During the development of rat muscles, the expression of PRiMA and AChET and the enzymatic activity increased dramatically; however, the proportion of G4AChE decreased.. In parallel, the
Trang 1(PRiMA) of globular form acetylcholinesterase at the
neuromuscular junctions – contribution and expression from motor neurons
K Wing Leung, Heidi Q Xie, Vicky P Chen, Mokka K W Mok, Glanice K Y Chu, Roy C Y Choi and Karl W K Tsim
Department of Biology and Center for Chinese Medicine, The Hong Kong University of Science and Technology, China
Acetylcholinesterase (AChE; EC 3.1.1.7) plays a
cru-cial role in terminating the synaptic transmission by
hydrolyzing the neurotransmitter acetylcholine at the
neuron-to-neuron synapses in the central nervous
sys-tem and at the neuromuscular junctions (NMJs) in the
peripheral nervous system AChE exists in different
molecular forms The formation of these molecular
forms depends on alternative splicing in the 3¢ region
of the primary transcript [1], which generates the
AChER (‘readthrough’), AChEH (‘hydrophobic’) and
AChET (‘tailed’) subunits, containing the same
cata-lytic domain but different carboxyl termini [1] In
mammals, the AChER variant produces a soluble monomer that is up-regulated in the brain during stress [2]; the AChEHvariant produces a glycosylphos-phatidylinositol-linked dimer and is expressed in blood cells; the AChET variant is the only subunit expressed
in the brain and muscle The AChET subunits form nonamphiphilic tetramers with a collagen tail as asym-metric AChE (A4, A8 and A12) in muscle In addition, the AChET variant produces monomers (G1), dimers (G2) and tetramers (G4) The amphiphilic tetramer (G4) is linked with a proline-rich membrane anchor (PRiMA) as a globular form of AChE (PRiMA-linked
Keywords
acetylcholinesterase; molecular form;
muscle fiber type; neuromuscular junction;
proline-rich membrane anchor
Correspondence
K W K Tsim, Department of Biology,
The Hong Kong University of Science and
Technology, Clear Water Bay Road,
Kowloon, Hong Kong SAR, China
Fax: +852 2358 1559
Tel: +852 2358 7332
E-mail: BOTSIM@UST.HK
(Received 21 November 2008, revised 11
March 2009, accepted 25 March 2009)
doi:10.1111/j.1742-4658.2009.07022.x
The expression and localization of the proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholines-terase (G4AChE), were studied at vertebrate neuromuscular junctions Both muscle and motor neuron contributed to this synaptic expression pattern During the development of rat muscles, the expression of PRiMA and AChET and the enzymatic activity increased dramatically; however, the proportion of G4AChE decreased G4AChE in muscle was recognized specifically by a PRiMA antibody, indicating the association of this enzyme with PRiMA Using western blot and ELISA, both PRiMA protein and PRiMA-linked G4AChE were found to be present in large amounts in fast-twitch muscle (e.g tibialis), but in relatively low abundance in slow-twitch muscle (e.g soleus) These results indicate that the expression level
of PRiMA-linked G4AChE depends on muscle fiber type In parallel, the expression of PRiMA, AChET and G4AChE also increased in the spinal cord during development Such expression in motor neurons contributed to the synaptic localization of G4 AChE After denervation, the expression of PRiMA, AChETand G4AChE decreased markedly in the spinal cord, and
in fast- and slow-twitch muscles
Abbreviations
AChE, acetylcholinesterase; AChR, acetylcholine receptor; BChE, butyrylcholinesterase; ChAT, choline acetyltransferase; GAPDH,
glyceraldehyde-3-phosphate dehydrogenase; GFAP, glial fibrillary acidic protein; NeuN, neuronal nuclei; NMJs, neuromuscular junctions; PRiMA, proline-rich membrane anchor; SNAP-25, synaptosomal-associated protein 25.
Trang 2G4AChE) in brain and muscle [3–5] Two PRiMA
isoforms (PRiMA I and PRiMA II) are generated
from the PRiMA gene by alternative splicing
PRi-MA I contains a longer C-terminal cytoplasmic
domain than does PRiMA II [6]
Although asymmetric AChE is the predominant
species at NMJs and its appearance in muscle
coin-cides with the establishment of neuromuscular contacts
during development and regeneration [7,8], G4AChE
also exists in muscles Several studies have revealed
that the level of G4AChE is controlled by the
dynamic activity of skeletal muscles The
transcrip-tional regulation of PRiMA is down-regulated during
myogenic differentiation and under the influence of
innervation [9] In line with the transcriptional
expres-sion of PRiMA, the proportion of G4AChE decreases
during myogenic differentiation and innervation [1,9]
In mammals, fast-twitch muscles contain a large
amount of G4AChE, whereas slow-twitch muscles
contain a much smaller amount [10]
The expression of different AChE forms at NMJs
raises the question of whether the synaptic enzyme is
produced by muscle, nerve or both under different
physiological states Both asymmetric and globular
forms of AChE are known to be produced by muscle
cells [11,12], and the presynaptic motor nerve terminals
synthesize and secrete AChE at NMJs [13,14] The
pre-dominant form of AChE expressed by motor neurons
in chick spinal cord is G4AChE [15]
In this article, we analyze the expression and
locali-zation of the PRiMA I-linked G4form of AChE in rat
muscles and motor neurons We prepared an antibody
against the cytoplasmic domain of PRiMA I, which allowed us to show that PRiMA-linked G4AChE is localized at NMJs in both presynaptic nerve terminals and postsynaptic muscle fiber It is expressed by motor neurons in the rat spinal cord: this expression increased during development, but decreased after denervation These data show that both presynaptic motor neuron and postsynaptic muscle fiber contribute
to the synaptic expression of PRiMA-linked G4AChE and illustrate its temporal and spatial expression at NMJs
Results
Regulation of G4AChE and PRiMA during muscle development
A rabbit polyclonal antibody against the C-terminus
of PRiMA I was generated To validate the PRiMA antibody, a full-length mouse PRiMA cDNA (corre-sponding to PRiMA I unless specified) and a C-termi-nal truncated mutant (PRiMADC-term) cDNA, both tagged with a FLAG epitope, were transfected into HEK293T cells In western blot analysis, a FLAG antibody recognized both PRiMA and PRiMADC-term with protein bands of approximately 20 and 16 kDa, respectively: these protein bands corresponded to the predicted size of the recombinant proteins (Fig 1A) The PRiMA antibody, however, recognized only the full-length PRiMA, but not the truncated PRiMADC-term construct In addition, the recognition was fully blocked by pre-incubation of the PRiMA
Fig 1 The specificity of the PRiMA antibody (A) Protein samples (40 lg) of HEK293T cells expressing FLAG-PRiMA or FLAG-PRiMADC-term were analyzed by 12% SDS–PAGE Both PRiMA and FLAG antibodies (Ab) were used to label the PRiMA proteins In the blocking experiment, excess amounts of recombinant PRiMA antigen (Ag) (from residues 114 to 153) at 5 lgÆmL)1were pre-incubated with the PRiMA antibody (0.5 lgÆmL)1) for 4 h at 4 C before it was used for western blotting (B) Transfected HEK293T cells were stained with PRiMA or FLAG antibody
as described in Materials and methods Bar, 10 lm.
Trang 3antibody with the antigen, i.e the PRiMA I
C-terminal peptide (Fig 1A) In the
immunocytofluo-rescent staining of transfected fibroblasts, the PRiMA
antibody also recognized FLAG-tagged
PRiMA-expressing cells (Fig 1B) In contrast, FLAG-tagged
PRiMADC-term-expressing cells were not recognized by
the antibody As a positive control, FLAG antibody
was used; it recognized both full-length and truncated
PRiMA in protein detection and immunostaining
(Fig 1A,B) Such recognition could not be blocked by
pre-incubation with PRiMA antigen These results
clearly indicate the specificity of the PRiMA antibody
in recognizing the cytoplasmic domain of PRiMA I
According to Perrier et al [6], two splicing variants
of PRiMA mRNAs are generated from the PRiMA
gene to produce different proteins (PRiMA I and
PRiMA II; Fig 2A) PRiMA I mRNA, which
posses-ses exons 4 and 5, produces a 40-residue-long
intra-cellular cytoplasmic tail, whereas PRiMA II mRNA, which possesses exons 4, 4b and 5, encodes a shorter intracellular motif These two PRiMA isoforms may
be distinguished by RT-PCR using primers flanking exons 4 and 5 In rat muscles, PRiMA I was found to
be present, whereas PRiMA II was barely detectable (Fig 2B) For precise quantification, we used real-time PCR with the same set of primers In agreement with the absence of PRiMA II in muscle, all the amplified products revealed by real-time PCR corresponded to PRiMA I The mRNA level of PRiMA I was up-regu-lated gradually in the early postnatal stages and dra-matically in the adult stage (Fig 2B) Meanwhile, the level of AChET mRNA increased gradually from the early postnatal stage to the adult Using PRiMA anti-body, the PRiMA protein was detected in the muscles
of embryonic rats; its level increased after postnatal day 10 to the adult (Fig 2C) As reported previously,
Fig 2 Developmental profiles of PRiMA, AChETand G4AChE in skeletal muscles (A) Splice variants of PRiMA mRNAs (PRiMA I and II) are illustrated PRiMA II contains an additional exon 4b Arrows show the location of primers used for qualitative and real-time PCR analyses (B) Total RNAs were extracted from rat leg muscles at different developmental stages to perform RT-PCR for PRiMA I (145 bp) PRiMA II (302 bp) and AChET(671 bp) Adult rat brain served as a positive control One representative result is shown (top) The bottom panel shows the results of real-time PCR analysis of the mRNA expression of PRiMA I and AChE T (C) Samples of extracts from the lower leg muscles
of rat (birth to adult stage) containing 40 lg of protein were loaded per lane for western blotting (top) The levels of PRiMA and AChET proteins were determined GAPDH served as a loading control The bottom panel shows the quantified data of protein bands AChE activity was determined by the Ellman assay (D) Samples of extracts from rat leg muscles containing equal amounts of AChE activity were loaded
on sucrose density gradients AChE activity was plotted as a function of the S value, estimated from the position of the sedimentation mark-ers Enzymatic activities are expressed in arbitrary units, and representative sedimentation profiles are shown (E) The specific activity of
G 4 AChE was quantified at different developmental stages Samples of muscle extracts at different developmental stages containing 600 lg
of protein were loaded on sucrose density gradients The peak area corresponding to G 4 AChE activity was determined The results are expressed as the ratio to the value obtained at E21 (basal), and are shown as means ± standard error of the mean (SEM), n = 4.
Trang 4AChETprotein and AChE enzymatic activity increased
during muscle development (Fig 2C) With regard to
the AChE molecular form, the AChE G1and G4forms
were predominant in embryonic muscles (Fig 2D) In
mature muscles, the relative proportion of the G1 and
G4 forms was reduced and the asymmetric form of
AChE (A12) was increased (Fig 2D) In order to
quan-tify the relative amount of PRiMA-linked G4AChE in
developing muscle, protein extracts at different
develop-mental stages were analyzed by sedimentation in sucrose
density gradients The proportion of G4AChE was
determined from the peak area, relative to the area of
the entire sedimentation profile, and its activity was
given by the product of this proportion with the total
AChE activity The amount of G4AChE in muscle
increased twofold from birth to adult (Fig 2E)
PRiMA-linked G4AChE therefore increased during
muscle development
Expression of PRiMA-linked G4AChE in
fast-twitch and slow-twitch muscles
In order to investigate the expression level of PRiMA
and PRiMA-linked G4AChE in different muscle fiber
types, fast-twitch (tibialis) and slow-twitch (soleus)
muscles from adult rats were collected and analyzed In
western blotting, PRiMA protein was detected in both
tissues, but its level was about threefold lower in the
soleus than in the tibialis (Fig 3A) The relative
abun-dance of PRiMA-linked G4AChE was determined by
ELISA using our PRiMA antibody Equal amounts of
AChE activity were loaded onto an ELISA plate
pre-coated with serial dilutions of PRiMA antibody The
retained AChE enzymatic activity, corresponding to
PRiMA-linked G4AChE, was measured after washing
We found larger amounts (over twofold) of
PRiMA-linked G4AChE in the tibialis than in the soleus
(Fig 3B) The higher expression of G4AChE in the
tibialis was further confirmed by sucrose density
gradi-ent analysis The PRiMA antibody was able to deplete
the G4 form of AChE in the tibialis, but this was not
obvious in the soleus (Fig 3C) In all cases, the brain
enzyme was used as a control
We analyzed the localization of PRiMA in sections
of tibialis and soleus muscle by
immunohistofluores-cence NMJs were visualized by labeling the
post-synaptic acetylcholine receptor (AChR) with
a-bungarotoxin (shown in red or pseudo-blue) and the
presynaptic nerve terminal with synaptotagmin (SV48;
shown in red) in both types of muscle (Fig 4) PRiMA
(shown in green) was expressed at the NMJs, and its
distribution was wider than that of AChE and AChR,
extending into a peri-junctional zone where neither
Fig 3 Expression of PRiMA and G4AChE in different muscles (A) Samples of extracts from adult rat soleus and tibialis containing
40 lg of protein were loaded per lane for western blotting of PRiMA protein Adult rat brain served as a positive control The bottom panel shows the quantification of PRiMA protein The results are expressed as the ratio to soleus (basal) equal to unity; means ± stan-dard error of the mean, n = 4 (B) The relative amount of PRiMA-linked G4AChE was quantified by ELISA Tissue lysates from rat brain, tibialis and soleus containing equal AChE activities were loaded onto an ELISA plate precoated with serial dilutions of PRiMA anti-body for 2 h The retained AChE activity was determined (C) For immunodepletion, 1 mL samples of extracts from adult rat brain, tibi-alis and soleus were incubated with PRiMA antibody (10 lgÆmL)1) and protein G-agarose before sucrose density gradient analysis AChE activity was plotted as a function of the S value, estimated from the position of the sedimentation markers Enzymatic activities are expressed in arbitrary units, and representative sedimentation profiles are shown.
Trang 5Fig 4 Localization of PRiMA and AChE at
NMJs Sections from adult rat tibialis (top)
and soleus (bottom) muscles were triple
stained with rhodamine-conjugated or Alexa
647-conjugated a-bungarotoxin (red or
pseudo-blue) for postsynaptic AChR,
anti-AChE T (pseudo-blue), anti-synaptotagmin
(SV48; red) for presynaptic nerve terminal
and anti-PRiMA (green), and examined by
confocal microscopy Merged images of
AChR⁄ SV48 and PRiMA are shown on the
right Representative images are shown,
n = 4 Bar, 20 lm.
Fig 5 Developmental evolution of PRiMA and G 4 AChE in the spinal cord (A) Total RNAs were extracted from spinal cord at different developmental stages for detection of transcripts encoding PRiMA I (145 bp), PRiMA II (302 bp) and AChE T (671 bp) Representative results are shown (B) Samples of extracts of rat spinal cord (from birth to adult stages) containing 40 lg of protein were loaded per lane for wes-tern blotting PRiMA and AChETproteins were determined GAPDH served as a loading control (C) Quantification of proteins (from B) and AChE activity during development (D) One milliliter samples of extract from adult rat spinal cord, with and without depletion by the PRiMA antibody (as in Fig 3C), were analyzed by sucrose density gradients AChE activity was plotted as a function of the S value, estimated from the position of the sedimentation markers Enzymatic activities are expressed in arbitrary units, and representative sedimentation profiles are shown (E) G 4 AChE specific activity in the spinal cord at different developmental stages was quantified as in Fig 2E The results are expressed as the ratio to the value obtained at P1 (basal) equal to unity; means ± standard error of the mean (SEM), n = 4.
Trang 6AChE nor AChR was present in either muscle fiber
type However, the precise localization of PRiMA has
yet to be determined
Presence of PRiMA-linked G4AChE in motor
neurons
At NMJs, AChE may originate from the muscle fiber
and⁄ or from the motor neuron In order to examine the
presence of PRiMA and PRiMA-linked G4AChE, rat
spinal cords were collected at early postnatal and adult
stages Qualitative PCR indicated that both PRiMA I
and II transcripts were expressed in the spinal cord: the
PRiMA I transcript decreased slightly after birth, but
increased dramatically thereafter and was the
predomi-nant form in the adult, the PRiMA II transcript first
increased but disappeared in the adult (Fig 5A) As a
result of the absence of a specific primer for PRiMA I,
the expression level of the PRiMA I transcript could
not be analyzed by real-time PCR The PRiMA I
pro-tein level in the spinal cord, determined in western blots
with the PRiMA antibody (recognizing the cytoplasmic
domain of PRiMA I), increased after birth, as did
AChE (Fig 5B,C) This was consistent with an increase
in total AChE activity (Fig 5C) and with the
observa-tion that G4was the predominant form of the enzyme in
the adult spinal cord (Fig 5D) The majority of
G4AChE was associated with PRiMA I, as more than
70% was immunoprecipitated with the PRiMA
anti-body The relative amount of G4AChE determined
from sedimentation profiles allowed us to evaluate its
activity: the specific activity of G4AChE per milligram
of protein reached a plateau in the spinal cord about
10 days after birth (Fig 5E)
To determine the origin of AChE in the spinal
cord, the lumbar region of the spinal cord was
sectioned and stained with the PRiMA antibody The
label was mostly present in the ventral horn
(Fig 6A) As expected, PRiMA was detected in
AChE-positive cells in the ventral horn (Fig 6B)
These PRiMA-stained cells were motor neurons, as
shown by their reactivity with an anti-choline
acetyl-transferase (anti-ChAT) antibody This identification
was further supported by double staining of neuronal
nuclei with a neuronal marker (NeuN) In contrast,
no PRiMA was found in glial cells that were labeled
specifically with an antibody against glial fibrillary
acidic protein (GFAP) (Fig 6B) These results clearly
show that PRiMA is synthesized by motor neurons in
the spinal cord
Although motor neurons are able to synthesize
PRiMA and produce G4AChE, the restricted
localiza-tion of PRiMA-linked G4AChE at NMJs could still
be derived from three sources: muscle, Schwann cells and⁄ or motor neurons In order to determine the local-ization of PRiMA-linked G4AChE, sections of tibialis muscle were triple stained for PRiMA, SV48 and AChR The staining of PRiMA was coincident with that of SV48, rather than with that of AChR (Fig 7, left panel) Similar results were obtained with another
Fig 6 Motor neurons in the spinal cord express PRiMA (A) Sche-matic diagram showing the lumbar region of the spinal cord (left) The dorsal horn and ventral horn are indicated The right panel shows PRiMA staining in the lumbar region on the same scale at low magnification The boxed area is shown at higher magnification
in (B) Bar, 100 lm (B) Spinal cord sections were double stained with anti-PRiMA (green) and with anti-AChET(red), anti-ChAT (red), anti-NeuN (red) or anti-GFAP (red), and examined by confocal microscopy PRiMA was co-localized with AChE T , ChAT and NeuN, but not with GFAP Representative images are shown, n = 4 Bar,
20 lm.
Trang 7presynaptic marker, synaptosomal-associated
pro-tein 25 (SNAP-25): PRiMA also showed a better
co-localization with SNAP-25 than with AChR
(Fig 7, right panel) Such overlapping of PRiMA
staining with presynaptic molecules indicates that
PRiMA at NMJs is mainly provided by motor
neurons
Innervation regulates the expression of
PRiMA-linked G4AChE in the spinal cord
The expression of PRiMA and the pattern of AChE
molecular forms in muscles are known to be modified
by denervation [7] In order to determine whether
PRiMA expression in the spinal cord was regulated by
a retrograde influence of the muscle, a portion of the
sciatic nerve was surgically removed After 7 days, we
examined the expression of PRiMA in both spinal
cord (lumbar region) and tibialis muscles by real-time
PCR analysis: PRiMA mRNA (PRiMA I) was not
modified significantly in the tibialis, but was reduced
by over 60% in the spinal cord (Fig 8A) In contrast,
the mRNA level of AChET was decreased in both the
spinal cord and tibialis when compared with that of
the sham-operated control (Fig 8A) At the protein
level, western blot analyses showed that PRiMA and
AChET were reduced by about 50% after denervation
in both tissues (Fig 8B) This is consistent with a
decrease in AChE enzymatic activity of about 50% in
the spinal cord and tibialis muscle (Fig 8B) Sucrose
density gradient analyses showed a significant
reduc-tion of G1and G4forms in the spinal cord and of G1,
G4 and A12 forms in the tibialis (Fig 8C) Thus,
denervation induced a decrease in PRiMA and
G4AChE in the spinal cord and muscle
To investigate the contribution of the motor neuron
to PRiMA-linked G4AChE at NMJs, we analyzed the effect of denervation on the localization of PRiMA The NMJs of the denervated tibialis and sham-oper-ated muscle were stained for PRiMA, together with the postsynaptic marker AChR (shown in pseudo-blue) and a presynaptic marker SV48 (shown in red) Pre-synaptic labeling essentially disappeared at the dener-vated NMJs and PRiMA labeling was considerably reduced This suggests that a significant proportion of PRiMA was provided by the presynaptic motor neuron (Fig 8D) However, a small amount of PRiMA could still be detected in the denervated muscles, possibly of muscle origin
Discussion
The muscles of mice in which the PRiMA gene is inac-tivated contain essentially no G4AChE, suggesting that this enzyme form is entirely associated with PRiMA Our results show that G4AChE is, indeed, largely immunoprecipitated with a PRiMA antibody However, a fraction of G4AChE was not immunodepleted (Fig 3C), even when the amount of antibody was increased or with a second round of immunodepletion (not shown) The interaction of this fraction with the antibodies may be prevented by the presence of partner(s) associated with the C-terminal region of PRiMA In addition, no G4AChE was found in muscles of PRiMA knockout mice, implying that all G4AChE in muscle is linked with the mem-brane-anchoring protein PRiMA During muscle devel-opment, the amount of PRiMA-linked G4AChE progressively increased from birth to the adult stage
In addition, the expression of PRiMA and G4AChE
Fig 7 Presynaptic localization of PRiMA at NMJs Adult rat tibialis sections were triple stained with Alexa 647-conjugated a-bungarotoxin (pseudo-blue), anti-synaptotagmin (SV48; red) or anti-SNAP-25 (red) antibodies, and anti-PRiMA (green), and examined by confocal micros-copy Merged images allow a comparison of PRiMA with presynaptic markers (PRiMA + SV48 ⁄ SNAP-25) and a postsynaptic marker
(PRi-MA + AChR) The distribution of PRi(PRi-MA overlapped with that of SV48 and SNAP-25 Representative images are shown, n = 4 Bar, 20 lm.
Trang 8was dependent on the fast or slow nature of muscle
fibers The strong expression of PRiMA protein and
G4AChE in fast-twitch muscles is consistent with
previous results on PRiMA mRNA expression, i.e the
tibialis contains an approximately 10-fold higher level
of PRiMA mRNA than the soleus [9] The
develop-mental change of PRiMA-linked G4AChE in muscle
correlates with an increase in muscular activity and
muscle loading [15–17], which leads to the
differentia-tion of fast-twitch and slow-twitch muscle fibers The
specific role of this AChE form at NMJs remains to be elucidated
Various forms of AChE exist in both developing and mature NMJs The major form is the asymmetric collagen-tailed AChE, which is attached to the synap-tic basal lamina [18] Our study and others have shown that G4AChE is linked by PRiMA and localized in the membranes of postsynaptic and presynaptic cells [9] At NMJs, three cell types can contribute to synap-tic AChE: the postsynapsynap-tic muscle cell, the presynapsynap-tic
Fig 8 Denervation reduces the expression of PRiMA and G 4 AChE in the spinal cord and in muscles (A) The sciatic nerve was sectioned
to examine the effect of muscle on the expression of PRiMA in motor neurons After 7 days, tibialis and spinal cord were collected for analy-sis The mRNA levels of denervated muscles (Den) corresponding to PRiMA (top) and AChE T (bottom) were determined by PCR and normal-ized to those of control (sham-operated) muscles (B) Samples of extracts from control and denervated muscles containing 50 lg of protein were loaded per lane for the western blotting of PRiMA and AChET GAPDH served as a loading control The bottom panel shows the ratios
of AChE enzymatic activity after nerve section to control values The results are expressed as the ratio to control values (sham-operated) equal to unity; means ± standard error of the mean (SEM), n = 3 (C) Effect of nerve section on AChE molecular forms in the spinal cord and tibialis muscles Samples containing equal amounts of protein were loaded onto sucrose gradients AChE activity was plotted as a func-tion of the S value, estimated from the posifunc-tion of the sedimentafunc-tion markers Enzymatic activities are expressed in arbitrary units, and representative sedimentation profiles are shown (D) Sections from adult rat tibialis after 7 days of denervation (right) and sham-operated (left) were triple stained with Alexa 647-conjugated a-bungarotoxin (pseudo-blue) for postsynaptic AChR, anti-synaptotagmin (SV48; red) for presynaptic nerve terminal, and anti-PRiMA (green), and examined by confocal microscopy Merged images allow a comparison of PRiMA with presynaptic (SV48 + PRiMA) and postsynaptic (AChR + PRiMA) markers The disappearance of the presynaptic nerve terminals in denervated muscle is verified by the absence of SV48 labeling PRiMA labeling was considerably reduced, but not completely absent Repre-sentative images are shown, n = 3 Bar, 10 lm.
Trang 9motor neuron and the Schwann cell During
develop-ment, the muscle is the primary source of all forms of
AChE [1] In contrast, the contribution of the
Schw-ann cell, if any, is limited [14]; however, the possible
presence of PRiMA in the Schwann cell membrane
could only be distinguished by electron microscopy In
this study, we confirmed the expression of PRiMA, as
well as of PRiMA-linked G4AChE, in the motor
neu-rons of the spinal cord using a PRiMA antibody The
level of AChE increased during development, and was
reduced after section of the sciatic nerve The current
results are in line with our previous observation that
chick motor neurons contain collagen-tailed AChE as
well as globular forms [15,19,20] In contrast, frog
motor axons have been shown to produce
collagen-tailed AChE, which could be deposited in the synaptic
basal lamina at NMJs [14] The production of
asym-metric AChE by motor neurons and its secretion by
the motor nerve terminals at frog NMJs could be
induced by damaged target muscles Indeed, the
capac-ity of a motor neuron to express asymmetric AChE at
an intact frog NMJ is still controversial
In this study, confocal microscopy showed that
PRiMA-linked G4AChE was found in both pre- and
postsynaptic membranes at NMJs The distribution of
PRiMA appeared to be more extensive than that of
AChE This may result from a higher sensitivity for
the detection of PRiMA Alternatively, a fraction of
PRiMA may not be associated with AChET catalytic
subunits For example, PRiMA can be associated with
butyrylcholinesterase (BChE) Indeed, the expression
of G4BChE, together with G4AChE, has been
revealed in brain and retina during development [21]
Our current and past results [15] indicate that motor
neurons represent the major cell type expressing
PRiMA and AChET in the spinal cord In line with
this observation, it has been shown that AChE is
expressed in both neurotube and myotomes [22] In
addition, previous studies have also shown that AChE
synthesized in the motor neuron is transported by
axo-nal flow to the presynaptic termiaxo-nal, as revealed by
enzymatic and microscopic studies [13] The function
of pre- and postsynaptic PRiMA-linked G4AChE
expressed by motor neuron and muscle, particularly
during early stages of development, is an open
ques-tion One of the proposed functions of two-sided
expression of AChE in both pre- and postsynaptic
membranes is to play an active role during
synapto-genesis through the adhesive function of AChE [23,24]
In addition, the decrease in PRiMA and AChE
expres-sion in the rat spinal cord after section of the sciatic
nerve could be the consequence of trauma or of the
loss of retrograde influence from the muscle cells
Indeed, muscle-derived factors control the expression
of presynaptic proteins by motor neurons at NMJs [17,25]
In previous studies, G4AChE could only be identi-fied by sucrose density gradients in the motor endplate region [16,26] In this study, we have provided the first analysis of the expression of PRiMA at NMJs, using
an antibody specific for the cytoplasmic domain of PRiMA I In both fast-twitch and slow-twitch NMJs, PRiMA was found in a peri-junctional region, suggest-ing that it is partly of muscle origin Such a peri-junc-tional distribution of G4AChE, which is more abundant in fast-twitch than slow-twitch muscles [16], may provide an AChE-rich environment embedding NMJs and control the diffusion of acetylcholine out of the synaptic cleft However, most PRiMA-linked
G4AChE was found to be located in the presynaptic membrane of the motor nerve terminal This is consis-tent with the presence of a significant amount of AChE activity in the presynaptic membrane at NMJs
of the rat lumbricalis muscle [27] The presence of AChE in the presynaptic membrane can facilitate the presynaptic re-uptake of choline resulting from the hydrolysis of acetylcholine
Materials and methods
Production of PRiMA antibody
The mouse PRiMA (amino acids 114–153)–glutathione S-transferase fusion protein was expressed in BL21 (DE3) pLysE Escherichia coli (Invitrogen, Carlsbad, CA, USA) and purified by glutathione bead chromatography (Amersham Biosciences, Piscataway, NJ, USA), according
to the manufacturer’s instructions After digestion by thrombin (Sigma, St Louis, MO, USA), the PRiMA (amino acids 114–153) antigen was purified by Superdex
75 10⁄ 300 gel filtration chromatography (Amersham Biosciences) Polyclonal antibodies were raised in a 2-kg male New Zealand White rabbit by immunization with
750 lg of antigen, mixed with an equal volume of complete Freund’s adjuvant (Sigma) The immunization was carried out with the same amount of antigen three times within 1 month The anti-PRiMA serum was col-lected and purified by protein G-Sepharose (Amersham Biosciences), according to the manufacturer’s instructions The amount of purified antibody was determined spectro-photometrically
DNA construction and transfection
The HEK293T cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and
Trang 10cultured in Dulbecco’s modified Eagle’s medium (DMEM)
supplemented with 10% fetal bovine serum Cultured cells
were incubated at 37C in a water-saturated 5% CO2
incu-bator All reagents for cell cultures were from Invitrogen
cDNAs encoding full-length mouse PRiMA (PRiMA I) and
a COOH-terminal truncated mutant (PRiMADC-term;
obtained by deleting the COOH-terminal region, residues
122–153, of PRiMA I) were tagged with a FLAG epitope
(obtained by inserting the FLAG epitope DYKDE at
posi-tion 36 between the putative signal sequence and the NH2
terminus) in pEF-BOS mammalian expression vector
Transfection in cultured HEK293T was performed by
calcium phosphate precipitation
Western blot analysis
HEK293T cultures or tissues were homogenized in lysis
buffer (10 mm HEPES, pH 7.5, 1 m NaCl, 1 mm EDTA,
1 mm EGTA, 0.5% Triton X-100 and 1 mgÆmL)1
bacitra-cin), followed by centrifugation at 12 000 g for 20 min at
4C Protein samples were denatured at 100 C for 5 min
in a buffer containing 1% SDS and 1% dithiothreitol,
and separated by 8% or 12% SDS–PAGE For western
blot analysis, our PRiMA polyclonal antibody (purified at
0.5 lgÆmL)1), an AChE antibody E19 (1 : 2000; Santa
Cruz Biotechnology Inc., Santa Cruz, CA, USA), a
mono-clonal FLAG antibody (1 : 1000; Sigma) and
anti-glyceral-dehyde-3-phosphate dehydrogenase (GAPDH) antibodies
(1 : 10 000; Sigma) were used The immune complexes
were visualized using the enhanced chemiluminescence
method (Amersham Biosciences) The intensities of the
bands in the control and stimulated samples, run on the
same gel and under strictly standardized enhanced
chemi-luminescence conditions, were compared on an image
analyzer using, in each case, a calibration plot constructed
from a parallel gel with serial dilutions of one of the
samples
Immunofluorescence analysis
Transfected cell cultures or tissue sections (16 lm) were
fixed by 4% paraformaldehyde in NaCl⁄ Pi for 15 min,
followed by 50 mm ammonium chloride (NH4Cl) treatment
for 25 min Samples were permeabilized by 0.2% Triton
X-100 in NaCl⁄ Pifor 10 min and blocked by 5% BSA in
NaCl⁄ Pi for 1 h at room temperature Cultures were
stained with PRiMA (2 lgÆmL)1) or FLAG (1 : 500,
Sigma) antibodies Tissue sections were double or triple
stained by rhodamine-conjugated or Alexa 647-conjugated
a-bungarotoxin (dilution 1 : 500; Molecular Probes,
Eugene, OR, USA), PRiMA antibody (2 lgÆmL)1), AChE
antibody (dilution 1 : 500, Santa Cruz Biotechnology),
anti-synaptotagmin (SV48) (1 : 500, BD Biosciences Clontech,
San Jose, CA, USA), SNAP-25 (1 : 200, Sigma),
anti-ChAT (1 : 200, Millipore, Bedford, MA, USA), anti-NeuN
(1 : 500, Millipore) and Cy3-conjugated anti-GFAP (1 : 500, Sigma) for 16 h at 4C, followed by the corre-sponding fluorescence-conjugated secondary antibodies (Alexa 488-conjugated anti-rabbit, Alexa 555- or Alexa 647-conjugated anti-mouse and anti-goat) for 2 h at room temperature The specificity of the PRiMA antibody was established by pre-incubation with the PRiMA antigen (10 lgÆmL)1) for 2 h at 4C Samples were dehydrated serially with 50%, 75%, 95% and 100% ethanol and mounted with fluorescence mounting medium (DAKO, Carpinteria, CA, USA) The samples were then examined using a Leica confocal microscope with excitation at
488 nm⁄ emission at 505–535 nm for green, excitation at
543 nm⁄ emission at 560–620 nm for red, and excitation
at 647 nm⁄ emission at 660–750 nm for pseudo-color
Sucrose density gradient analyses
Separation of the various molecular forms of AChE was performed by sucrose density gradient analysis, as described previously [28] In brief, sucrose gradients (5% and 20%) in lysis buffer were prepared in 12 mL polyallo-mer ultracentrifugation tubes with a 0.4 mL cushion of 60% sucrose at the bottom Sample extracts (0.2 mL) mixed with sedimentation markers (alkaline phosphatase, 6.1S; b-galactosidase, 16S) were loaded onto the gradients and centrifuged at 175 000 g in a Sorvall TH 641 rotor at
4C for 16 h Approximately 45 fractions were collected and AChE enzymatic activity was determined according to the method of Ellman [29]; the reaction medium contained 0.1 mm tetra-isopropylpyrophosphoramide, an inhibitor of BChE Absorbance at 410 nm was recorded as a function
of the reaction time The proportions of the various AChE forms were determined by summation of the enzy-matic activities corresponding to the peaks of the sedimen-tation profile In the immunoprecipisedimen-tation of G4AChE by PRiMA antibody, 1 mL samples of tissue extracts were incubated for 4 h at 4C with purified PRiMA antibody (10 lgÆmL)1) Then, 50 lL of washed protein-G agarose gel (Santa Cruz Biotechnology) was added and incubated for 1 h at 4C After centrifugation, the supernatants were loaded onto sucrose gradients for sedimentation analysis
ELISA for PRiMA-linked G4AChE
Fifty microliter samples of serially diluted PRiMA antibody were coated in a 96-well ELISA plate (Nunc Maxisorp Immunoplate, Roskilde, Denmark) for 16 h The antibody was removed and the plate was washed twice with 200 lL NaCl⁄ Picontaining 0.1% Tween-20 The plate was blocked
by NaCl⁄ Pi with 5% fetal bovine serum for 2 h at room temperature Tissue lysates containing equal AChE activity were loaded onto the precoated ELISA plate and incubated for 2 h The plate was washed three times with 200 lL