From the Department of Cellular and Molecular
Medicine and Centre for Neuromuscular Disease, Faculty of Medicine,
University of Ottawa, Ottawa, Ontario K1H 8M5, Canada and the
¶ Department of Neurosciences, University of New Mexico School of
Medicine, Albuquerque, New Mexico 87131-5223
Received for publication, September 12, 2002, and in revised form, December 1, 2002
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ABSTRACT |
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Expression of acetylcholinesterase (AChE) is
greatly enhanced during neuronal differentiation, but the nature of the
molecular mechanisms remains to be fully defined. In this study, we
observed that nerve growth factor treatment of PC12 cells leads to a
progressive increase in the expression of AChE transcripts, reaching
~3.5-fold by 72 h. Given that the AChE 3'-untranslated region
(UTR) contains an AU-rich element, we focused on the potential role of
the RNA-binding protein HuD in mediating the increase in AChE mRNA
seen in differentiating neurons. Using PC12 cells engineered to stably
express HuD or an antisense to HuD, our studies indicate that HuD can
regulate the abundance of AChE transcripts in neuronal cells.
Furthermore, transfection of a reporter construct containing the AChE
3'-UTR showed that this 3'-UTR can increase expression of the reporter gene product in cells expressing HuD but not in cells expressing the
antisense. RNA gel shifts and Northwestern blots revealed an increase
in the binding of several protein complexes in differentiated neurons.
Immunoprecipitation experiments demonstrated that HuD can bind directly
AChE transcripts. These results show the importance of
post-transcriptional mechanisms in regulating AChE expression in
differentiating neurons and implicate HuD as a key
trans-acting factor in these events.
Acetylcholinesterase
(AChE)1 is the enzyme
responsible for the rapid hydrolysis of acetylcholine in the central
and peripheral nervous systems (see, for review, Refs. 1-4). The
enzyme exists in multiple molecular forms that differ in their C
terminus and mode of anchoring to subcellular structures. The different
C termini of the protein are generated through alternative splicing of
a single gene, and three different mature mRNAs, referred to as the
T (tail), H (hydrophobic), and R (readthrough) transcripts, can be
produced. The pattern of expression of these transcripts is known to be
tissue-specific because, for example, the T transcript is abundantly
expressed in excitable cells such as skeletal muscle and neurons,
whereas the H transcript is found predominantly in the hematopoietic
lineage. Two polyadenylation signals can be used in the 3'-untranslated
region (UTR) to produce a ~2.4- or 3.2-kb transcripts. Choice of the
polyadenylation signal is also tissue-specific because neurons appear
to preferentially express the shorter form of the transcript whereas
skeletal muscle express both species but in different amounts
(5-7).
In addition to its role in cholinergic neurotransmission, converging
lines of evidence indicate that it likely fulfills additional, non-catalytic functions within the nervous system (see, for review, Ref. 8). For example, Northern blot analysis and in situ
hybridization experiments have revealed the presence of AChE mRNAs
in non-cholinergic areas of the brain such as the cerebellum (9-12).
Moreover, AChE expression in the brain is known to precede the
establishment of synaptic transmission and coincides with the period of
neurite outgrowth (13, 14). Finally, several studies in which AChE levels have been experimentally manipulated directly support the notion
that AChE is indeed involved in neurite outgrowth (see, for example,
Refs. 13-15 and 17-22).
In recent years, there have been several studies that have examined the
basic molecular events that preside over AChE expression in developing
and adult skeletal muscles (see, for example, Refs. 23-30). By
contrast, there is relatively little information concerning the
mechanisms regulating AChE expression in neurons. In addition, of the
few available reports, there also appear to be some contradictory findings. In particular, Greene and Rukenstein (31) have provided evidence indicating that differentiation of PC12 cells, which leads to
an increase in AChE expression, induces an increase in AChE gene
transcription. Alternatively, embryonic P19 carcinoma cells induced to
differentiate into neurons via retinoic acid treatment failed to
increase the transcriptional activity of the AChE gene, thereby
indicating that post-transcriptional mechanisms represent key events in
regulating the abundance of AChE transcripts during neuronal
development (32). Given the diverse and key functions of AChE within
the nervous system, it appears important to gain a more complete
understanding of the molecular mechanisms that control AChE expression
in neurons. Accordingly, we have initiated a series of experiments in
attempts to characterize some of the molecular events involved in AChE
expression during neuronal differentiation. Specifically, we have
examined the importance of transcriptional and post-transcriptional
mechanisms in the regulation of AChE during NGF-induced differentiation
of PC12 cells.
Cell Culture--
PC12 cells, a rat pheochromocytoma-derived
cell line (33), were cultured on culture dishes coated with type I
collagen (Sigma) in Dulbecco's modified Eagle's medium
(Invitrogen) supplemented with 10% horse serum, 5% fetal
bovine serum, and 100 units/ml penicillin-streptomycin, in a humidified
chamber at 37 °C containing 5% CO2. Stably transfected
PC12 cells were maintained as described elsewhere (34). These lines
were transfected with the pcDNA3 vector alone (pcDNA) or with
plasmids containing the human HuD sequence in sense (pcHuD) or
antisense (pDuH) orientation. All cells were plated at a density of
1-2 × 105 cells/cm2 and induced to
differentiate by adding 100 ng/ml 7S-NGF (Sigma) to the culture medium.
Culture media were changed every 72 h.
AChE Enzymatic Assay--
Cultures of undifferentiated and
differentiated PC12 cells (three 35-mm wells) were washed with cold
phosphate-buffered saline (PBS), scraped, and homogenized on ice with a
glass Kontes homogenizer in 0.5 ml of a high salt detergent buffer
containing anti-proteolytic agents (10 mM Tris-HCl, pH 7.0, 10 mM EDTA, 1 M NaCl, 1% Triton X-100, 1 mg/ml
bacitracin (Sigma), and 25 units/ml aprotinin (Sigma)). Following
centrifugation of the homogenates (20,000 × g for 15 min at 4 °C), the supernatant was removed and stored immediately at
RNA Extraction and RT-PCR--
Total RNA was extracted from
undifferentiated and differentiated PC12 cell cultures (three 35-mm
wells) using 0.5-1 ml of TRIzol reagent (Invitrogen) according to the
instructions from the manufacturer. Briefly, the cells were scraped
from the plates and disrupted by vigorous pipetting, followed by
addition of chloroform. The resulting solution was mixed vigorously and
centrifuged (12,000 × g for 15 min at 4 °C). The
aqueous layer was then transferred to a fresh tube and combined with an
equal volume of isopropanol. The RNA was precipitated by
centrifugation, and the resulting pellet was washed twice with 75%
ice-cold ethanol and resuspended in RNase-free water. All samples were
stored in
RNA from each sample was quantified using the Amersham
Biosciences Gene Quant II RNA/DNA spectrophotometer and adjusted
to a final concentration of 80 ng/µl. Reverse transcription of RNA was performed using 2 µl of each RNA sample at 42 °C for 45 min, followed by 5 min at 99 °C, as previously described elsewhere (37-39). Negative controls consist of the same RT mixture in which the
RNA was replaced with 2 µl of RNase-free water. PCR was used to
amplify cDNAs corresponding to AChE and S12 rRNA as described in
detail elsewhere (23, 37-39). Primers for AChE (see Ref. 12) and S12
rRNA (used as an internal control; see Ref. 40) were synthesized based
on available sequences that have been previously described, and they
amplified products of 670 and 368 bp, respectively. PCR cycling
parameters for AChE and S12 rRNA consisted of denaturation for 1 min at
94 °C, followed by primer annealing and extension for 3 min at
70 °C for AChE and primer annealing for 1 min at 54 °C and
extension for 2 min at 72 °C for S12 rRNA, followed by a 10-min
elongation step at 72 °C. PCR products were visualized and
quantified on ethidium bromide-stained 1.5% agarose gels. Quantitation
of the labeling intensity of the PCR products was performed using the
Kodak Digital Science Image Station 440 CF and related Kodak Digital
Science 1D Image Analysis Software (Eastman Kodak Co.). All values
obtained for AChE were corrected according to the corresponding level
of S12 rRNA present in the sample.
All RT-PCR experiments aimed at determining the relative abundance of
AChE transcripts were performed using cycle numbers that fell within
the linear range of amplification (37-39). The cycle numbers were
between 24 and 27 for AChE and 22 for S12 rRNA. RT-PCR conditions
(primer concentration, input RNA, choice of RT primer, cycling
conditions) were initially optimized, and these were identical for all
experiments. Appropriate precautions (use of sterile filtered tips and
gloves) were taken to prevent contamination of the samples and
degradation of the RNA. Samples, including the negative control, were
always prepared using the same RT and PCR reagents and master mixes,
and were run in parallel. In all experiments, PCR products were never
detected in the negative controls.
Nuclear Run-on Assays--
Nuclear run-on assays were performed
as described in detail elsewhere (23, 41, 42). Briefly, nuclei were
isolated from undifferentiated and differentiated PC12 cells (two
250-ml flasks) and resuspended in a transcription buffer containing
GTP, ATP, CTP, and 25 µCi of [ Reporter Constructs and Transfection Studies--
The recently
described 5.3-kb AChE promoter fragment termed GRAP (25) was subcloned
into a LacZ reporter vector. In addition, the 3'-UTR from the mouse
~2.4-kb AChE transcript, which contains the shorter 3'-UTR in
comparison to the ~3.2-kb AChE mRNA (see Introduction), was
amplified by RT-PCR and first inserted into the pGL3 vector. For these
experiments, we focused on the short 3'-UTR as opposed to the longer
one because (i) previous studies showed that it appears to contain
important cis-acting regulatory elements (24, 43, 44), and
(ii) the ~2.4-kb AChE transcript is considerably more abundant in
nervous tissues (12) including PC12 cells (45, 46). The 3'-UTR was
subsequently cloned into a luciferase reporter construct driven by the
thymidine kinase promoter (phRG-TK) (Promega).
Plasmid DNA was prepared using the Mega-Prep procedure (Qiagen,
Chatsworth, CA). DNA pellets were resuspended in 10 mM
Tris-HCl, pH 8.5. Transfections were performed using the LipofectAMINE
reagent kit (Invitrogen) according to the instructions from the
manufacturer. Undifferentiated cells (1-2 × 105
cells/cm2) were transfected with 0.5 µg of the
appropriate reporter gene construct and 0.5 µg of the constitutively
expressed chloramphenicol acetyltransferase (CAT) plasmid driven by the
SV40 promoter used, in this case, to control for transfection
efficiency. Transfected cells were induced to differentiate 24 h
later by the addition of NGF.
To determine reporter gene activity, undifferentiated and
differentiated cells were washed with cold PBS and lysed in
Reporter-Lysis buffer (Promega) following two cycles of freezing and
thawing. The extracts were then centrifuged (15,000 × g for 2 min at 4 °C), and the resulting supernatants were
assayed for In Vitro Transcription--
cDNAs encoding different lengths
of the AChE 3'-UTR were obtained by PCR amplification of the plasmid
template pGL3-3'-UTR (see above). The PCR primers employed to amplify:
1) the full-length AChE 3'-UTR, 2) a truncated fragment in which the
AU-rich element was absent ( Electrophoretic Mobility Shift Assay and Northwestern
Analyses--
RNA-based electrophoretic mobility shift assays (REMSAs)
and Northwestern blots were performed using total protein extracts obtained from undifferentiated and differentiated cells (two 250-ml flasks). The cells were washed with cold PBS, scraped, and lysed in 300 µl of homogenization buffer (0.3 M sucrose, 60 mM NaCl, 15 mM Tris, pH 8.0, 10 mM
EDTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM
benzamidine, 10 µg/µl leupeptin, 10 µg/µl pepstatin A, 1 µg/µl aprotinin, pH 7.4). The samples were centrifuged (15,000 × g for 15 min at 4 °C), and the resulting supernatant
was stored at
REMSAs were performed as described elsewhere in detail (47-49). Forty
µg of protein extract were incubated for 20 min at room temperature
with 1 × 105 cpm of 32P-labeled AChE
3'-UTR fragments in 2× binding buffer (20 mM Hepes, pH
7.9, 3 mM MgCl2, 50 mM KCl, 1 mM DTT, 5% glycerol, 0.2 µg/µl yeast tRNA) in a total
volume of 20 µl. The unbound RNA was digested with ribonuclease T1
(Calbiochem, San Diego, CA) for 20 min at 37 °C, and the samples
were then incubated at room temperature for 10 min with heparin (2.5 mg/ml). This mixture was separated by 4 or 6% native polyacrylamide
gel electrophoresis with 0.5× TBE (Tris borate-EDTA) running buffer.
The gels were subsequently dried under vacuum at 80 °C for 1 h
and exposed to x-ray film at
Northwestern analyses were performed according to the procedure
described elsewhere (50, 51). Fifty µg of protein extract diluted in
SDS buffer (50 mM Tris-HCl, pH 6.8, 100 mM DTT,
2% SDS, 0.1% bromphenol blue, 10% glycerol) were denatured at
100 °C for 3 min and separated by 8% SDS-PAGE. After separation,
proteins were electroblotted onto a polyvinylidene difluoride membrane (Schleicher & Schuell). The membrane was then incubated in renaturation buffer (15 mM Hepes, pH 7.9, 50 mM KCl, 0.1 mM MnCl2, 0.1 mM ZnCl2, 0.1 mM EDTA, 0.5 µM DTT, 0.1% (w/v) Ficoll
400 D-L, 0.1% (w/v) polyvinylpyrrolidone, and 0.01% (v/v)
Igepal CA-630 (a Nonidet P-40 substitute) in RNase-free water) at
4 °C overnight. Following pre-hybridization at room temperature for
1 h in renaturation buffer containing 0.2 mg/ml yeast tRNA, the
membrane was incubated for 4 h with 1 × 106
cpm/ml of a probe corresponding to the radiolabeled AChE 3'-UTR RNA
dissolved in renaturation buffer containing 0.2 mg/ml yeast tRNA and 5 mg/ml heparin at room temperature. After several 5-min washes, the
membrane was put to x-ray film at Immunoprecipitation and AChE mRNA Analysis--
The
RNA-binding protein HuD was immunoprecipitated from a total protein
extract as described (52). Total protein was extracted from PC12 cells
stably transfected to express HuD (8 × 100-mm plate). To this
end, the cells were pelleted, resuspended in 0.3 ml of
immunoprecipitation (IP) buffer (1% Igepal CA-630, 10 mM Tris-HCl, pH 7.5, 1% bovine serum albumin, 150 mM NaCl, 2 mM EDTA, 25 µg/µl pepstatin A, 2.5 µg/µl aprotinin,
10 units of RNase inhibitor), and sonicated (10-s pulse at 50% duty
cycle and a power output of 1 using the Branson Sonifier 450). Protein
samples (200 µg) were incubated for 1 h at 4 °C, with an
affinity-purified antibody to HuD previously described (34) or with
normal rabbit IgG (Jackson Immunoresearch Laboratories, West Grove, PA)
in IP buffer. This reaction mixture was subsequently added to 20 µl
of pre-washed A/G chimera Sepharose beads (Pierce) and incubated by
gentle shaking at 4 °C for 1 h. The mixture was centrifuged
(10,000 × g for 20 s), and the supernatant was
removed. After several washes with IP buffer, the RNA was extracted
from the pellet using the TRIzol reagent and analyzed by RT-PCR as
described above.
Western Blot--
Cultured cells were washed in 1× PBS;
resuspended in a homogenization buffer containing 0.3 M
sucrose, 60 mM NaCl, 15 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0.1 mM
For Western blotting, 50 µg of protein extracts were denaturated in
SDS loading buffer and subjected to SDS-PAGE using a 10% gel. The
proteins were then transferred onto a polyvinylidene difluoride
membrane (Sigma). Following transfer, the membranes were incubated with
antibodies directed against HuD (34) and revealed using a commercially
available ECL kit from Pierce.
Statistical Analysis--
An analysis of variance was performed
to evaluate the effects of NGF-induced neuronal differentiation on AChE
expression. The Fisher's Least Square Difference test was used to
determine whether the differences seen between group means were
significant. The level of significance was set at p < 0.05. Data are expressed as mean ± S.E. throughout.
AChE Expression during Neuronal Differentiation--
In a first
series of experiments, we examined the expression of AChE during the
process of NGF-induced neuronal differentiation of PC12 cells.
Initially, we compared the level of cell-associated AChE between
undifferentiated and 24-, 48-, and 72-h differentiated PC12 cells.
Previous studies have demonstrated that undifferentiated PC12 cells
express a basal level of AChE activity that increases upon NGF
stimulation (31, 53, 54). In agreement with these earlier reports, Fig.
1A shows that AChE activity
increases considerably during differentiation. In fact, the
cell-associated activity increased significantly by ~2.5-fold
(p < 0.01) within the first 48 h, and reached a
maximal 5-fold induction (p < 0.0001) following 72 h of NGF treatment.
We next examined the impact of NGF on the relative abundance of AChE
transcripts in PC12 cells. We focused on the level of the T transcript
because this transcript is the predominant splice variant found in
nervous tissues (12) and PC12 cells (45, 46). As illustrated in Fig.
1B, AChE mRNAs could be detected in undifferentiated
cells. However, treatment of PC12 cells with NGF led to a pronounced
increase in the levels of AChE mRNA. These increases were highly
significant, reaching more than 2-fold (p < 0.002) and
3.5-fold (p < 0.0001) by 48 and 72 h,
respectively (Fig. 1C). The relative amount of S12 rRNA,
which was used as an internal control for these assays, did not change
during differentiation. The observed increase in transcript level was
directly related to the NGF treatment and not to the length of time in
culture or cell density because, in separate studies, both of these
factors had no effect on AChE expression (data not shown). In addition, NGF removal from the culture medium 24 h after the initial
treatment resulted in a gradual decrease in AChE mRNA levels (data
not shown).
Because recent studies demonstrated the importance of transcriptional
events in regulating AChE expression at the early stages of muscle
differentiation (23, 30), we determined whether the increase in the
abundance of AChE transcripts seen in differentiating PC12 cells could
be linked to an increase in transcription. To this end, we performed
transfection studies using an AChE promoter-reporter gene construct
that contained a ~5.3-kb promoter fragment termed GRAP (24). This
fragment contains, in addition to the basal promoter region, intronic
elements previously shown to be important for muscle expression (23,
25). We observed, using this approach, a small but significant increase
(1.5-fold; p < 0.0001) in reporter gene expression
during the early phases of differentiation of PC12 cells (Fig.
2). Nuclear run-on assays performed with
nuclei isolated from undifferentiated and 24-h differentiated PC12
cells confirmed these findings (data not shown). This transcriptional increase parallels the initial 2-fold increase in AChE mRNA levels, which occurs during the initial phase of neuronal differentiation. However, it is important to emphasize that this increase in
transcription is transient and that, therefore, it fails to account for
the increase in AChE transcript level seen at later time points,
i.e. 72 and 96 h. Indeed, at these later time points,
transcription of the AChE gene had returned to the level seen in
undifferentiated cells (data not shown).
Post-transcriptional Mechanisms Regulate AChE Transcript Levels
during Neuronal Differentiation--
Recent studies have demonstrated
that NGF can act indirectly to stabilize neuron-specific transcripts
via the ELAV-like RNA-binding protein HuD (52, 55). Interestingly,
examination of the sequence of the AChE 3'-UTR revealed the presence of
a conserved element known to be recognized by HuD (see Fig.
4A). Therefore, to ascertain whether post-transcriptional
mechanisms are indeed involved in regulating AChE expression during
neuronal differentiation and to explore the possibility that this
effect involves HuD, we next examined the expression of AChE
transcripts in PC12 cells stably transfected with a HuD construct
(pcHuD), with an antisense sequence to HuD (pDuH), or with the empty
plasmid (pcDNA) acting in this case, as a control (34). In
comparison to the control levels of HuD seen in pcDNA cells, levels
of HuD in pcHuD cells are 2-3-fold higher, whereas, in pDuH cells, HuD
expression is reduced by ~70% (34).
As observed in non-transfected PC12 cells, AChE transcripts are
expressed, albeit at different levels, in these undifferentiated stable
cells. Specifically, undifferentiated pcHuD cells expressed twice as
much AChE transcripts as undifferentiated pcDNA cells, whereas the
pDuH cells express ~80% less transcript than the pcDNA cells in
the undifferentiated state (Fig.
3A). Interestingly, these data
parallel the changes in HuD expression seen in the stable cell lines
(see above).
In response to NGF, however, AChE mRNA levels showed an increase
only in the PC12 cells transfected with pcDNA and pcHuD and not in
the cells expressing the antisense sequence to HuD (Fig. 3). In fact,
AChE transcript levels in cells transfected with pcDNA increased by
a maximum of ~3-fold (p < 0.006) following NGF
treatment, demonstrating that these cells display a pattern of AChE
mRNA expression similar to that seen in non-transfected PC12. NGF
treatment of pcHuD cells dramatically increased AChE transcripts to a
level greater than that observed in the pcDNA cells. This increase
reached more than 2.5- and 5-fold within 24 and 48 h of NGF
stimulation (p < 0.02), respectively (Fig. 3B). Additionally, the maximal increase in AChE mRNA
expression in pcHuD cells was reached earlier during differentiation
such that, after 48 h of NGF treatment, pcHuD cells contained
significantly (p < 0.006) more AChE mRNAs than
pcDNA cells. By contrast, PC12 cells engineered to express an
antisense sequence to HuD (pDuH cells) did not exhibit any significant
changes in the relative abundance of AChE mRNA during the course of differentiation.
We next considered whether the effect of HuD occurred directly on AChE
transcripts. To this end, we used an approach similar to that used
recently by others (56, 57). Therefore, we transfected pcDNA,
pcHuD, and pDuH cells with a luciferase reporter construct in which the
3'-UTR of AChE transcript was inserted. This 3'-UTR contains the
conserved ARE, which, as mentioned, is known to be a
cis-acting element recognized by HuD (Fig.
4A). These experiments were
performed on undifferentiated cells in attempts to minimize the
potential confounding impact of an increased level of endogenous AChE
caused by NGF stimulation. In comparison to cells stably transfected
with pcDNA, the activity of the reporter gene was significantly
higher (p < 0.05) in cells expressing HuD.
Importantly, this effect was completely abolished in pDuH cells,
indicating that HuD is indeed an important factor regulating AChE
expression via the 3'-UTR.
Changes in the Pattern of RNA-Protein Interactions during Neuronal
Differentiation--
Based on these findings, we also examined the
pattern of proteins that could bind to the AChE 3'-UTR by both REMSA
and Northwestern analyses. For these experiments, we used three probes:
one corresponding to the full-length AChE 3'-UTR (245 nucleotides); a
truncated version of the full-length probe (90 nucleotides) in which
the ARE was deleted (
Northwestern analyses were also performed to confirm that the pattern
of RNA-protein interactions was indeed altered following NGF
stimulation. In comparison to REMSA, Northwestern analyses allow the
determination of the molecular mass of specific proteins that interact
with the RNA probe. In agreement with our REMSA data, several proteins
appeared to interact with the AChE 3'-UTR (Fig. 5C).
Importantly, NGF stimulation markedly increased the relative abundance
of these proteins. The greatest increase appeared at the level of a
protein of ~42 kDa. Together with our functional data (see above),
the presence of this ~42-kDa protein (approximate mass of HuD) in
PC12 cells exposed to NGF for 72 h raised the possibility that
this protein may in fact correspond to HuD. Western blot analysis
confirmed the molecular mass of HuD at ~42 kDa (Fig. 5C).
In control experiments, the truncated AChE 3'-UTR probe, i.e.
In a final series of experiments, we verified that HuD is able to
interact directly and bind with the AChE 3'-UTR in PC12 cells. To this
end, we first immunoprecipitated HuD protein from a total protein
extract obtained from 48-h differentiated pcHuD cells, isolated total
RNA from the immunoprecipitate, and performed RT-PCR to detect AChE
transcripts. This procedure has previously been successfully used to
show the interaction between other ELAV/Hu family proteins and neuronal
transcripts (34, 58). As shown in Fig. 5D, we were able to
specifically detect the presence of AChE transcripts from the
immunoprecipitate obtained using the affinity-purified anti-HuD
antibody. By contrast, no AChE PCR product could be visualized in total
RNA isolated from immunoprecipitates obtained using either normal
rabbit IgG or A/G chimera Sepharose beads alone (34). In these assays,
the identity of the AChE PCR product was confirmed by sequencing.
In recent years, there has been significant progress in
characterizing some of the transcriptional and post-transcriptional mechanisms involved in the regulation of the AChE gene in skeletal muscle (23, 25, 27, 30, 43, 59, 60). By contrast, there are only a few
reports available that have examined the molecular events controlling
AChE expression in neurons. Given its central role in terminating
neurotransmission at cholinergic synapses, its additional non-catalytic
function in the control, for example, of neurite outgrowth (see
Introduction), and its involvement in a variety of neurological
conditions such as Alzheimer's disease, post-traumatic stress disorder
(61), and brain tumors (62), it appears important to gain a better
understanding of the molecular pathways ultimately controlling the
expression and localization of AChE in neurons. In the present study,
we have examined some of the molecular events involved in regulating
expression of the AChE gene during neuronal differentiation. Our
findings indicate that, although transcription provides an initial
boost necessary to rapidly increase AChE mRNA levels at the onset
of neuronal differentiation, post-transcriptional events involving the
3'-UTR and the RNA-binding protein HuD are also involved and largely
account for the pronounced and sustained increase in AChE transcripts
seen at later stages of differentiation.
Several years ago, a series of experiments focusing on P19 cells
induced to differentiate into neurons via retinoic acid treatment led
Coleman and Taylor (31) to suggest that changes in mRNA stability
was a key mechanism controlling the relative abundance of AChE
transcripts during neuronal differentiation. However, this initial
study provided little insight into the nature of the cis-
and trans-acting elements that could be involved. In this
context, cis-elements contained within the 3'-UTR have been shown to be critical for regulating the turnover rate of
pre-synthesized mRNAs in a variety of cells. In particular, the
element known as the AU-rich element, which is known to exist in
different classes (63, 64), has received considerable attention because
of its key role in mRNA metabolism. Several studies have shown that
this element can either stabilize or destabilize transcripts depending on the number of sequential repeats, variations of the basic sequence, and the identity of the trans-acting factor that binds (65, 66). Among the proteins known to interact with the ARE is the ELAV-like
family of Hu proteins. This group of RNA-binding proteins consists of
HuR, HuB (Hel-N1), HuC, and HuD, of which HuB, -C, and -D are
neuron-specific and HuR is more ubiquitously expressed (65, 67).
Several studies from different laboratories have identified HuD as a
trans-acting factor binding and stabilizing a variety of
different cellular transcripts including c-Myc (68), neuroserpin (69),
tau (70), and GAP-43 (34).
Because AChE contains in its 3'-UTR a conserved ARE, we examined in the
present studies whether HuD plays a functional role in regulating AChE
expression in neurons. To this end, we used a series of distinct, yet
complementary, approaches to determine whether indeed HuD could affect
AChE mRNA expression. Using PC12 cells stably transfected to
express HuD or an antisense sequence to its mRNA, we found that the
relative abundance of AChE transcripts varied with the amount of HuD
expressed by these cells. Moreover, HuD expression induced a faster and
greater increase of AChE mRNA levels during NGF-induced
differentiation of these cells. Importantly, this differentiating
effect of NGF on AChE gene expression was completely blocked in cells
expressing the HuD antisense. Furthermore, we also observed that
expression of HuD could increase the expression of reporter transcripts
engineered to contain the AChE 3'-UTR. Finally, immunoprecipitation
experiments revealed that, indeed, HuD can directly interact with AChE
transcripts. Taken together, these results strongly implicate HuD and
the AChE 3'-UTR in the regulation of AChE mRNA expression during
neuronal differentiation.
In comparable studies, HuD has been shown recently to be capable of
regulating the stability and localization of transcripts encoding
GAP-43 and tau in neurons (34, 55, 70, 71). Interestingly, these
proteins are known to be essential for the process of neurite outgrowth
during neuronal differentiation. Because AChE has also been implicated
in the events regulating neuritogenesis (see Introduction), it appears
reasonable to speculate that HuD may, in fact, directly control neurite
outgrowth by regulating the abundance and localization of a variety of
transcripts encoding proteins that are key to this process. In
agreement with this view, several studies have demonstrated that
neurite extension is completely inhibited in PC12 cells stably
expressing an antisense to HuD (34, 70-72). Such mechanism would
confer HuD a central organizing role and could provide neurons with an
efficient post-transcriptional regulatory pathway. In one basic
scenario, neurons could therefore simply induce HuD expression to
trigger the process of neurite outgrowth, as opposed to activating
multiple signaling cascades that would need to culminate in the
activation of the transcriptional machinery of important genes via
distinct promoter elements.
Although the data presented here strongly indicate that HuD and the
AChE 3'-UTR are indeed important elements in controlling AChE mRNA
levels in differentiating neurons, our findings do not imply that these
elements act alone. It is well established, for example, that the
RNA-binding protein AUF1 forms a complex with several other proteins
involved in transcript elongation or stability (73). Similarly, it has
become increasingly apparent that the precise regulation of mRNA
stability, localization, and translation depends on the presence of
multiple cis-acting elements that interact with several
distinct trans-acting factors (65, 66). Our analysis of
RNA-protein interactions using both REMSA and Northwestern blotting is
consistent with this notion because we observed several complexes and
proteins that could interact with the AChE 3'-UTR. In future studies,
it will be important to characterize these factors to gain a complete
understanding of the cis- and trans-acting factors contributing to the regulation of AChE expression in neurons.
Our experimental approach has also allowed us to examine whether
differentiation of PC12 cells was accompanied by a change in the
transcriptional activity of the AChE gene. Our promoter-reporter studies and nuclear run-on assays both revealed that NGF initially induces a modest but significant increase in transcription. These results suggest therefore that, during the earlier stages of neuronal differentiation, transcriptional regulation plays a key role in the
initial up-regulation of AChE mRNA expression as previously suggested by Greene and Rukenstein (31). In fact, these findings fit
nicely with the recent demonstration that transcription, indeed, is
involved in regulating AChE expression in neuronal cells (46, 74) and
with the transient transcriptional induction of GAP-43 following NGF
treatment of PC12 cells (75, 76). Together with the observation that,
during the early phases of myogenic differentiation, transcriptional
changes can also account for the initial increase in AChE mRNA
levels (23), these findings are therefore consistent with a model in
which developmental changes in AChE mRNA expression are initially
regulated at the transcriptional level with post-transcriptional mechanisms becoming more important at later time points to ensure the
adequate supply of AChE to differentiating muscle and neuron.
Because differentiation of PC12 cells is dependent on the continuous
presence of NGF in the culture media, it appears reasonable to argue
that NGF acts indirectly through HuD to regulate AChE expression in a
manner similar to that described previously for the regulation of
GAP-43 (see above). In addition to directly affecting transcription of
target genes (77, 78), these findings indicate therefore that some of
the pleiotropic effects of neurotrophins on various neuronal
populations may occur through RNA-binding proteins, which in turn
affect, via post-transcriptional mechanisms, the expression of key
mRNAs. In this context, previous animal studies have shown, for
example, that treatment of axotomized facial motoneurons and
rubrospinal neurons with neurotrophins leads to an increase in the
expression of both GAP-43 and AChE transcripts (16, 79-81). Based on
the foregoing discussion, we can speculate that some of these
neurotrophin-induced changes in gene expression observed in injured
neurons may in fact be caused by post-transcriptional mechanisms
operating at the level of transcript stability, localization, and
translation. Future studies should therefore make an attempt at
unraveling the importance of these post-transcriptional events in
vivo because, ultimately, these could lead to the design of
additional therapeutic strategies aimed at promoting neuronal
regeneration and survival.
INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
80 °C. AChE activity was measured using a modified version of the
spectrophotometric method of Ellman et al. (35) as described previously (36). The total amount of protein present in the extracts
was determined by the bicinchoninic acid assay (BCA; Pierce).
80 °C until used.
-32P]UTP. RNA was
transcribed for 60 min at 30 °C in the presence of an RNase
inhibitor (Promega, Madison, WI). Following a 30-min RQ1 DNase I
(Promega) treatment, the nascent radiolabeled RNA was extracted using
TRIzol reagent (see above) and hybridized for 48 h to 10 µg of
linearized AChE cDNA (2 kb) immobilized on a Protran pure
nitrocellulose membrane (Schleicher & Schuell). After hybridization the
membranes were washed thoroughly at 42 °C in a 1× saline-sodium
citrate (SSC), 0.1% sodium dodecyl sulfate (SDS) solution and
subjected to autoradiography. The intensity of the resulting signals
was quantified using a STORM PhosphorImager and the accompanying
ImageQuant software (Amersham Biosciences). The signals
corresponding to AChE were standardized relative to the signal obtained
from genomic DNA.
-galactosidase, luciferase, or CAT activities using
available kits (Promega). The
-galactosidase and luciferase
activities were normalized to CAT levels. Background values, obtained
by transfecting promoterless LacZ and luciferase plasmids, were
subtracted from the activities obtained with the reporter constructs.
ARE), and 3) a small fragment
encompassing the ARE (see Fig. 5A), were designed to include a T7
promoter. An in vitro T7 transcription system (Promega) was
used to synthesize radiolabeled AChE 3'-UTR fragments. Briefly, the
transcription reaction containing 0.5 µg of PCR fragment, 5 µCi of
[
-32P]UTP, nucleotides, RNase inhibitor, and T7
polymerase, was carried out at 37 °C for 1 h. The template PCR
fragments were digested with 1 unit of RQ1 DNase I (Promega) for 30 min
at 37 °C. The resulting radiolabeled RNA was purified on an
RNase-free G-25 RNA purification column (Roche Diagnostics Corp.,
Indianapolis, IN). The integrity of the RNA was confirmed by gel
electrophoresis. Unlabeled RNA probes were generated by the same method
and used in cold competition assays.
80 °C until used. The total amount of protein
present in the extracts was determined by the BCA method (see above).
70 °C. Competition assays were
performed by incubating a 25 M excess of cold probe with
the protein extract for 10 min prior to the incubation with the
radiolabeled probe.
70 °C. To ensure that equivalent
amounts of proteins were loaded for each sample, membranes were also
stained with Ponceau S (Sigma), following exposure to x-ray films.
-mercaptoethanol, 0.01 mM phenylmethylsulfonyl fluoride, 0.01 mM
benzamidine, 1 µg of leupeptin, 10 µg of pepstatin A, and 1 µg of
aprotinin; and sonicated (see above). Following centrifugation, the
supernatant was recovered, aliquoted, and stored at
80 °C. The
concentration of proteins in each sample was determined using the BCA
method (see above).
RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (22K):
[in a new window]
Fig. 1.
AChE activity and transcript levels increase
in differentiating PC12 cells. A, AChE activity was
determined from protein extracts obtained from untreated and
NGF-treated cells for different time periods. The activity is expressed
as a percentage of the activity seen in the untreated (control) cells.
Symbols indicate significant differences from control cells
(*, p < 0.01; #,
p < 0.0001; n = 5 independent
experiments). B, examples of ethidium bromide-stained
agarose gels displaying AChE and S12 rRNA PCR products from control
(C) and NGF-treated (24, 48, or 72 h) PC12 cells. The
negative control lane (water, no RNA) is shown with a .
C, quantitation of AChE mRNA levels in NGF-treated (24, 48, and 72 h) PC12 cells expressed as a percentage of untreated
(control) cells. Symbols indicate significant differences
from control cells (¤, p < 0.002;
#, p < 0.0001; n = 5 independent experiments).
View larger version (17K):
[in a new window]
Fig. 2.
Transcriptional activity of the ACHE gene
increases early in differentiating PC12 cells. Quantitation of
normalized -galactosidase activity expressed in arbitrary units,
obtained from undifferentiated and NGF-induced differentiated (24 and
48 h) PC12 cells transfected with the GRAP promoter-reporter
construct (top). Symbols indicate significant
differences from undifferentiated cells (*, p < 0.0001; n = 4 independent experiments).
View larger version (60K):
[in a new window]
Fig. 3.
PC12 cells expressing the RNA-binding protein
HuD have higher levels of AChE transcripts. A, examples
of ethidium bromide-stained agarose gels displaying AChE and S12 rRNA
PCR products from control (C), NGF-treated (24, 48, or
72 h) PC12 cells stably transfected to express HuD (pcHuD), an
antisense to HuD (pDuH), or the empty vector (pcDNA). The negative
control lane (water, no RNA) is shown with a .
B, quantitation of AChE mRNA levels in NGF-treated (24, 48, and 72 h) PC12 cells expressed as a percentage of the
untreated (control) pcDNA cells. Symbols indicate
significant differences (§, p < 0.006; #,
p < 0.01; ¤, p < 0.02;
n = 4 independent experiments) from control.
* indicates a significant difference (p < 0.006) from 48-h treated pcDNA cells.
View larger version (26K):
[in a new window]
Fig. 4.
The AChE 3'-UTR increases expression of a
reporter construct in PC12 cells. A, alignment of the
AChE 3'-UTR from mouse, rat, and human shows the presence of a
conserved ARE found within an AU-rich domain (underlined)
and poly(A) signal (PAS). B, undifferentiated
pcDNA, pcHuD, and pDuH cells were co-transfected with a reporter
construct containing the full-length AChE 3'-UTR and with a
constitutively expressed CAT plasmid. The luciferase activity was
normalized to that seen with CAT and is expressed as a percentage of
the activity found in the control pcDNA cells. Symbol
indicates a significant difference (*, p < 0.05;
n = 5 independent experiments) from control
cells.
ARE), thereby eliminating the putative HuD
binding region; and a smaller probe encompassing the ARE (Fig.
5A). In REMSA, four distinct
protein complexes could be visualized using the full-length AChE 3'-UTR
probe (Fig. 5B). Although the relative abundance of all four
complexes appeared to increase in PC12 cells stimulated with NGF for
72 h, two, in particular, showed dramatic increases in the binding
intensity. The appearance of these complexes began after 48 h of
differentiation (data not shown). By contrast, the formation of these
complexes could not be observed in REMSA using
ARE probe. Specificity
of the protein complexes for the AChE 3'-UTR was demonstrated in
competition assays using a 25-fold molar excess of unlabeled,
full-length AChE 3'-UTR probe. In addition, REMSA using the small
fragment of the AChE 3'-UTR that encompasses the ARE (see Fig.
5A) showed that indeed a protein complex could directly
interact with this specific region (Fig. 5B). This latter complex appeared to correspond to one of the larger complexes seen with
the full-length probe. It is important to note that, in all these
experiments, both yeast tRNA and heparin were used to eliminate the
possibility of nonspecific interactions.
View larger version (69K):
[in a new window]
Fig. 5.
Changes in the pattern of RNA-protein
interactions in the AChE 3'-UTR in differentiating PC12 cells.
A, schematic displaying the ~2.4-kb AChE transcript
depicting important elements within the 3'-UTR, i.e. the ARE
and poly(A) signal. The locations of the full-length (245 nucleotides
(nt)), a truncated probe (90 nucleotides) lacking the ARE
( ARE), and a probe (64 nucleotides) encompassing the ARE
are also shown. B, REMSAs were performed using protein
extracts from control (C) untreated and 72-h NGF-treated
(72) PC12 cells. Representative autoradiograms demonstrating
the interaction between RNA and protein complexes using the full-length
(FL) probe are shown. Closed arrows
indicate specific RNA-protein complexes, and open
arrow points to the free probe. Competition experiments
using a 25 M excess of unlabeled full-length
(FL) probe is also shown (Cold). Note the
increase in the relative abundance of the protein complexes in
differentiated cells and the absence of these complexes in REMSAs
performed with the truncated probe (
ARE). REMSA performed
using the ARE probe shows that a single protein complex can directly
interact with the ARE. C, Northwestern blots
(left panel) were performed using protein
extracts from control untreated (C) and 72-h NGF-treated
(72) PC12 cells. A representative autoradiogram shows the
increase in the amount of proteins interacting with the AChE 3'-UTR and
a particular strong induction of a ~42-kDa protein. A Western blot
(right panel) shows that, indeed, HuD migrates at
~42 kDa (see arrow). D, example of an ethidium
bromide-stained agarose gel displaying AChE PCR product obtained from
an immunoprecipitate isolated from differentiated pcHuD cells using an
antibody against HuD, normal serum IgG, and A/G chimera Sepharose beads
(Bds). The negative control lane (water, no RNA) is shown
with a
. Note the presence of a PCR product corresponding
to AChE only in the immunoprecipitate obtained with the HuD
antibody.
ARE probe, could not bind any proteins in these
Northwestern assays (data not shown).
DISCUSSION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
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FOOTNOTES |
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* This work was supported in part by operating grants from the Canadian Institutes of Health Research (CIHR) and the Ontario Neurotrauma Foundation (ONF) (to B. J. J.) and by National Institutes of Health Grant NS-30255 (to N. P. B.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
§ Supported by a studentship from the ONF during the course of this work.
CIHR Investigator. To whom correspondence should be addressed:
Dept. of Cellular and Molecular Medicine, Faculty of Medicine, University of Ottawa, 451 Smyth Rd., Ottawa, Ontario K1H 8M5, Canada.
Tel.: 613-562-5800 (ext. 8383); Fax: 613-562-5636; E-mail: jasmin@uottawa.ca.
Published, JBC Papers in Press, December 4, 2002, DOI 10.1074/jbc.M209383200
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ABBREVIATIONS |
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The abbreviations used are: AChE, acetylcholinesterase; NGF, nerve growth factor; RT, reverse transcription; GRAP, giant rat acetylcholinesterase promoter; CAT, chloramphenicol acetyltransferase; 3'-UTR, 3'-untranslated region; REMSA, RNA-based electrophoretic mobility shift assay; DTT, dithiothreitol; ELAV, embryonic lethal abnormal vision; ARE, AU-rich element; PBS, phosphate-buffered saline; IP, immunoprecipitation.
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---|
1. | Massoulie, J., Pezzementi, L., Bon, S., Krejci, E., and Vallette, F. M. (1993) Prog. Neurobiol. 41, 31-91[CrossRef][Medline] [Order article via Infotrieve] |
2. | Legay, C. (2000) Microsc. Res. Tech. 49, 56-72[CrossRef][Medline] [Order article via Infotrieve] |
3. | Taylor, P., and Radic, Z. (1994) Annu. Rev. Pharmacol. Toxicol. 34, 281-320[CrossRef][Medline] [Order article via Infotrieve] |
4. | Soreq, H., and Seidman, S. (2001) Nat. Rev. Neurosci. 2, 294-302[CrossRef][Medline] [Order article via Infotrieve] |
5. | Legay, C., Huchet, M., Massoulie, J., and Changeux, J. P. (1995) Eur. J. Neurosci. 7, 1803-1809[Medline] [Order article via Infotrieve] |
6. |
Li, Y.,
Camp, S.,
and Taylor, P.
(1993)
J. Biol. Chem.
268,
5790-5797 |
7. | Rachinsky, T. L., Camp, S., Li, Y., Ekstrom, T. J., Newton, M., and Taylor, P. (1990) Neuron 5, 317-327[Medline] [Order article via Infotrieve] |
8. | Layer, P. G., and Willbold, E. (1995) Prog. Histochem. Cytochem. 29, 1-94[Medline] [Order article via Infotrieve] |
9. | Bernard, V., Legay, C., Massoulie, J., and Bloch, B. (1995) Neuroscience 64, 995-1005[CrossRef][Medline] [Order article via Infotrieve] |
10. |
Hammond, P.,
Rao, R.,
Koenigsberger, C.,
and Brimijoin, S.
(1994)
Proc. Natl. Acad. Sci. U. S. A.
91,
10933-10937 |
11. | Landwehrmeyer, B., Probst, A., Palacios, J. M., and Mengod, G. (1993) Neuroscience 57, 615-634[Medline] [Order article via Infotrieve] |
12. | Legay, C., Bon, S., Vernier, P., Coussen, F., and Massoulie, J. (1993) J. Neurochem. 60, 337-346[Medline] [Order article via Infotrieve] |
13. | Brimijoin, S., and Hammond, P. (1996) Neuroscience 71, 555-565[CrossRef][Medline] [Order article via Infotrieve] |
14. |
Robertson, R. T.,
and Yu, J.
(1993)
News Physiol. Sci.
8,
266-272 |
15. | Blasina, M. F., Faria, A. C., Gardino, P. F., Hokoc, J. N., Almeida, O. M., De, Mello, F. G., Arruti, C., and Dajas, F. (2000) Cell Tissue Res. 299, 173-184[CrossRef][Medline] [Order article via Infotrieve] |
16. | Tetzlaff, W., Alexander, S. W., Miller, F. D., and Bisby, M. A. (1991) J. Neurosci. 11, 2528-2544[Abstract] |
17. | Dupree, J. L., and Bigbee, J. W. (1996) J. Neurocytol. 25, 439-454[Medline] [Order article via Infotrieve] |
18. | Jones, S. A., Holmes, C., Budd, T. C., and Greenfield, S. A. (1995) Cell Tissue Res. 279, 323-330[CrossRef][Medline] [Order article via Infotrieve] |
19. | Koenigsberger, C., Chiappa, S., and Brimijoin, S. (1997) J. Neurochem. 69, 1389-1397[Medline] [Order article via Infotrieve] |
20. | Sharma, K. V., and Bigbee, J. W. (1998) J. Neurosci. Res. 53, 454-464[CrossRef][Medline] [Order article via Infotrieve] |
21. | Srivatsan, M., and Peretz, B. (1997) Neuroscience 77, 921-931[CrossRef][Medline] [Order article via Infotrieve] |
22. |
Sternfeld, M.,
Ming, G.,
Song, H.,
Sela, K.,
Timberg, R.,
Poo, M.,
and Soreq, H.
(1998)
J. Neurosci.
18,
1240-1249 |
23. |
Angus, L. M.,
Chan, R. Y.,
and Jasmin, B. J.
(2001)
J. Biol. Chem.
276,
17603-17609 |
24. | Boudreau-Lariviere, C., Chan, R. Y., Wu, J., and Jasmin, B. J. (2000) J. Neurochem. 74, 2250-2258[CrossRef][Medline] [Order article via Infotrieve] |
25. |
Chan, R. Y.,
Boudreau-Lariviere, C.,
Angus, L. M.,
Mankal, F. A.,
and Jasmin, B. J.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
4627-4632 |
26. |
Krejci, E.,
Legay, C.,
Thomine, S.,
Sketelj, J.,
and Massoulie, J.
(1999)
J. Neurosci.
19,
10672-10679 |
27. |
Luo, Z. D.,
Wang, Y.,
Werlen, G.,
Camp, S.,
Chien, K. R.,
and Taylor, P.
(1999)
Mol. Pharmacol.
56,
886-894 |
28. | Pregelj, P., and Sketelj, J. (2002) J. Neurosci. Res. 67, 114-121[CrossRef][Medline] [Order article via Infotrieve] |
29. | Rimer, M., and Randall, W. R. (1999) Biochem. Biophys. Res. Commun. 260, 251-255[CrossRef][Medline] [Order article via Infotrieve] |
30. |
Siow, N. L.,
Choi, R. C.,
Cheng, A. W.,
Jiang, J. X.,
Wan, D. C.,
Zhu, S. Q.,
and Tsim, K. W.
(2002)
J. Biol. Chem.
277,
36129-36136 |
31. |
Greene, L. A.,
and Rukenstein, A.
(1981)
J. Biol. Chem.
256,
6363-6367 |
32. |
Coleman, B. A.,
and Taylor, P.
(1996)
J. Biol. Chem.
271,
4410-4416 |
33. | Greene, L. A., Aletta, J. M., Ruckenstein, A., and Greene, S. H. (1986) Methods Enzymol. 147, 207-216 |
34. |
Mobarak, C. D.,
Anderson, K. D.,
Morin, M.,
Beckel-Mitchener, A.,
Rogers, S. L.,
Furneaux, H.,
King, P.,
and Perrone-Bizzozero, N. I.
(2000)
Mol. Biol. Cell
11,
3191-3203 |
35. | Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. (1961) Biochem. Pharmacol. 7, 88-95[CrossRef][Medline] [Order article via Infotrieve] |
36. | Jasmin, B. J., and Gisiger, V. (1990) J. Neurosci. 10, 1444-1454[Abstract] |
37. | Boudreau-Lariviere, C., Sveistrup, H., Parry, D. J., and Jasmin, B. J. (1996) Neuroscience 73, 613-622[CrossRef][Medline] [Order article via Infotrieve] |
38. | Michel, R. N., Vu, C. Q., Tetzlaff, W., and Jasmin, B. J. (1994) J. Cell Biol. 127, 1061-1069[Abstract] |
39. | Jasmin, B. J., Lee, R. K., and Rotundo, R. L. (1993) Neuron 11, 467-477[Medline] [Order article via Infotrieve] |
40. | Forster, E., Otten, U., and Frotscher, M. (1993) Neurosci. Lett. 155, 216-219[Medline] [Order article via Infotrieve] |
41. | Boudreau-Lariviere, C., and Jasmin, B. J. (1999) FEBS Lett. 444, 22-26[CrossRef][Medline] [Order article via Infotrieve] |
42. |
Chan, R. Y.,
Adatia, F. A.,
Krupa, A. M.,
and Jasmin, B. J.
(1998)
J. Biol. Chem.
273,
9727-9733 |
43. | Fuentes, M. E., and Taylor, P. (1993) Neuron 10, 679-687[Medline] [Order article via Infotrieve] |
44. |
Sketelj, J.,
Crne-Finderle, N.,
Strukelj, B.,
Trontelj, J. V.,
and Pette, D.
(1998)
J. Neurosci.
18,
1944-1952 |
45. |
Li, Y.,
Liu, L.,
Kang, J.,
Sheng, J. G.,
Barger, S. W.,
Mrak, R. E.,
and Griffin, W. S.
(2000)
J. Neurosci.
20,
149-155 |
46. |
Meshorer, E.,
Erb, C.,
Gazit, R.,
Pavlovsky, L.,
Kaufer, D.,
Friedman, A.,
Glick, D.,
Ben Arie, N.,
and Soreq, H.
(2002)
Science
295,
508-512 |
47. | Alterio, J., Mallet, J., and Biguet, N. F. (2001) Mol. Cell Neurosci. 17, 179-189[CrossRef][Medline] [Order article via Infotrieve] |
48. |
Hew, Y.,
Lau, C.,
Grzelczak, Z.,
and Keeley, F. W.
(2000)
J. Biol. Chem.
275,
24857-24864 |
49. | Wilson, G. M., and Brewer, G. (1999) Methods 17, 74-83[CrossRef][Medline] [Order article via Infotrieve] |
50. |
Erondu, N. E.,
Nwankwo, J.,
Zhong, Y.,
Boes, M.,
Dake, B.,
and Bar, R. S.
(1999)
Mol. Endocrinol.
13,
495-504 |
51. |
Sagesser, R.,
Martinez, E.,
Tsagris, M.,
and Tabler, M.
(1997)
Nucleic Acids Res.
25,
3816-3822 |
52. |
Chung, S.,
Eckrich, M.,
Perrone-Bizzozero, N.,
Kohn, D. T.,
and Furneaux, H.
(1997)
J. Biol. Chem.
272,
6593-6598 |
53. | Inestrosa, N. C., Reiness, C. G., Reichardt, L. F., and Hall, Z. W. (1981) J. Neurosci. 1, 1260-1267[Abstract] |
54. | Lucas, C. A., Czlonkowska, A., and Kreutzberg, G. W. (1980) Neurosci. Lett. 18, 333-337[Medline] [Order article via Infotrieve] |
55. |
Tsai, K. C.,
Cansino, V. V.,
Kohn, D. T.,
Neve, R. L.,
and Perrone-Bizzozero, N. I.
(1997)
J. Neurosci.
17,
1950-1958 |
56. |
Rodriguez-Pascual, F.,
Hausding, M.,
Ihrig-Biedert, I.,
Furneaux, H.,
Levy, A. P.,
Forstermann, U.,
and Kleinert, H.
(2000)
J. Biol. Chem.
275,
26040-26049 |
57. |
Yeap, B. B.,
Voon, D. C.,
Vivian, J. P.,
McCulloch, R. K.,
Thomson, A. M.,
Giles, K. M.,
Czyzyk-Krzeska, M. F.,
Furneaux, H.,
Wilce, M. C.,
Wilce, J. A.,
and Leedman, P. J.
(2002)
J. Biol. Chem.
277,
27183-27192 |
58. |
Antic, D., Lu, N.,
and Keene, J. D.
(1999)
Genes Dev.
13,
449-461 |
59. |
Getman, D. K.,
Mutero, A.,
Inoue, K.,
and Taylor, P.
(1995)
J. Biol. Chem.
270,
23511-23519 |
60. |
Luo, Z.,
Fuentes, M. E.,
and Taylor, P.
(1994)
J. Biol. Chem.
269,
27216-27223 |
61. | Kaufer, D., Friedman, A., Seidman, S., and Soreq, H. (1998) Nature 393, 373-377[CrossRef][Medline] [Order article via Infotrieve] |
62. | Karpel, R., Aziz-Aloya, R., 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] |
63. | Peng, S. S., Chen, C. Y., and Shyu, A. B. (1996) Mol. Cell. Biol. 16, 1490-1499[Abstract] |
64. | Xu, N., Chen, C. Y., and Shyu, A. B. (1997) Mol. Cell. Biol. 17, 4611-4621[Abstract] |
65. | Guhaniyogi, J., and Brewer, G. (2001) Gene (Amst.) 265, 11-23[CrossRef][Medline] [Order article via Infotrieve] |
66. | Malter, J. A. (2001) J. Neurosci. Res. 66, 311-316[CrossRef][Medline] [Order article via Infotrieve] |
67. | Perrone-Bizzozero, N., and Bolognani, F. (2002) J. Neurosci. Res. 68, 121-126[CrossRef][Medline] [Order article via Infotrieve] |
68. |
Manohar, C. F.,
Short, M. L.,
Nguyen, A.,
Nguyen, N. N.,
Chagnovich, D.,
Yang, Q.,
and Cohn, S. L.
(2002)
J. Biol. Chem.
277,
1967-1973 |
69. |
Cuadrado, A.,
Navarro-Yubero, C.,
Furneaux, H.,
Kinter, J.,
Sonderegger, P.,
and Munoz, A.
(2002)
Nucleic Acids Res.
30,
2202-2211 |
70. |
Aranda-Abreu, G. E.,
Behar, L.,
Chung, S.,
Furneaux, H.,
and Ginzburg, I.
(1999)
J. Neurosci.
19,
6907-6917 |
71. | Anderson, K. D., Sengupta, J., Morin, M., Neve, R. L., Valenzuela, C. F., and Perrone-Bizzozero, N. I. (2001) Exp. Neurol. 168, 250-258[CrossRef][Medline] [Order article via Infotrieve] |
72. | Dobashi, Y., Shoji, M., Wakata, Y., and Kameya, T. (1998) Biochem. Biophys. Res. Commun. 244, 226-229[CrossRef][Medline] [Order article via Infotrieve] |
73. |
Laroia, G.,
Cuesta, R.,
Brewer, G.,
and Schneider, R. J.
(1999)
Science
284,
499-502 |
74. | Wan, D. C., Choi, R. C., Siow, N. L., and Tsim, K. W. (2000) Neurosci. Lett. 288, 81-85[CrossRef][Medline] [Order article via Infotrieve] |
75. | Perrone-Bizzozero, N. I., Neve, R. L., Irwin, N., Lewis, S. E., Fischer, I., and Benowitz, L. I. (1991) Mol. Cell Neurosci. 2, 402-409 |
76. |
Federoff, H. J.,
Grabczyk, E.,
and Fishman, M. C.
(1988)
J. Biol. Chem.
263,
19290-19295 |
77. | Huang, E. J., and Reichardt, L. F. (2001) Annu. Rev. Neurosci. 24, 677-736[CrossRef][Medline] [Order article via Infotrieve] |
78. | Sofroniew, M. V., Howe, C. L., and Mobley, W. C. (2001) Annu. Rev. Neurosci. 24, 1217-1281[CrossRef][Medline] [Order article via Infotrieve] |
79. |
Fernandes, K. J.,
Kobayashi, N. R.,
Jasmin, B. J.,
and Tetzlaff, W.
(1998)
J. Neurosci.
18,
9936-9947 |
80. | Palacios, G., Mengod, G., Sarasa, M., Baudier, J., and Palacios, J. M. (1994) Brain Res. Mol. Brain Res. 24, 107-117[Medline] [Order article via Infotrieve] |
81. | Tetzlaff, W., Zwiers, H., Lederis, K., Cassar, L., and Bisby, M. A. (1989) J. Neurosci. 9, 1303-1313[Abstract] |