Restricted localization of proline-rich membrane anchor (PRiMA) of globular form acetylcholinesterase at the neuromuscular junctions – contribution and expression from motor neurons

Authors


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

Abstract

The expression and localization of the proline-rich membrane anchor (PRiMA), an anchoring protein of tetrameric globular form acetylcholinesterase (G4 AChE), 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 G4 AChE decreased. G4 AChE 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 G4 AChE 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 G4 AChE depends on muscle fiber type. In parallel, the expression of PRiMA, AChET and G4 AChE 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, AChET and G4 AChE 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

Acetylcholinesterase (AChE; EC 3.1.1.7) plays a crucial role in terminating the synaptic transmission by hydrolyzing the neurotransmitter acetylcholine at the neuron-to-neuron synapses in the central nervous system 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 catalytic 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 AChEH variant produces a glycosylphosphatidylinositol-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 asymmetric 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 G4 AChE) in brain and muscle [3–5]. Two PRiMA isoforms (PRiMA I and PRiMA II) are generated from the PRiMA gene by alternative splicing. PRiMA 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 coincides with the establishment of neuromuscular contacts during development and regeneration [7,8], G4 AChE also exists in muscles. Several studies have revealed that the level of G4 AChE is controlled by the dynamic activity of skeletal muscles. The transcriptional regulation of PRiMA is down-regulated during myogenic differentiation and under the influence of innervation [9]. In line with the transcriptional expression of PRiMA, the proportion of G4 AChE decreases during myogenic differentiation and innervation [1,9]. In mammals, fast-twitch muscles contain a large amount of G4 AChE, 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 predominant form of AChE expressed by motor neurons in chick spinal cord is G4 AChE [15].

In this article, we analyze the expression and localization of the PRiMA I-linked G4 form 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 G4 AChE 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 G4 AChE and illustrate its temporal and spatial expression at NMJs.

Results

Regulation of G4 AChE 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 (corresponding to PRiMA I unless specified) and a C-terminal truncated mutant (PRiMAΔC-term) cDNA, both tagged with a FLAG epitope, were transfected into HEK293T cells. In western blot analysis, a FLAG antibody recognized both PRiMA and PRiMAΔC-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 PRiMAΔC-term construct. In addition, the recognition was fully blocked by pre-incubation of the PRiMA antibody with the antigen, i.e. the PRiMA I C-terminal peptide (Fig. 1A). In the immunocytofluorescent staining of transfected fibroblasts, the PRiMA antibody also recognized FLAG-tagged PRiMA-expressing cells (Fig. 1B). In contrast, FLAG-tagged PRiMAΔC-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.

Figure 1.

 The specificity of the PRiMA antibody. (A) Protein samples (40 μg) of HEK293T cells expressing FLAG-PRiMA or FLAG-PRiMAΔC-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 μg·mL−1 were pre-incubated with the PRiMA antibody (0.5 μg·mL−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 μm.

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 possesses exons 4 and 5, produces a 40-residue-long intracellular 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-regulated gradually in the early postnatal stages and dramatically in the adult stage (Fig. 2B). Meanwhile, the level of AChET mRNA increased gradually from the early postnatal stage to the adult. Using PRiMA antibody, 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, AChET protein and AChE enzymatic activity increased during muscle development (Fig. 2C). With regard to the AChE molecular form, the AChE G1 and G4 forms 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 quantify the relative amount of PRiMA-linked G4 AChE in developing muscle, protein extracts at different developmental stages were analyzed by sedimentation in sucrose density gradients. The proportion of G4 AChE 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 G4 AChE in muscle increased twofold from birth to adult (Fig. 2E). PRiMA-linked G4 AChE therefore increased during muscle development.

Figure 2.

 Developmental profiles of PRiMA, AChET and G4 AChE 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 AChET. (C) Samples of extracts from the lower leg muscles of rat (birth to adult stage) containing 40 μg 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 markers. Enzymatic activities are expressed in arbitrary units, and representative sedimentation profiles are shown. (E) The specific activity of G4 AChE was quantified at different developmental stages. Samples of muscle extracts at different developmental stages containing 600 μg of protein were loaded on sucrose density gradients. The peak area corresponding to G4 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.

Expression of PRiMA-linked G4 AChE in fast-twitch and slow-twitch muscles

In order to investigate the expression level of PRiMA and PRiMA-linked G4 AChE 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 abundance of PRiMA-linked G4 AChE was determined by ELISA using our PRiMA antibody. Equal amounts of AChE activity were loaded onto an ELISA plate precoated with serial dilutions of PRiMA antibody. The retained AChE enzymatic activity, corresponding to PRiMA-linked G4 AChE, was measured after washing. We found larger amounts (over twofold) of PRiMA-linked G4 AChE in the tibialis than in the soleus (Fig. 3B). The higher expression of G4 AChE in the tibialis was further confirmed by sucrose density gradient 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.

Figure 3.

 Expression of PRiMA and G4 AChE in different muscles. (A) Samples of extracts from adult rat soleus and tibialis containing 40 μg 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 ± standard error of the mean, n = 4. (B) The relative amount of PRiMA-linked G4 AChE 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 antibody for 2 h. The retained AChE activity was determined. (C) For immunodepletion, 1 mL samples of extracts from adult rat brain, tibialis and soleus were incubated with PRiMA antibody (10 μg·mL−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.

We analyzed the localization of PRiMA in sections of tibialis and soleus muscle by immunohistofluorescence. NMJs were visualized by labeling the postsynaptic acetylcholine receptor (AChR) with α-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 AChE nor AChR was present in either muscle fiber type. However, the precise localization of PRiMA has yet to be determined.

Figure 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 α-bungarotoxin (red or pseudo-blue) for postsynaptic AChR, anti-AChET (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 μm.

Presence of PRiMA-linked G4 AChE 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 G4 AChE, 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 predominant 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 protein 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 observation that G4 was the predominant form of the enzyme in the adult spinal cord (Fig. 5D). The majority of G4 AChE was associated with PRiMA I, as more than 70% was immunoprecipitated with the PRiMA antibody. The relative amount of G4 AChE determined from sedimentation profiles allowed us to evaluate its activity: the specific activity of G4 AChE per milligram of protein reached a plateau in the spinal cord about 10 days after birth (Fig. 5E).

Figure 5.

 Developmental evolution of PRiMA and G4 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 AChET (671 bp). Representative results are shown. (B) Samples of extracts of rat spinal cord (from birth to adult stages) containing 40 μg of protein were loaded per lane for western blotting. PRiMA and AChET proteins 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) G4 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.

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 acetyltransferase (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.

Figure 6.

 Motor neurons in the spinal cord express PRiMA. (A) Schematic 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 μm. (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 AChET, ChAT and NeuN, but not with GFAP. Representative images are shown, n = 4. Bar, 20 μm.

Although motor neurons are able to synthesize PRiMA and produce G4 AChE, the restricted localization of PRiMA-linked G4 AChE at NMJs could still be derived from three sources: muscle, Schwann cells and/or motor neurons. In order to determine the localization of PRiMA-linked G4 AChE, 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 presynaptic marker, synaptosomal-associated protein 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.

Figure 7.

 Presynaptic localization of PRiMA at NMJs. Adult rat tibialis sections were triple stained with Alexa 647-conjugated α-bungarotoxin (pseudo-blue), anti-synaptotagmin (SV48; red) or anti-SNAP-25 (red) antibodies, and anti-PRiMA (green), and examined by confocal microscopy. Merged images allow a comparison of PRiMA with presynaptic markers (PRiMA + SV48/SNAP-25) and a postsynaptic marker (PRiMA + AChR). The distribution of PRiMA overlapped with that of SV48 and SNAP-25. Representative images are shown, n = 4. Bar, 20 μm.

Innervation regulates the expression of PRiMA-linked G4 AChE 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 reduction of G1 and G4 forms in the spinal cord and of G1, G4 and A12 forms in the tibialis (Fig. 8C). Thus, denervation induced a decrease in PRiMA and G4 AChE in the spinal cord and muscle.

Figure 8.

 Denervation reduces the expression of PRiMA and G4 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 analysis. The mRNA levels of denervated muscles (Den) corresponding to PRiMA (top) and AChET (bottom) were determined by PCR and normalized to those of control (sham-operated) muscles. (B) Samples of extracts from control and denervated muscles containing 50 μg 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 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. (D) Sections from adult rat tibialis after 7 days of denervation (right) and sham-operated (left) were triple stained with Alexa 647-conjugated α-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. Representative images are shown, n = 3. Bar, 10 μm.

To investigate the contribution of the motor neuron to PRiMA-linked G4 AChE at NMJs, we analyzed the effect of denervation on the localization of PRiMA. The NMJs of the denervated tibialis and sham-operated muscle were stained for PRiMA, together with the postsynaptic marker AChR (shown in pseudo-blue) and a presynaptic marker SV48 (shown in red). Presynaptic labeling essentially disappeared at the denervated 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 inactivated contain essentially no G4 AChE, suggesting that this enzyme form is entirely associated with PRiMA. Our results show that G4 AChE is, indeed, largely immunoprecipitated with a PRiMA antibody. However, a fraction of G4 AChE 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 G4 AChE was found in muscles of PRiMA knockout mice, implying that all G4 AChE in muscle is linked with the membrane-anchoring protein PRiMA. During muscle development, the amount of PRiMA-linked G4 AChE progressively increased from birth to the adult stage. In addition, the expression of PRiMA and G4 AChE was dependent on the fast or slow nature of muscle fibers. The strong expression of PRiMA protein and G4 AChE 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 developmental change of PRiMA-linked G4 AChE in muscle correlates with an increase in muscular activity and muscle loading [15–17], which leads to the differentiation 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 synaptic basal lamina [18]. Our study and others have shown that G4 AChE is linked by PRiMA and localized in the membranes of postsynaptic and presynaptic cells [9]. At NMJs, three cell types can contribute to synaptic AChE: the postsynaptic muscle cell, the presynaptic motor neuron and the Schwann cell. During development, the muscle is the primary source of all forms of AChE [1]. In contrast, the contribution of the Schwann 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 G4 AChE, in the motor neurons 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 asymmetric AChE by motor neurons and its secretion by the motor nerve terminals at frog NMJs could be induced by damaged target muscles. Indeed, the capacity 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 G4 AChE 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 G4 BChE, together with G4 AChE, 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 axonal flow to the presynaptic terminal, as revealed by enzymatic and microscopic studies [13]. The function of pre- and postsynaptic PRiMA-linked G4 AChE expressed by motor neuron and muscle, particularly during early stages of development, is an open question. One of the proposed functions of two-sided expression of AChE in both pre- and postsynaptic membranes is to play an active role during synaptogenesis through the adhesive function of AChE [23,24]. In addition, the decrease in PRiMA and AChE expression 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, G4 AChE could only be identified 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, suggesting that it is partly of muscle origin. Such a peri-junctional distribution of G4 AChE, 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 G4 AChE was found to be located in the presynaptic membrane of the motor nerve terminal. This is consistent 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 μg 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 collected and purified by protein G-Sepharose (Amersham Biosciences), according to the manufacturer’s instructions. The amount of purified antibody was determined spectrophotometrically.

DNA construction and transfection

The HEK293T cell line was obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum. Cultured cells were incubated at 37 °C in a water-saturated 5% CO2 incubator. All reagents for cell cultures were from Invitrogen. cDNAs encoding full-length mouse PRiMA (PRiMA I) and a COOH-terminal truncated mutant (PRiMAΔC-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 position 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 mg·mL−1 bacitracin), followed by centrifugation at 12 000 g for 20 min at 4 °C. 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 μg·mL−1), an AChE antibody E19 (1 : 2000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), a monoclonal FLAG antibody (1 : 1000; Sigma) and anti-glyceraldehyde-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 chemiluminescence 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 μm) 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/Pi for 10 min and blocked by 5% BSA in NaCl/Pi for 1 h at room temperature. Cultures were stained with PRiMA (2 μg·mL−1) or FLAG (1 : 500, Sigma) antibodies. Tissue sections were double or triple stained by rhodamine-conjugated or Alexa 647-conjugated α-bungarotoxin (dilution 1 : 500; Molecular Probes, Eugene, OR, USA), PRiMA antibody (2 μg·mL−1), AChE antibody (dilution 1 : 500, Santa Cruz Biotechnology), anti-synaptotagmin (SV48) (1 : 500, BD Biosciences Clontech, San Jose, CA, USA), anti-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 4 °C, followed by the corresponding 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 μg·mL−1) for 2 h at 4 °C. 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 polyallomer 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; β-galactosidase, 16S) were loaded onto the gradients and centrifuged at 175 000 g in a Sorvall TH 641 rotor at 4 °C 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 enzymatic activities corresponding to the peaks of the sedimentation profile. In the immunoprecipitation of G4 AChE by PRiMA antibody, 1 mL samples of tissue extracts were incubated for 4 h at 4 °C with purified PRiMA antibody (10 μg·mL−1). Then, 50 μL of washed protein-G agarose gel (Santa Cruz Biotechnology) was added and incubated for 1 h at 4 °C. After centrifugation, the supernatants were loaded onto sucrose gradients for sedimentation analysis.

ELISA for PRiMA-linked G4 AChE

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 μL NaCl/Pi containing 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 μL NaCl/Pi containing 0.1% Tween-20, and the retained AChE activity was measured.

Real-time PCR analysis

Total RNA from rat tissues was isolated with TRIzol reagent (Invitrogen), and 5 μg of RNA was reverse transcribed by Moloney Murine Leukemia Virus Reverse Transcriptase (Invitrogen), according to the manufacturer’s instructions. Real-time PCR of PRiMA, AChET and 18S transcripts was performed on equal amounts of reverse-transcribed products, using SYBR Green Master mix and Rox reference dye, according to the manufacturer’s instructions (Applied Bioscience, Foster City, CA, USA). The primers were as follows: 5′-TCTGACTGTCCTGGTCATCATTTGCTAC-3′ and 5′-TCACACCACCGCAGCGTTCAC-3′ for mouse PRiMA I and II (GenBank numbers NM 133364 and NM 178023); 5′-CTGGGGTGCGGATCGGTGTACCCC-3′ and 5′-TCACAGGTCTGAGCAGCGTTCCTG-3′ for mouse AChET [30]; 5′-TGTGATGCCCTTAGATGTCC-3′ and 5′-GATAGTCAAGTTCGACCGTC-3′ for rat 18S ribosomal RNA. The SYBR green signal was detected by an Mx3000p™ multiplex quantitative PCR machine (Stratagene, La Jolla, CA, USA). Transcript expression levels were quantified using the ΔΔCt value method [31], where values were normalized to 18S rRNA as an internal control in the same sample. PCR products were analyzed by gel electrophoresis and the specificity of amplification was confirmed by the melting curves.

Sciatic nerve section

Two-month-old Sprague–Dawley rats weighing approximately 250 g were anesthetized by isoflurane. A portion of approximately 3 mm of the sciatic nerve located around the upper thigh was removed by an aseptic surgical technique [13]. The rats were sacrificed according to the instructions of the Animal Care Facility of The Hong Kong University of Science and Technology. Spinal cord (lumbar) and tibialis muscles were collected 7 days after denervation. Samples were frozen in liquid nitrogen immediately after dissection and stored at −80 °C for RNA and protein extraction, and for confocal microscopy. Control experiments were performed by sham operation on different rats.

Other assays

Protein concentrations were measured by Bradford’s method [32] with a kit from Bio-Rad Laboratories (Hercules, CA, USA). Statistical tests were performed by the primer program, version 1 [33]: differences from basal or control values (as shown in the plots) were classified as significant for P < 0.05 and P < 0.01 and highly significant for P < 0.001.

Acknowledgements

This work was supported by the Research Grants Council of Hong Kong (HKUST 6404/05M, 6419/06M, 662407, 662608) to KWKT.

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