6-10
hr period revealed that (AChE-E6+) neurites indeed exhibit a higher
growth rate. As shown in Table 1, in the
same group of cultures, the average growth rate was 12.9 ± 2.7 µm/hr (SEM; n = 33) for noninjected control neurons
but was elevated to 37.9 ± 5.2 µm/hr (SEM; n = 32) for (AChE-E6+) neurons, the difference being highly significant
(p < 0.005; two-tailed t test). In
contrast, neurons expressing the read-through form AChE-I4 did not show
any difference in growth rate compared with the noninjected controls.
The enhanced growth rate of (AChE-E6+) neurons was sufficient to
account for the difference in the overall neurite length described above, although earlier neurite initiation could also have occurred. In
addition to the growth rate, we also measured the number of neuritic
branches in each group of neurons. As shown in Table 1, no significant
difference was found between any of the groups.
Growth promotion is unrelated to ACh hydrolytic activity
Whether the growth-promoting effect on Xenopus neurons
was partially or entirely caused by the catalytic activity of the
enzyme in hydrolyzing ACh was tested by expressing AChE-E6-IN, the
insertion-inactivated form of AChE-E6, which lacks ACh hydrolytic
activity (see above). As shown in Table 1, (AChE-E6+) and (ACHE-E6-IN+)
neurites were similar both in the total neurite length after 9-10 hr
in culture and in the rate of neurite growth during this period. Thus,
the ability of hAChE to promote growth was associated with the presence of the E6-derived C terminus, regardless of the catalytic activity.
The E6-derived C terminus is essential for growth promotion
The ability of AChE-E6 but not AChE-I4 to promote neurite growth
could be attributable to a dominant-positive effect of the AChE-E6 C
terminus (conferred by a sequence and/or structural element present in
this domain), or to a dominant-negative effect exerted by the
I4-encoded C terminus (i.e., the I4 peptide could interfere with the
growth-promoting properties of other domain(s) in the AChE core
protein). This problem was examined by expressing the truncated AChE-E4
form of AChE, which retains the ACh hydrolytic activity yet lacks
either the E6- or the I4-derived natural C-terminal peptides. The
inability of AChE-E4 to promote growth (Table 1) proved the first
option correct. Therefore, growth promotion of Xenopus
spinal neurons by AChE did not require ACh hydrolytic activity; neither
was it affected by the presence or absence of the I4-derived C
terminus. Rather, to exert this growth-promoting activity the
E6-derived synapse-characteristic C terminus must be present.
The growth promotion activity of AChE is associated with
membrane interaction
Despite their sequence and biochemical similarities, AChE-E6 and
AChE-I4 have previously been shown to be differentially localized in
Xenopus embryos, both intra- and intercellularly (Seidman et al., 1995). AChE-E6 was found to be associated with NMJs and AChE-I4 localized in epidermis and secreted therefrom. To compare the hydrodynamic and membrane association properties of the different recombinant hAChE variants in relation to their growth-promoting capacities, we microinjected ACHE-E4, ACHE-E6, or ACHE-I4/E5 DNAs into
in vitro fertilized Xenopus eggs. Sequential
extractions of injected embryos into low-salt, low-salt-detergent, and
high-salt buffers yielded hAChE-containing homogenates from 1-, 2-, and 3-d-old injected Xenopus embryos. AChE catalytic activities
measured in these homogenates ranged 2- to 10-fold higher than those of homogenates from uninjected embryos (data not shown) (Seidman et al.,
1995). Most importantly, the recombinant hAChE variants differed in
their membrane association. Catalytically active AChE in homogenates
from ACHE-E4-injected embryos was 92-95% soluble in low-salt buffer,
and AChE-I4 was 74-91% low-salt soluble (Fig. 4), as compared with 33-53% for
low-salt-soluble AChE-E6. A major fraction (20-50%) of hAChE-E6, but
no other active variant, partitioned into the low-salt-detergent
fraction, reconfirming our previous reports (Seidman et al., 1995) and
resembling the solubility pattern of the endogenous Xenopus
enzyme (Fig. 4). Thus, although AChE-E6 could be membrane-associated,
AChE-E4 and AChE-I4 appear to be soluble proteins that could be
secreted from the cells expressing them. Moreover, in all cases except
AChE-E4, but including the endogenous Xenopus enzyme, there
seemed to be a shift in solubility with development, from the low-salt
fraction to the detergent and high-salt fractions. This shift may
reflect a progressive increase in membrane interaction and association
with other components, such as the extracellular matrix, at the time
these components are being formed and neurons extend their neurites
in vivo.
View larger version (84K):
[in this window]
[in a new window]
|
Figure 4.
AChE-E6 exhibits developmentally increased
membrane association in vivo. Cleaving
Xenopus embryos were injected with the various ACHE DNA
vectors or with buffer (C), and sequential
extractions into low-salt-soluble (LSS),
low-salt-detergent-soluble (DS), and high-salt-soluble
(HSS) fractions were performed. Endogenous Xenopus AChE activities were subtracted from activities
of all other embryo samples. Slices therefore represent the net
relative fractions of the total summed activities for the host enzyme
and each hAChE variant. Note that AChE-E6 is similar to
Xenopus AChE in its lower solubility under low-salt
extraction, whereas AChE-E4 and AChE-I4 are both predominantly low-salt
soluble. Top, Schematic drawings of 1,- 2-, and 3-d-old
Xenopus embryos modeled after those of Deuchar
(1966).
|
|
The synaptogenic and neurite growth-promoting activities of AChE
are distinct
To compare the synaptogenic effect of AChE (Seidman et al., 1995)
with its neurite growth-promoting capacity, we examined neuromuscular
junctions (NMJs) from myotomes of 2-d-old embryos injected with the
above four vectors. AChE activity staining (Seidman and Soreq, 1996)
followed by transmission electron microscopy was used to assess the
in vivo localization of AChE and its synaptogenic activity
in Xenopus NMJs. As is apparent from the representative images of NMJs presented in Figure 5,
both AChE-E6 and the truncated AChE-E4 accumulated in NMJs in amounts
exceeding those in control NMJs. High levels of catalytically active
AChE, most likely of endogenous Xenopus origin, were also
detected in NMJs from embryos expressing AChE-E6-IN. In contrast,
AChE-I4 was absent from NMJs, confirming previous observations (Seidman
et al., 1995). These analyses demonstrated that although the E6-encoded
C terminus may promote the interaction of AChE with NMJ components, it
is not obligatory for NMJ accumulation of catalytically active AChE. Rather, the I4-encoded C terminus appeared to interfere dominantly with
the accumulation of AChE in NMJs. Moreover, postsynaptic length
measurements in 2-d-old embryos showed that transgenic expression of
both AChE-E6 and the soluble AChE-E4 enlarged NMJs from an average
length of 1.82 ± 0.16 µm (SEM; n = 55) to
significantly larger synapses with averages of 2.29 ± 0.16 µm
(SEM; n = 66) and 2.68 ± 0.36 µm (SEM;
n = 16), respectively (p < 0.05; two-tailed t test). In contrast, NMJs from embryos
expressing AChE-I4 or AChE-E6-IN displayed average postsynaptic lengths
that were somewhat smaller yet not significantly different from that of
control NMJs (1.69 ± 0.19 µm, SEM, n = 43, and
1.41 ± 0.23 µm, SEM, n = 31, respectively). The
columns in Figure 5 depict this change, which is reflected by a shift
in NMJ distribution between two groups: synapses with postsynaptic
lengths smaller or larger than 2.5 µm. Thus, both the synaptic
accumulation and the hydrolytic capacity of the transgenic enzyme were
found to be obligatory requirements for its ability to enhance NMJ
development. In contrast, the synaptogenic activity of AChE was found
to be unrelated to membrane association, unlike its neuritic
growth-promoting function.
View larger version (170K):
[in this window]
[in a new window]
|
Figure 5.
AChE-E6 and AChE-E4 enhance NMJ length, whereas
AChE-I4 and AChE-E6-IN do not. Two-day-old DNA-injected and control
uninjected Xenopus embryos were stained for
catalytically active AChE and examined by electron microscopy.
Representative images of NMJs from embryos injected with each of the
vectors are shown. Note the enhanced staining apparent as dark
electron-dense deposits in NMJs from AChE-E6-, AChE-E6-IN-, and
AChE-E4-injected embryos as compared with controls. T,
Nerve terminal; M, muscle cell; arrows
points at synaptic clefts. Bottom right panel, NMJ
population analysis. Electron microscope NMJ images (16, 31, 43, and 66 sections from AChE-E4, -E6-IN, -I4, and -E6 injected and 55 sections
from control uninjected embryos, respectively) were used for
postsynaptic length measurements. The percentage of synapses with
lengths shorter or longer than 2.5 µm are presented for NMJs from
embryos injected with each vector. Note that expression of AChE-E6 and
AChE-E4 increases postsynaptic length as compared with controls.
|
|
| |
DISCUSSION |
We used four recombinant hAChE variants to demonstrate that the
neurite growth-promoting activity of AChE in cultured
Xenopus neurons depends on the E6-encoded C terminus but not
on catalytic activity, whereas its synaptogenic property in live
Xenopus embryos depends both on its ability to accumulate
within the synapse and on its hydrolytic capacity. Thus, AChE plays two
distinct roles, with different mechanistic requirements, during nervous
system development.
That the C terminus of hAChE modifies the electrophoretic migration
properties of the enzyme under both native and denaturing conditions
demonstrated that it constitutes an independent domain in the AChE
protein, affecting its Stokes radius and surface charge. Human AChE-E6,
AChE-I4, and AChE-E4, all of which differ in their C termini, are
catalytically active (this report) and enzymatically indistinguishable
(Schwarz et al., 1995a). Cleavage of the E6-encoded C terminus does not
affect the catalytic activity of Torpedo AChE (Duval et al.,
1992), which demonstrates that the C terminus is not necessary for
substrate hydrolysis in vitro. Our current results further
reveal that the intact catalytic activity of AChE in Xenopus laevis is C terminus independent also in vivo.
Furthermore, an in-frame insertion of a foreign septapeptide near the
active site serine abolished the catalytic activity of the enzyme,
consistent with predictions of others (Taylor and Radic, 1994). Neither
of these alterations had an apparent effect on the amounts or the immunoreactivity of the enzyme produced in Xenopus oocytes
or embryos.
The minor fast-migrating immunoreactive band produced from the hAChE-E6
and hAChE-E6-IN proteins implies partial cleavage of the C terminus
encoded by E6, yielding a protein that is identical to hAChE-E4. The
electrophoretic heterogeneity of hAChE-I4 and the fact that part of the
hAChE-I4 protein migrated similarly to hAChE-E4 under nondenaturing gel
electrophoresis further suggested that the I4-encoded C terminus could
be positioned in several orientations relative to the core domain and
be accessible to proteases, consistent with findings of others (Sussman
et al., 1991). However, because only minor parts of hAChE-E6 or
hAChE-I4 co-migrate with hAChE-E4, we conclude that most of the former proteins indeed possessed their complete C termini.
Sequential extraction experiments demonstrated that the endogenous
Xenopus enzyme and hAChE-E6 partitioned mainly between the
low-salt and the low-salt-detergent fractions, whereas hAChE-E4 and
hAChE-I4 were almost completely solubilized in low salt. These observations extend previous findings (Seidman et al., 1995) that unlike hAChE-E6, hAChE-I4 is secreted to the medium by
Xenopus embryos and imply that the C terminus encoded by I4
does not contribute to the membrane interaction properties of this
enzyme. Furthermore, these results are in agreement with the well known
membrane association of AChE-E6 (for review, see Massoulie et al.,
1993). Therefore, of all AChE forms tested, only proteins containing
the E6-derived C terminus could associate with neuronal membranes and
support growth through such association.
The endogenous Xenopus enzyme, which has not yet been
extensively characterized or molecularly cloned, displayed hydrodynamic properties and subcellular interactions similar to those of transgenic hAChE-E6 and resembles the synaptic form of mammalian AChE encoded by
ACHE-E6. Xenopus AChE is more resistant to heat and to the anticholinesterase echothiophate than the human enzyme (this report) and does not form heteromeric multimers with hAChE (Seidman et al.,
1994). However, despite these distinctions, Xenopus neurons and NMJs respond to the morphogenic activities of hAChE in an evolutionarily conserved manner. This emphasizes the importance of
these functions and may allude to their early emergence in evolution.
In addition to its extracellular function in the hydrolysis of synaptic
ACh, we demonstrate here that when intracellularly expressed, the
membrane-associated AChE-E6 protein but not the alternatively spliced
AChE-I4-secreted protein promotes autologous neurite extension of
Xenopus neurons. Similar neurite growth activity can be
induced by mutation-inactivated AChE-E6 but not by the enzymatically
active truncated AChE-E4 enzyme. In cultures of chick sympathetic
neurons, rat hippocampus, or retinal ganglion cells, AChE inhibitors
interacting with the peripheral site of the enzyme prevented the
neurite growth effect and fasciculation exerted by externally added
AChE (Layer et al., 1993; Jones et al., 1995; Small et al., 1995).
However, the active site organophosphate inhibitor echothiophate, which
totally inhibits cholinesterase activity, did not block AChE-induced
growth. This indicated that the neurite promotion effects were not
caused by enzyme activity per se. However, the effect of these
pharmacological agents could be unrelated to their binding to AChE,
whereas elevation of the levels of intracellularly produced AChE
protein unequivocally establishes a novel noncatalytic function for
this protein. Like the process extension in rat glioma cells
microinjected with ACHE-E6 DNA (Karpel et al., 1996), the neurite
growth-promoting effect was spatially limited to those cells expressing
the enzyme and did not extend to adjacent, non-hAChE-expressing
neurons. This suggests that it involves the detergent-extractable
fraction of AChE-E6 and that it is associated with membrane protein
signaling.
How does AChE promote neurite growth? A group of adhesion molecules,
such as neurotactin, neuroligin, and gliotactin, contain extracellular
domains showing a uniformly distributed homology to cholinesterases.
Their cysteine positions correspond to those involved in the formation
of AChE intramolecular disulfide bonds, suggesting that these adhesion
molecules may resume tertiary structure similar to that of
cholinesterases. Although none of these proteins is a catalytically
active esterase, replacement of the extracellular domain of
Drosophila neurotactin with the core domain of AChE created
a chimeric protein promoting cell adhesion (Darboux et al., 1996).
Drosophila AChE itself or neurotactin lacking most of its
intracellular or extracellular domains failed to do so, suggesting
membrane-associated signaling operable with the AChE core domain. Thus,
AChE may promote neurite extension by modulating the adhesion capacity
of neurites.
 -Neurexins have been identified as the neuronal membrane partners
interacting with neuroligins (Ichtchenko et al., 1995). This indicates
that the core AChE domain encoded by exons 2-4 and corresponding to
the cholinesterase-like domains of neurotactin and neuroligins could
also operate by supporting recognition of neurexins and related
ligand(s). In mammals, it has been postulated that association between
-neurexins and neuroligins contributes to axon growth and cell-cell
and cell-extracellular matrix interactions (Puschel and Betz, 1995).
Unlike the core polypeptide of AChE, the E6-derived C terminus does not
share homology with the neurotactin family members (Seidman et al.,
1995) but could associate the enzyme with the cell membrane. Both
hAChE-E6 and hAChE-E6-IN, but not hAChE-E4 or hAChE-I4, could
potentially associate with the cell membrane through the E6 C terminus
and interact, through the core AChE domain, with a -neurexin-like
ligand expressed on the surface of the same or other cells or
associated with the extracellular matrix. This would elicit signal
transduction by the intracellular domain of the ligand, which can
induce neurite extension. Likewise, the process extension effect
exerted by hAChE-E6 on glia (Karpel et al., 1996) can be attributed to
interaction with the corresponding ligand of the AChE homologous
protein gliotactin (Auld et al., 1995). Moreover, the recent discovery
of the novel Neurexin 4 expressed in epithelial cells (Baumgartner et
al., 1996) extends this hypothesis also to non-neuronal sites. This theory is strengthened further by our recent observation that transgenic expression of hAChE in mice modulates the production of
-neurexins in the mouse spinal cord (Andres et al., 1997). Molecular
cloning of Xenopus neurexins would be required to further investigate this mechanism.
The novel function of AChE in promoting neurite growth explains the
early developmental involvement for this protein before synaptogenesis,
supporting descriptive theories based on its spatiotemporal expression
pattern in avian embryogenesis (Layer et al., 1995). It presents an
interesting example of multiple, seemingly unrelated functions for one
protein. That such functional duality in various tissues may be a more
general phenomenon than we are currently aware of is indicated from
findings with other proteins, such as lactate dehydrogenase (LDH),
which is both a hepatic enzyme (Baumgart et al., 1996) and a structural
crystallin protein in the lens (Chiou et al., 1991; Voorter et al.,
1993). Although ACh hydrolysis was the first and foremost identified
function of AChE, distinct elements on the surface of this protein
might have been preserved during evolution because of their interaction capacities with specific diverse molecules, which serve for different cellular functions.
Unlike its neurite growth-promoting activity, our current findings
demonstrate that the synaptogenic activity of AChE is tightly related
with its catalytic activity yet not dependent on the E6-derived C
terminus. Thus the synaptic accumulation of the catalytically active,
truncated AChE-E4 sufficed to enhance NMJ development in
vivo, whereas the inert AChE-E6-IN did not enlarge these synapses, although its structural properties suggest distribution similar to that
of the native enzyme. This in turn suggests that the I4-derived C
terminus is actively involved in the exclusion of AChE-I4 from the
synaptic cleft and attributes an important role to the effective termination of cholinergic neurotransmission in synapse development. Altogether, one should view AChE as a modular macromolecule, designed to transduce neurite growth signals as well as synapse development ones
by virtue of a concerted combination of its core protein domain,
alternative C termini and its ACh hydrolytic capacity.
| |
FOOTNOTES |
Received Oct. 2, 1997; revised Nov. 21, 1997; accepted Nov. 26, 1997.
This work was supported by grants from National Institutes of Health
(NS 31923) to M-m.P., and U.S. Army Medical Research and Development
Command (DAMD 17-97-1-7007), the Israeli Ministry of Defense, and the
Binational Science Foundation United States-Israel (96/00110/1) to H.S.
We thank Dr. U. Brodbeck, Bern, Switzerland, for anti-AChE antibodies,
and Ms. Daniela Kaufer for help with experiments.
M.S. and G.M. contributed equally to this work.
Correspondence should be addressed to Hermona Soreq, Department of
Biological Chemistry, The Life Sciences Institute, The Hebrew
University of Jerusalem, 91904, Israel.
| |
REFERENCES |
-
Anderson MJ,
Cohen MW,
Zorychta E
(1977)
Effects of innervation on the distribution of acetylcholine receptors on cultured muscle cells.
J Physiol (Lond)
268:731-756[Abstract/Free Full Text].
-
Andres C,
Beeri R,
Friedman A,
Lev-Lehman E,
Henis S,
Timberg R,
Shani M,
Soreq H
(1997)
Acetylcholinesterase-transgenic mice display embryonic modulations in spinal cord choline acetyltransferase and neurexin I gene expression followed by late-onset neuromotor degeneration.
Proc Natl Acad Sci USA
95:8173-8178.
-
Auld VJ,
Fetter RD,
Broadie K,
Goodman CS
(1995)
Gliotactin, a novel transmembrane protein on peripheral glia, is required to form the blood-nerve barrier in Drosophila.
Cell
81:757-767[Web of Science][Medline].
-
Baumgart E,
Fahimi H,
Stich A,
Volki A
(1996)
L-lactate dehydrogenase A4- and A3B isoforms are bona fide peroxisomal enzymes in rat liver. Evidence for involvement in intraperoxisomal NADH reoxidation.
J Biol Chem
271:3846-3855[Abstract/Free Full Text].
-
Baumgartner S,
Littleton JT,
Broadie K,
Bhat MA,
Harbecke R,
Lengyel JA,
Chiquet-Ehrismann R,
Prokop A,
Bellen HJ
(1996)
A Drosophila neurexin is required for septate junction and blood-nerve barrier formation and function.
Cell
87:1059-1068[Web of Science][Medline].
-
Ben Aziz-Aloya R,
Seidman S,
Timberg R,
Sternfeld M,
Zakut H,
Soreq H
(1993)
Expression of a human acetylcholinesterase promoter-reporter construct in developing neuromuscular junctions of Xenopus embryos.
Proc Natl Acad Sci USA
90:2471-2475[Abstract/Free Full Text].
-
Chiou SH,
Lee HJ,
Huang SM,
Chang GG
(1991)
Kinetic comparison of caiman epsilon-crystallin and authentic lactate dehydrogenases of vertebrates.
J Protein Chem
10:161-166[Web of Science][Medline].
-
Darboux I,
Barthalay Y,
Piovant M,
Hipeau-Jaczquotte R
(1996)
The structure-function relationships in Drosophila neurotactin show that cholinesterasic domains may have adhesive properties.
EMBO J
15:4835-4843[Web of Science][Medline].
-
de La Escalera S,
Bockamp EO,
Moya F,
Piovant M,
Jimenez F
(1990)
Characterization and gene cloning of neurotactin, a Drosophila transmembrane protein related to acetylcholinesterase.
EMBO J
9:3593-3601[Web of Science][Medline].
-
Deuchar EM
(1966)
In: Biochemical aspects of amphibian development. London: Methuen.
-
Duval N,
Massoulie J,
Bon S
(1992)
H and T subunits of acetylcholinesterase from Torpedo, expressed in COS cells generate all types of globular forms.
J Cell Biol
118:641-653[Abstract/Free Full Text].
-
Higuchi R
(1990)
Recombinant PCR.
In: PCR protocols (Innis MA,
Gellfand DH,
Sninsky JJ,
White TJ,
eds), pp 177-183. San Diego: Academic.
-
Ichtchenko K,
Hata Y,
Nguyen T,
Ullrich B,
Missler M,
Moomaw C,
Sudhof TC
(1995)
Neuroligin 1: a splice site-specific ligand for -neurexins.
Cell
81:435-443[Web of Science][Medline].
-
Jones SA,
Holmes C,
Budd TC,
Greenfield SA
(1995)
The effect of acetylcholinesterase on outgrowth of dopaminergic neurons in organotypic slice culture of rat midbrain.
Cell Tissue Res
279:323-330[Web of Science][Medline].
-
Karpel R,
Ben Aziz-Aloya R,
Sternfeld M,
Ehrlich G,
Ginzberg D,
Tarroni P,
Clementi F,
Zakut H,
Soreq H
(1994)
Expression of three alternative acetylcholinesterase messenger RNAs in human tumor cell lines of different tissue origins.
Exp Cell Res
210:268-277[Web of Science][Medline].
-
Karpel R,
Sternfeld M,
Ginzberg D,
Guhl E,
Graessman A,
Soreq H
(1996)
Overexpression of alternative human acetylcholinesterase forms modulates process extensions in cultured glioma cells.
J Neurochem
66:114-123[Web of Science][Medline].
-
Layer PG,
Willbold E
(1995)
Novel functions of cholinesterases in development, physiology and disease.
Prog Histochem Cytochem
29:1-99[Web of Science][Medline].
-
Layer PG,
Weikert T,
Alber R
(1993)
Cholinesterases regulate neurite growth of chick nerve cells in vitro by means of a non-enzymatic mechanism.
Cell Tissue Res
273:219-226[Web of Science][Medline].
-
Massoulie J,
Pezzementi L,
Bon S,
Krejci E,
Vallette F-M
(1993)
Molecular and cellular biology of cholinesterases.
Prog Neurobiol
41:31-91[Web of Science][Medline].
-
Neville LF,
Gnatt A,
Padan R,
Seidman S,
Soreq H
(1990)
Anionic site interactions in human butyrylcholinesterase disrupted by two adjacent single point mutations.
J Biol Chem
265:20735-20738[Abstract/Free Full Text].
-
Nieuwkoop PD,
Faber J
(1967)
In: Normal table of Xenopus laevis, Ed 2. Amsterdam: North Holland.
-
Puschel AW,
Betz H
(1995)
Neurexins are differentially expressed in the embryonic nervous system of mice.
J Neurosci
15:2849-2856[Abstract].
-
Salpeter M
(1967)
Electron microscope radioautography as a quantitative tool in enzyme cytochemistry. I. The distribution of acetylcholinesterase at motor endplates of a vertebrate twitch muscle.
J Cell Biol
32:379-389[Abstract/Free Full Text].
-
Schwarz M,
Loewenstein-Lichtenstein Y,
Glick D,
Liao J,
Norgaard-Pedersen B,
Soreq H
(1995a)
Successive organophosphate inhibition and oxime reactivation reveals distinct responses of recombinant human cholinesterase variants.
Mol Brain Res
31:101-110[Medline].
-
Schwarz M,
Glick D,
Loewenstein Y,
Soreq H
(1995b)
Engineering of human cholinesterases explains and predicts diverse consequences of administration of various drugs and poisons.
Pharmacol Ther
67:283-322[Web of Science][Medline].
-
Seidman S,
Soreq H
(1996)
In: Transgenic Xenopus microinjection methods and developmental neurobiology. Neuromethods series (Boulton A, Baker GB, eds), pp 1-198. Totowa, NJ: Humana.
-
Seidman S,
Ben Aziz-Aloya R,
Timberg R,
Loewenstein Y,
Velan B,
Shaffeman A,
Liao J,
Norgaard-Pedersen B,
Brodbeck U,
Soreq H
(1994)
Overexpressed monomeric human acetylcholinesterase induces subtle ultrastructural modifications in developing neuromuscular junctions of Xenopus laevis embryos.
J Neurochem
62:1670-1681[Web of Science][Medline].
-
Seidman S,
Sternfeld M,
Ben Aziz-Aloya R,
Timberg R,
Kaufer-Nachum D,
Soreq H
(1995)
Synaptic and epidermal accumulation of human acetylcholinesterase are encoded by alternative 3'-terminal exons.
Mol Cell Biol
15:2993-3002[Abstract].
-
Small DH,
Reed G,
Whitefield B,
Nurcombe V
(1995)
Cholinergic regulation of neurite outgrowth from isolated chick sympathetic neurons in culture.
J Neurosci
15:144-151[Abstract].
-
Soreq H,
Ben Aziz R,
Prody CA,
Seidman S,
Gnatt A,
Neville L,
Lieman-Hurwitz J,
Lev-Lehman E,
Lapidot-Lifson Y,
Zakut H
(1990)
Molecular cloning and construction of the coding region for human acetylcholinesterase reveals a G+C-rich attenuating structure.
Proc Natl Acad Sci USA
87:9688-9692[Abstract/Free Full Text].
-
Spitzer NC,
Lamborghini JC
(1976)
The development of the action potential mechanism of amphibian neurons isolated in culture.
Proc Natl Acad Sci USA
73:1641-1645[Abstract/Free Full Text].
-
Sussman JL,
Harel M,
Frolow F,
Oefner C,
Goldman A,
Toker L,
Silman I
(1991)
Atomic structure of acetylcholinesterase from Torpedo californica: a prototypic acetylcholine-binding protein.
Science
253:872-879[Abstract/Free Full Text].
-
Tabti N,
Poo M-m
(1995)
Study on the induction of spontaneous transmitter release at early nerve-muscle contacts in Xenopus cultures.
Neurosci Lett
173:21-26.
-
Taylor P,
Radic Z
(1994)
Cholinesterases: from genes to proteins.
Annu Rev Pharmacol Toxicol
34:281-320[Web of Science][Medline].
-
Voorter CE,
Wintjes LT,
Heinstra PW,
Bloemendal H,
De-Jong WW
(1993)
Comparison of stability properties of lactate dehydrogenase B4/epsilon-crystalline from different species.
Eur J Biochem
211:643-648[Web of Science][Medline].
Copyright © 1998 Society for Neuroscience 0270-6474/98/1841240-10$05.00/0
This article has been cited by other articles:
|
|
|
|
 
T. Evron, B. C. Geyer, I. Cherni, M. Muralidharan, J. Kilbourne, S. P. Fletcher, H. Soreq, and T. S. Mor
Plant-derived human acetylcholinesterase-R provides protection from lethal organophosphate poisoning and its chronic aftermath
FASEB J,
September 1, 2007;
21(11):
2961 - 2969.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Gilboa-Geffen, P. P. Lacoste, L. Soreq, G. Cizeron-Clairac, R. Le Panse, F. Truffault, I. Shaked, H. Soreq, and S. Berrih-Aknin
The thymic theme of acetylcholinesterase splice variants in myasthenia gravis
Blood,
May 15, 2007;
109(10):
4383 - 4391.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
H. Q. Xie, R. C. Y. Choi, K. W. Leung, N. L. Siow, L. W. Kong, F. T. C. Lau, H. B. Peng, and K. W. K. Tsim
Regulation of a Transcript Encoding the Proline-rich Membrane Anchor of Globular Muscle Acetylcholinesterase: THE SUPPRESSIVE ROLES OF MYOGENESIS AND INNERVATING NERVES
J. Biol. Chem.,
April 20, 2007;
282(16):
11765 - 11775.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Deschenes-Furry, K. Mousavi, F. Bolognani, R. L. Neve, R. J. Parks, N. I. Perrone-Bizzozero, and B. J. Jasmin
The RNA-Binding Protein HuD Binds Acetylcholinesterase mRNA in Neurons and Regulates its Expression after Axotomy
J. Neurosci.,
January 17, 2007;
27(3):
665 - 675.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
E. Huchard, M. Martinez, H. Alout, E. J.P Douzery, G. Lutfalla, A. Berthomieu, C. Berticat, M. Raymond, and M. Weill
Acetylcholinesterase genes within the Diptera: takeover and loss in true flies
Proc R Soc B,
October 22, 2006;
273(1601):
2595 - 2604.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Pick, C. Perry, T. Lapidot, C. Guimaraes-Sternberg, E. Naparstek, V. Deutsch, and H. Soreq
Stress-induced cholinergic signaling promotes inflammation-associated thrombopoiesis
Blood,
April 15, 2006;
107(8):
3397 - 3406.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
C. A. Rosenfeld and L. G. Sultatos
Concentration-Dependent Kinetics of Acetylcholinesterase Inhibition by the Organophosphate Paraoxon
Toxicol. Sci.,
April 1, 2006;
90(2):
460 - 469.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Grisaru, M. Pick, C. Perry, E. H. Sklan, R. Almog, I. Goldberg, E. Naparstek, J. B. Lessing, H. Soreq, and V. Deutsch
Hydrolytic and Nonenzymatic Functions of Acetylcholinesterase Comodulate Hemopoietic Stress Responses
J. Immunol.,
January 1, 2006;
176(1):
27 - 35.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
A. Dori, J. Cohen, W. F. Silverman, Y. Pollack, and H. Soreq
Functional Manipulations of Acetylcholinesterase Splice Variants Highlight Alternative Splicing Contributions to Murine Neocortical Development
Cereb Cortex,
April 1, 2005;
15(4):
419 - 430.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. X. S. Jiang, R. C. Y. Choi, N. L. Siow, H. H. C. Lee, D. C. C. Wan, and K. W. K. Tsim
Muscle Induces Neuronal Expression of Acetylcholinesterase in Neuron-Muscle Co-culture: TRANSCRIPTIONAL REGULATION MEDIATED BY cAMP-DEPENDENT SIGNALING
J. Biol. Chem.,
November 14, 2003;
278(46):
45435 - 45444.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
J. Deschenes-Furry, G. Belanger, N. Perrone-Bizzozero, and B. J. Jasmin
Post-transcriptional Regulation of Acetylcholinesterase mRNAs in Nerve Growth Factor-treated PC12 Cells by the RNA-binding Protein HuD
J. Biol. Chem.,
February 14, 2003;
278(8):
5710 - 5717.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
T. BRENNER, Y. HAMRA-AMITAY, T. EVRON, N. BONEVA, S. SEIDMAN, and H. SOREQ
The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis
FASEB J,
February 1, 2003;
17(2):
214 - 222.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
N. Farchi, H. Soreq, and B. Hochner
Chronic acetylcholinesterase overexpression induces multilevelled aberrations in mouse neuromuscular physiology
J. Physiol.,
January 1, 2003;
546(1):
165 - 173.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. B. Abou-Donia, L. B. Goldstein, and W. A. Khan
Reply
Toxicol. Sci.,
August 1, 2002;
68(2):
517 - 519.
[Full Text]
[PDF]
|
|
|
|
|
|
|
R. P Beliles
Concordance across species in the reproductive and developmental toxicity of tetrachloroethylene
Toxicology and Industrial Health,
March 1, 2002;
18(2):
91 - 106.
[Abstract]
[PDF]
|
|
|
|
|
|
|
M. B. Abou-Donia, A. M. Dechkovskaia, L. B. Goldstein, S. L. Bullman, and W. A. Khan
Sensorimotor Deficit and Cholinergic Changes following Coexposure with Pyridostigmine Bromide and Sarin in Rats
Toxicol. Sci.,
March 1, 2002;
66(1):
148 - 158.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. B. Abou-Donia, L. B. Goldstein, K. H. Jones, A. A. Abdel-Rahman, T. V. Damodaran, A. M. Dechkovskaia, S. L. Bullman, B. E. Amir, and W. A. Khan
Locomotor and Sensorimotor Performance Deficit in Rats following Exposure to Pyridostigmine Bromide, DEET, and Permethrin, Alone and in Combination
Toxicol. Sci.,
April 1, 2001;
60(2):
305 - 314.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Sternfeld, S. Shoham, O. Klein, C. Flores-Flores, T. Evron, G. H. Idelson, D. Kitsberg, J. W. Patrick, and H. Soreq
Excess "read-through" acetylcholinesterase attenuates but the "synaptic" variant intensifies neurodeterioration correlates
PNAS,
July 5, 2000;
(2000)
140004597.
[Abstract]
[Full Text]
|
|
|
|
|
|
|
Y. Li, L. Liu, J. Kang, J. G. Sheng, S. W. Barger, R. E. Mrak, and W. S. T. Griffin
Neuronal-Glial Interactions Mediated by Interleukin-1 Enhance Neuronal Acetylcholinesterase Activity and mRNA Expression
J. Neurosci.,
January 1, 2000;
20(1):
149 - 155.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Kaufer, A. Friedman, and H. Soreq
Review : The Vicious Circle of Stress and Anticholinesterase Responses
Neuroscientist,
May 1, 1999;
5(3):
173 - 183.
[Abstract]
[PDF]
|
|
|
|
|
|
|
G. Feng, E. Krejci, J. Molgo, J. M. Cunningham, J. Massoulie, and J. R. Sanes
Genetic Analysis of Collagen Q: Roles in Acetylcholinesterase and Butyrylcholinesterase Assembly and in Synaptic Structure and Function
J. Cell Biol.,
March 22, 1999;
144(6):
1349 - 1360.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
D. Grisaru, E. Lev-Lehman, M. Shapira, E. Chaikin, J. B. Lessing, A. Eldor, F. Eckstein, and H. Soreq
Human Osteogenesis Involves Differentiation-Dependent Increases in the Morphogenically Active 3' Alternative Splicing Variant of Acetylcholinesterase
Mol. Cell. Biol.,
January 1, 1999;
19(1):
788 - 795.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Grifman, N. Galyam, S. Seidman, and H. Soreq
Functional redundancy of acetylcholinesterase and neuroligin in mammalian neuritogenesis
PNAS,
November 10, 1998;
95(23):
13935 - 13940.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
|
|
M. Sternfeld, S. Shoham, O. Klein, C. Flores-Flores, T. Evron, G. H. Idelson, D. Kitsberg, J. W. Patrick, and H. Soreq
Excess "read-through" acetylcholinesterase attenuates but the "synaptic" variant intensifies neurodeterioration correlates
PNAS,
July 18, 2000;
97(15):
8647 - 8652.
[Abstract]
[Full Text]
[PDF]
|
|
|
|
|
Top
Abstract
Introduction
