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Originally published In Press as doi:10.1074/jbc.M406896200 on July 23, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40601-40608, September 24, 2004
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Inhibition of SNAP-25 Phosphorylation at Ser187 Is Involved in Chronic Morphine-induced Down-regulation of SNARE Complex Formation*

Nan-Jie Xu{ddagger}§, Yong-Xin Yu{ddagger}§, Jian-Mei Zhu{ddagger}, Hua Liu{ddagger}, Li Shen{ddagger}, Rong Zeng{ddagger}, Xu Zhang||, and Gang Pei{ddagger}**

From the {ddagger}Laboratory of Molecular Cell Biology, Institute of Biochemistry and Cell Biology, ||Laboratory of Sensory System, Institute of Neuroscience, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Graduate School of the Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, People's Republic of China

Received for publication, June 21, 2004 , and in revised form, July 19, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Opiate abuse has been shown to cause adaptive changes in presynaptic release and protein phosphorylation-mediated synaptic plasticity, but the underlying mechanisms remain unclear. Neuronal SNARE proteins serve as important regulatory molecules underlying neural plasticity in view of their major role in the process of neurotransmitter release. In the present study, the expression of SNAP-25, a t-SNARE protein essential for vesicle release, was found to be dramatically regulated in hippocampus after chronic morphine treatment, which was visualized with two-dimensional gel electrophoresis. The spots of SNAP-25 in the gel were shifted along the dimension of isoelectric point, indicating a likely change of the post-transcriptional modification. Immunoblotting analysis with specific antibody to Ser187, a protein kinase C (PKC) phosphorylation site of SNAP-25, revealed that the specific phosphorylation was correspondingly decreased, which was correlated with morphine-induced inhibition of PKC activity. Moreover, the level of ternary complex of SNARE proteins in either synaptosomes or PC12 cells was significantly reduced after chronic morphine treatment. This suggests a causal relationship between the inhibition of PKC-dependent SNAP-25 phosphorylation and the down-regulation of SNARE complex formation after chronic morphine treatment. Further analysis of SNARE complex formed by transfection of the wild-type or Ser187 mutants of SNAP-25 showed that only wild-type-formed complex was inhibited by morphine treatment. Thus, these results indicate that chronic morphine treatment inhibits phosphorylation of SNAP-25 at Ser187 and leads to a down-regulation of SNARE complex formation, which presents a potential molecular mechanism for the alteration of exocytotic process and neural plasticity during opiate abuse.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Opiate abuse causes extensive neural adaptive changes in the brain (1–3), which may be involved in a formation of aberrant learning (2). Accumulating evidences demonstrate that opiates significantly alter synaptic transmission and neural plasticity in the hippocampus, a center of learning and memory (4–8). Importantly, presynaptic neurotransmitter release, which can be modulated by phosphorylation, serves as a critical element in the regulation of synaptic transmission during opiate abuse. Many protein kinases involved in modulation of transmitter release and synaptic plasticity, such as cAMP-dependent protein kinase (PKA),1 protein kinase C (PKC), and Ca2+/calmodulin-dependent protein kinase II (CaMKII), also participate in cellular and synaptic adaptation mediating opiate dependence (3, 9). However, the protein substrates of these kinases involved in opiate abuse still need to be further elucidated.

Regulated membrane fusion of synaptic vesicles and subsequent transmitter release involves the assembly of ternary complexes from soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) proteins (10). The ternary complex is formed by the plasma membrane proteins synaptosomal-associated protein of 25kDa (SNAP-25) and syntaxin, and vesicle-associated SNARE synaptobrevin 2 (synaptobrevin) (11). The SNARE proteins have been shown to be phosphorylated by various kinases (12), and accumulating evidences demonstrate that phosphorylation of SNAREs by various protein kinases plays an important role in modulating the molecular interactions between synaptic vesicles and the presynaptic membrane. For instance, phosphorylation of SNAP-25 by PKC regulates SNAP-25 localization (13, 14), affects interaction of SNAP-25 with syntaxin and synaptotagmin (15, 16), and potentiates the recruitment of vesicles to the plasma membrane (17, 18), whereas syntaxin1A phosphorylation by death-associated protein kinase (19) regulates its binding to Munc-18, a syntaxin1A-binding protein that regulates SNARE complex formation. In addition, many SNARE regulatory proteins have been shown to be phosphorylated in vitro, which may alter the interactions of the proteins with their effectors and binding partners and hence modulate neurotransmitter release (12). However, whether phosphorylation of SNARE proteins is modulated directly by opiate remains unclear, and the significance of phosphorylation of SNARE proteins in vivo during opiate abuse is still poorly understood.

In the current study, we showed that chronic morphine treatment inhibited the phosphorylation of SNAP-25, and this was attributable to the down-regulation of PKC activity. Moreover, the inhibition of PKC-dependent phosphorylation participated in a down-regulation of SNARE complex formation during morphine treatment. These results suggest that the phosphorylation of SNARE protein can be modulated in vivo, which may serve as a potential mechanism for the modulation of elementary steps of exocytotic process under opiate abuse.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Preparations of Mice Brain Tissue—Male C57BL/6J mice (20–22 g) were obtained from the Shanghai Laboratory Animal Center, Chinese Academy of Sciences (Shanghai, China). Mice were housed in groups and maintained on a 12-h light/dark cycle with food and water available ad libitum. All treatments were strictly in accordance with the National Institutes of Heath Guide for the Care and Use of Laboratory Animals. Mice were treated by repeated intraperitoneal injection of morphine (or saline in control) at an interval of 12 h, over 6 days (20 mg/kg on day 1 to 100 mg/kg on days 5 and 6) (20). Then the mice were decapitated at the time mentioned in the results. Withdrawal was precipitated by injecting naloxone (1 mg/kg, subcutaneously) 2 h after the last morphine administration (20). Then the brains were removed, the hippocampus, prefrontal cortex, cerebellum, and brain stem were rapidly dissected, and the tissue was prepared for two-dimensional gel electrophoresis (2-DE) as mentioned below. The further subcellular fractions were prepared, respectively, according to the standard methods as described previously (21, 22). Briefly, brain samples were homogenized in ice-cold 0.32 M sucrose, 5 mM HEPES (pH 7.4), 0.1 mM EDTA, and protease inhibitors mixture (Roche Applied Science) in a glass/Teflon homogenizer. Homogenates were centrifuged (1,000 x gx 10 min; 4 °C), and the supernatants were spun at 10,000 x gx 20 min in a microcentrifuge at 4 °C. The pellets constituted the crude synaptosomal fraction (P2). Then the fractions were resuspended in 500 µl of 1x P buffer (5.4 mM KCl, 0.8 mM MgSO4, 5.5 mM glucose, 50 mM HEPES, 130 mM choline chloride, 1 mM bovine serum albumin, and 0.01% CHAPS). Synaptosomes were then solubilized with 3.5 ml of buffer (150 mM NaCl, 10 mM HEPES, and 1.5% CHAPS, pH 7.4), stirred at 4 °C for 1.5 h, and further centrifuged at 12,000 x g for 1 h. After given a protein concentration of ~0.5 mg/ml that was determined by Bradford methods (23), this suspension was used in Western blot analysis as described below.

Two-dimensional Gel Electrophoresis (2-DE) for Protein Identification—Brain samples were homogenized in ice-cold 2-DE sample buffer (4% CHAPS, 8 M urea, 40 mM Tris, 65 mM dithioerythritol) with protease inhibitors mixture (Roche Applied Science), in a glass/Teflon homogenizer. The homogenates were sonicated on ice and centrifuged (10,000 x gx 1h) at 4 °C, and the supernatants were collected. Two-DE was performed according to the previous studies (24, 25). 100 µg of total protein for analytical 2-DE gel and 1 mg for preparative gel were loaded on nonlinear immobilized pH gradient (IPG) strips (NL pH 3–10; Amersham Biosciences), respectively, and hydrated in hydration buffer (8 M urea, 2% CHAPS, 18 mM dithiothreitol, 0.5% IPG buffer, bromphenol blue trace) for 10 h. Isoelectric focusing was performed, at 20 °C, for 1 h at 500 V, for 1 h more at 1,000 V, and then for 12 h at 8,000 V in an IPGphor apparatus (Amersham Biosciences) with a limiting current of 70 µA per strip. Prior to the second dimension, strips were incubated in 50 mM dithioerythritol buffer (6 M urea, 2% SDS, 30% glycerol, 50 mM Tris-HCl, pH 6.8) for 15 min to reduce S–S bonds. The -SH groups were subsequently blocked for 15 min with the same solution except that the dithioerythritol was replaced by iodoacetamide (2.5%) and a trace of bromphenol blue. Equilibrated strips were inserted onto 12.5% SDS-PAGE gels, sealed with 0.5% agarose, and SDS-PAGE gels were run under constant current 10 mA/gel for 0.5 h and 30 mA/gel for 3.5 h at 10 °C (Bio-Rad). Analytical gels were visualized by silver staining or were electroblotted onto nitrocellulose and then incubated with the monoclonal antibody of SNAP-25 as described below. Protein spots excised from 2-DE gels in preparative gel were identified by Western blot analysis visualized with SNAP-25 antibody or were subjected to in-gel digestion with trypsin, and the resulting peptides were characterized by matrix-assisted laser desorption/ionization-time of flightmass spectrometry (MALDI-TOF-MS) and data base searching.

PC12 Cell Culture and Drug Treatment—PC12 cells were grown in RPMI 1640 media (Invitrogen) supplemented with 5% horse serum/10% fetal bovine serum/50 units/ml penicillin, and 50 µg/ml streptomycin. Cells (106) were incubated in a humidified atmosphere of 5% CO2. After chronic treatment with morphine (0.01–10 µM) or saline for the indicated time (1–24 h), the cells were lysed and analyzed by Western blot analysis as described below.

Transfection—Enhanced green fluorescent protein (EGFP)-SNAP-25 (Wild-type), EGFP-SNAP-25E (S187E), and EGFP-SNAP-25A (S187A) were kindly provided by Dr. Masakazu Kataoka (Dept. of Environmental Science and Technology, Shinshu University, Nagano, Japan) (14). Transfection of PC12 cells with eukaryotic expression plasmids was performed by using LipofectAMINE 2000 (Invitrogen) as previously described (26). After 24 h of transfection, the cells were treated with morphine as mentioned above and analyzed by Western blotting.

Electrophoretic Procedure and Western Blot Analysis—The samples (20 µg of protein) from brain tissue or cultured cells of control and morphine-treated groups were incubated at 100 °C or 22 °C for 5 min, respectively, before electrophoresis. Then the samples were loaded and subjected to a discontinuous gel, with the upper two-thirds being 8% and the lower one-third being 15% acrylamide, respectively, and were electroblotted onto nitrocellulose membrane by using a minigel and mini transblot apparatus (Bio-Rad). The membranes were blocked in blocking buffer (20 mM Tris-HCl, pH 7.5, 137 mM NaCl, and 0.1% Tween 20, 15% nonfat milk) at room temperature for 1 h. Then the membranes were incubated with antibodies of mouse anti-SNAP-25 (1:10,000) or mouse anti-syntaxin1A (1:10,000), or mouse anti-synaptobrevin (1:10,000) from Synaptic Systems (Göttingen, Germany), or rabbit antiphosphorylated SNAP-25 (1:1,000; a kind gift from Masami Takahashi, Kitasato University, Kanagawa, Japan), or mouse anti-GFP (1:1000; Roche Applied Science), respectively, overnight at 4 °C. The membranes were washed and incubated with horseradish peroxidase-conjugated anti-mouse or anti-rabbit secondary antibody for 1 h at room temperature (Sigma). Finally, the blots were visualized with enhanced chemiluminescence (Amersham Biosciences). For the quantification of the Western blot data, the developed films were scanned, the immunoreactive bands were digitized, and the densitometry was performed using Scion Image for Windows (Scion, Frederick, MD).

The re-electrophoresis in a second dimension for detecting SNARE complexes was carried out as described previously (27). In brief, after the first dimension, the whole lane containing the separated proteins was excised, soaked for 20 min in 10% (v/v) acetic acid and 25% (v/v) isopropanol, washed with H2O for 5 min, and then incubated for 20 min in SDS sample buffer. After heating for 5 min at 100 °C, the strip was mounted on top of a 15% gel, re-electrophoresed, and analyzed by Western blotting as mentioned above.

Protein Kinase Activity Assay—PKC and PKA activity was determined essentially according to the method described previously (28). Animal was rapidly decapitated, and hippocampus was dissected rapidly and the hippocampal synaptosomal fraction was isolated as described above. The synaptosomes were assayed for PKC and PKA activity using PepTag Nonradioactive PKC/PKA assay kit (Promega, Madison, WI) according to the manufacturer's instruction. All reaction components were added on ice in a final volume of 25 µl of the following mixture: 5 µl of PepTag PKC/PKA reaction 5x buffer, 5 µl of PepTag C1/A1 peptide (0.4 µg/µl), 5 µl of PKC/PKA activator 5x solution, 1 µl of peptide protection solution, and 1 µg of synaptosome for PKC/PKA activity assay. The mixture was incubated for 10 min at 37 °C for the detection of PKC activity and 30 min at 25 °C for that of PKA activity. The reaction was stopped by placing the tube into a boiling water bath for 10 min, and the samples were loaded onto the gel for electrophoresis. Before loading samples, 1 µl of 80% glycerol was added to the sample to ensure that it remained in the well. The specific peptide substrates used in this experiment were PLSRTLSVAAK for PKC and LRRASLG for PKA, respectively. The assay was based on the changes in the net charge of the fluorescent PKC/PKA substrates before and after phosphorylation. This change allowed the phosphorylated and unphosphorylated versions of the substrate to be rapidly separated on an agarose gel at neutral pH. The phosphorylated species migrated toward the positive electrode, whereas the nonphosphorylated substrate migrated toward the negative electrode. The intensity of fluorescence of phosphorylated peptides, which reflected the activity of PKC/PKA, was scanned by using a bioimaging system (Syngene, Cambridge, UK) and further quantified by using Scion Image for Windows (Scion).


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Regulation of SNAP-25 following Chronic Morphine Treatment—To test the potential changes of protein expression in the hippocampus during opiate abuse, mice were injected with morphine in an increasing dose for 6 days, a procedure known to produce significant morphine dependence (20), and then the proteome analysis of hippocampal protein expression was conducted. The hippocampal extracts were separated by high resolution 2-DE, and SNAP-25, a t-SNARE protein located within presynaptic terminals and essential for vesicle release (29, 30), was identified with MALDI-TOF-MS followed by data base searching and further confirmed by immunoblotting with antibody to SNAP-25 (Fig. 1, A and B). Interestingly, the spots of SNAP-25 in the lower isoelectric points (pIs) were dramatically redistributed to higher pI after the treatment, indicating a likely change of the post-transcriptional modification (Fig. 1A). To further investigate the expression pattern of SNAP-25, the 2-DE gel was electroblotted onto nitrocellulose membrane and visualized with antibody to SNAP-25. The expression of phosphorylated SNAP-25 in the 2-DE membrane was also detected with a specific antibody to Ser187 (13, 17), a specific phosphorylation site in the C-terminal (15). The result of Fig. 1C showed that, following chronic morphine treatment, the relative intensity of the immunoblotting spots visualized with antibody to SNAP-25 was considerably altered. Concretely, the labeling of spots on the left (low pI) became weaker, whereas the spots on the right (high pI) were more intensely labeled (Fig. 1C). No significant change was observed in the total level of SNAP-25 expression. The phosphorylated SNAP-25 mainly distributed in the spots of left part and was significantly decreased in morphine-treated group (Fig. 1D), which was consistent with the redistribution of the spots visualized with antibody to SNAP-25. The expression pattern of SNAP-25 on the blots of the 2-DE was similar to that shown in the previous study in which spots were demonstrated to represent different states of phosphorylation of SNAP-25 by PKC but not by casein kinase II or CaMKII (24). We also detected the expression pattern of syntaxin1A or synaptobrevin, other SNARE proteins, by using their specific antibodies, and no significant changes were observed after chronic morphine treatment under the same conditions (data not shown).



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  		FIG. 1.
Regulation of SNAP-25 in chronic morphine treated mice. A, the proteome analysis of the hippocampal protein expression was conducted in the mice following treatment of morphine in an increasing dose (from 20 mg/kg to 100 mg/kg, intraperitoneal) for 6 days. The high resolution two-dimensional electrophoresis (2-DE) gel was visualized with silver staining. Arrows indicate the altered proteins. A relative decrease in the optical density of the left spots (lower isoelectric point) and an increase in that of the right spots (higher isoelectric point) were observed after morphine treatment. These spots were identified as SNAP-25 by using MALDI-TOF-MS and data base searching. B, these spots in the two-dimensional gel were also excised, respectively, and further confirmed by immunoblotting with antibody to SNAP-25. Spots 1–10 as indicated by arrows in the two-dimensional gel (the upper panel) were excised for immunoblotting, and spots 4, 5, 6, and 7 corresponding to SNAP-25 on the gel were identified (the lower panel). The data were pooled from two independent experiments. The slight labeling of spot 8 might be caused by the contamination of spots 4 or 5, because the density was much lower than other spots, and the position of molecular weight was higher. TL, total lysates; Sal, saline treatment; Mor, morphine treatment. C, the optical density of the spots visualized with antibody to SNAP-25 on the left decreased, while that on the right increased following chronic morphine treatment. The relative density of SNAP-25 spots on the left and right is expressed as the percentage of the summation of the measures for all spots in the right panel. Data are presented as mean ± S.E. values of the results obtained in three separate experiments. D, the phosphorylated SNAP-25 was mainly distributed in the spots on the left and was significantly decreased in the morphine-treated group. Group data (mean ± S.E.) of SNAP-25 and phosphorylated-SNAP-25 in C and D for hippocampal homogenates of morphine-treated mice were expressed as percentages of that of saline-treated rats in the right panel (mean ± S.E.). *, p < 0.01, Student's t test; as compared with saline group. Sal, saline treatment; Mor, morphine treatment.

 
Inhibition of PKC Activity and PKC-dependent Phosphorylation of SNAP-25—To examine the regulation of SNAP-25 at synapses, the synaptosomal fraction of the hippocampus was prepared and the protein expression of total and phosphorylated SNAP-25 was detected, respectively. In agreement with the results of 2-DE gel, the level of phosphorylated SNAP-25 was decreased after chronic morphine treatment, whereas the total SNAP-25 expression was unchanged (Fig. 2A). Moreover, when the morphine-treated mice were injected with naloxone (1 mg/kg), a nonspecific opioid receptor antagonist documented to lead to morphine withdrawal (20), 2 h after the last morphine administration, the decreased level of phosphorylated SNAP-25 was restored to the normal level (Fig. 2B). We also detected expression of phosphorylated SNAP-25 in synaptosomal fraction of other brain regions under the same condition, and a similar decrease was observed in the brain stem but not in the prefrontal cortex and the cerebellum (Fig. 2, C and D), suggesting differential sensitivity of brain regions to chronic use of opiate.



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  		FIG. 2.
Differential regulation of SNAP-25 phosphorylation in several brain regions following chronic morphine treatment. A, SNAP-25 phosphorylation in the hippocampus (Hip) was decreased after chronic morphine treatment (n = 6). B, naloxone (Nal)-initiated morphine withdrawal 2 h after the last morphine administration restored the phosphorylation of SNAP-25 in Hip to the normal level (n = 6). C, the decrease in SNAP-25 phosphorylation was also observed in the brain stem (BS; n = 6) but not in the prefrontal cortex (PC; n = 6) and the cerebellum (Cer; n = 6). No significant change of total level of SNAP-25 was observed in each brain region after chronic morphine treatment. D, the density of phosphorylated SNAP-25 of morphine groups was expressed as the percentage of that of control group. Data are presented as mean ± S.E. values of the results obtained in three separate experiments. *, p < 0.05, Student's t test; as compared with saline group. Sal, saline treatment; Mor, morphine treatment.

 
It is known that SNAP-25 is phosphorylated by PKC (15), and PKC expression in hippocampus decreases after chronic morphine treatment in the brain (31, 32), but how the activity of PKC is regulated in the hippocampus by morphine remains unknown. We found that the PKC activity in hippocampal synaptosomes was significantly decreased 2 h after the last morphine administration, and then recovered 8 h later (Fig. 3B), whereas a significant increase in PKA activity was observed after chronic morphine treatment (Fig. 3D). The time course of changes in PKC activity was parallel with that in levels of phosphorylated SNAP-25 (Fig. 3, F and H). We also detected the effect of acute morphine treatment (10–100 mg/kg) on PKC activity and SNAP-25 phosphorylation, and no obvious change was observed (Fig. 3, A, C, E, and G).



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  		FIG. 3.
Correlated decrease in PKC activity and SNAP-25 phosphorylation. A and B, PKC activity of hippocampal synaptosomes in acute morphine-treated (100 mg/kg intraperitoneal, A) and chronic morphine-treated (B) mice was measured, respectively. The activity of PKC in chronic morphine-treated mice was decreased 2 h after the last injection of morphine and restored to the normal level 8 h later, whereas no significant change could be observed after acute morphine treatment. 10 mg/kg morphine was also used, and the results were similar to that of 100 mg/kg. The upper band indicates unphosphorylated substrates, and the lower band shows the fluorescence of phosphorylated peptides that reflects the activity of PKC. C and D, PKA activity of hippocampal synaptosomes in acute morphine-treated (C) and chronic morphine-treated (D) mice was measured, respectively. A significant increase could be observed in chronic morphine-treated groups compared with control groups, whereas no significant change could be observed in the acute morphine-treated group. The data of PKC and PKA were pooled from five independent experiments, respectively. E, no significant change in SNAP-25 phosphorylation was observed after acute morphine treatment as compared with the saline control group. F, phosphorylation of SNAP-25 was decreased 2 h after the last injection of morphine in chronic morphine-treated mice and was restored 8 h later, whereas no change was observed in total SNAP-25 expression in synaptosomes following chronic morphine treatment. The immunoblots were representative of two independent experiments. G and H, activities of PKA and PKC reflected by fluorescence and the density of phosphorylated SNAP-25 in acute (G) and chronic (H) treated groups were quantitated. The data of morphine groups are expressed as the percentage of that of saline control groups (mean ± S.E.). *, p < 0.05; Student's t test; as compared with the activity of PKA/PKC in the saline group. Sal, saline treatment; Mor, morphine treatment.

 
Down-regulation of SNARE Ternary Complex after Chronic Morphine Treatment—Because SNAP-25 is a key component of the synaptic vesicle docking/fusion machinery that binds with synaptobrevin and syntaxin to form the SNARE complex, we want to know whether the abundance of the ternary complex of SNARE proteins was also altered after chronic exposure to morphine. The ternary complex is highly stable, such that it resists denaturation by SDS and is disrupted only upon boiling of sample (33). Therefore, it is possible to detect the signal of SNARE complex by comparing boiled and nonboiled samples. Fig. 4A shows that a high molecular mass band of ternary complex in ~100 kDa was detected in nonboiled samples. The band disappeared after boiling the samples, which was accompanied with a corresponding increase in the level of monomeric SNAP-25. The band at ~100 kDa was also detected using antibodies to syntaxin or synaptobrevin (Fig. 4A). Moreover, all the three proteins could be detected when the gel in the position of the band was excised, boiled, and reloaded (data not shown), indicating that the band containing all three proteins is one of the forms of ternary complex. As shown in Fig. 4B, after chronic morphine treatment, a significant decrease in the high molecular mass band at 100 kDa and an increase of monomeric SNAP-25 band was observed, whereas the total expression level of SNAP-25 remained unchanged (Fig. 4C). Interestingly, naloxone-initiated withdrawal could restore morphine-altered SNAP-25 to a high level (Fig. 4B), suggesting that the change of the complex is correlated to the modulation of morphine level in mice. Similar results were also obtained when the antibodies to syntaxin1A and synaptobrevin was applied, respectively (data not shown).



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  		FIG. 4.
Chronic morphine-induced reduction in the ternary complex of SNARE proteins. A, the high molecular mass bands of SNAP-25, syntaxin1A, and synaptobrevin were detected at ~100 kDa in nonboiled samples and disappeared after boiling the samples, indicating the expression of SNARE complex. B, after chronic morphine treatment, a significant decrease in the ~100-kDa band and an increase of monomeric band was detected with antibody to SNAP-25. Naloxone (Nal)-initiated withdrawal 2 h after the last morphine administration restored the alteration of SNARE complex in the chronic morphine-treated mice. C, immunoblotting of the boiled sample showed that no significant change of total level of SNAP-25 could be detected after morphine treatment. D, SDS-resistant complexes was significant down-regulated as compared with the saline group. After completion of the first dimension, the gel lane in saline and morphine groups was excised respectively, heated to 100 °C to disrupt the complexes, and re-electrophoresed under identical conditions in a second dimension on a 15% gel. The immunoblots were representative of two independent experiments. Sal, saline treatment; Mor, morphine treatment.

 
SDS-resistant complexes contain large forms with distinct molecular weight (27), and anti-SNAP-25 antibody may not recognize all the forms of these complexes in the hippocampal samples. Thus it is essential to examine whether the other high molecular weight bands are also altered by chronic morphine treatment. In the present study, the gel lane in saline and morphine groups was excised, respectively, after electrophoresis, heated to 100 °C to disrupt the complexes, and re-electrophoresed under identical conditions in a second dimension. Fig. 4D shows that the SDS-resistant complexes were visualized with any one of the three antibodies. After chronic morphine treatment, the expression level of complexes was significantly down-regulated, which was accompanied with an up-regulation of monomeric proteins, as compared with the saline group (Fig. 4D). This was consistent with previous observations.

Regulation of SNARE Complex Formation by PKC-dependent Phosphorylation of SNAP-25—To clarify the possible involvement of PKC-dependent SNAP-25 phosphorylation in the regulation of SNARE complex formation by chronic morphine treatment, PC12 cells were used as a cellular model, in which both µ opioid receptor and {delta} opioid receptor were expressed (34–37), to further detect the effect of morphine. As shown in Fig. 5A, the phosphorylation of SNAP-25 at Ser187 in PC12 cells was markedly reduced in a dose-dependent manner (Fig. 5A), and the decrease appeared 1 h after morphine incubation (Fig. 5B).



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  		FIG. 5.
Decrease of SNAP-25 phosphorylation in chronic morphine-treated-PC12 cells. A, SNAP-25 phosphorylation was markedly reduced by morphine in a dose-dependent manner after treatment with morphine 0.01–10 µM for 12 h. No significant change was observed in total level of SNAP-25. B, morphine treatment (1 µM) decreased SNAP-25 phosphorylation 1 h after morphine incubation in PC12 cells. The immunoblots were representative of three independent experiments. Sal, saline treatment; Mor, morphine treatment.

 
In addition to the SNAP-25 phosphorylation, the high molecular mass band at ~100 kDa was also detected in PC12 cells using antibodies to SNAP-25, syntaxin 1A, and synaptobrevin, respectively. Consistent with the data in hippocampal fraction, the band at ~100 kDa was recognized and could be abolished by boiling samples (Fig. 6A). After morphine treatment, the high molecular mass band was significantly down-regulated in a dose- and time-dependent manner (Fig. 6, B and C). However, the total expression level of SNAP-25 in PC12 cells remained unchanged after morphine treatment in the indicated time and dose (Fig. 5, A and B).



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  		FIG. 6.
Down-regulation of SNARE complex formation in PC12 cells after chronic morphine treatment. A, the high molecular weight band at ~100 kDa, which represented SNARE complex, could be detected in PC12 cells with antibodies to SNAP-25, syntaxin1A, and synaptobrevin respectively, and could be abolished by boiling samples. B, chronic treatment of PC12 cells with morphine 0.01–10 µM for 12 h decreased the expression of SNAP-25 at ~100 kDa in a dose-dependent manner. C, chronic preincubation with morphine 1 µM for 1–12 h attenuated the SNAP-25 expression at ~100 kDa in a time-dependent manner. The immunoblots in B and C were representative of three independent experiments.

 
The next question we asked was whether the phosphorylation of SNAP-25 was implicated in the regulation of SNARE complex formation. Wild-type (WT) and mutant constructs of SNAP-25 (S187E and S187A), in which Ser187 was replaced by Glu or Ala, were fused with EGFP at the N terminus, expressed in PC12 cells, and analyzed with an anti-GFP antibody. S187E was used to mimic the constitutively phosphorylated state, whereas S187A to mimic the nonphosphorylated form of serine. As shown in Fig. 7, two high molecular mass bands at ~100 kDa and ~130 kDa were recognized by anti-GFP antibody after transfection of EGFP-tagged SNAP-25 in PC12 cells (Fig. 7A), but only the ~130-kDa band could resist the high temperature incubation up to 95 °C (Fig. 7B). This suggests that the ~130-kDa band is the ternary complex formed by EGFP-tagged SNAP-25 in transfected PC12 cells. Moreover, S187E expression potentiated the level of the formed ternary complex, whereas S187A expression attenuated it (Fig. 7A). Twenty-four hours after incubation of PC12 cells with morphine, the expression level of ternary complex at ~130 kDa containing WT was attenuated, whereas the expression patterns of the ternary complex containing S187E or S187A were not changed (Fig. 7, C and D). This indicates that SNAP-25 phosphorylation at Ser187 is involved in the down-regulation of SNARE complex formation following chronic morphine treatment.



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  		FIG. 7.
Phosphorylation of SNAP-25 participated in the regulation of SNARE complex formation during chronic morphine treatment. A, high molecular mass band at ~130 kDa, which represented SNARE complex, could be observed in PC12 cells transfected with EGFP-tagged SNAP-25 but not in untransfected cells or hippocampal synaptosomes (Hip) after immunoblotting with anti-GFP antibody. The complex signal at ~130 kDa was stronger in the group transfected with S187E and was weaker with S187A than that with WT. Immunoblotting of the boiled sample detected with anti-GFP antibody shows the expression of total transfected EGFP-SNAP-25. B, the complex signal at ~130 kDa could not be abolished until the sample was treated above 95 °C. C, after morphine treatment for 24 h, the complex signal at ~130 kDa was attenuated in the WT group, whereas no significant change could be observed in either S187E or S187A groups. Immunoblots were representative of three independent experiments. D, the complex signal of SNARE complex was quantitated, and the density in the morphine groups was expressed as the percentage of that in the saline group transfected with WT (mean ± S.E.). *, p < 0.05 Student's t test; as compared with the saline group. Sal, saline treatment; Mor, morphine treatment.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Studies on opiate dependence have been focused on protein phosphorylation-mediated synaptic plasticity, which are generally thought to be part of the neural basis of an addictive state (38, 39). Neuronal SNARE proteins serve as important regulatory molecules underling neural plasticity (24, 40, 41) in view of their major role in the process of neurotransmitter release. In this report we demonstrated that phosphorylation of SNARE protein SNAP-25 was inhibited by long term treatment of morphine, and this was attributable to a down-regulation of PKC activity, suggesting alteration in the presynaptic functions after opiate abuse. This is in agreement with our previous studies showing that chronic opiate treatment modulates hippocampal long term potentiation (7), and provides a molecular target of PKC that plays a critical role in the development of morphine dependence (42, 43).

It is known that SNAP-25 is synthesized as a soluble protein in the cytosol and is targeted to the plasma membrane. The plasma membrane targeting of SNAP-25 increases its local concentration and is necessary for SNARE complex formation (44), which is thought to form a protein bridge between an incoming vesicle and the acceptor compartment (45, 46). The present study revealed that the abundance of SNARE complex in synaptosomal fraction of the hippocampus and PC12 cells was significantly down-regulated by chronic morphine exposure. The down-regulation in the level of SNARE complex induced by morphine was attributable to PKC-dependent phosphorylation, because the level of SNARE complex formed with SNAP-25 WT but not that with the mutants of Ser187, a PKC phosphorylation site, could be reduced by chronic morphine treatment. This indicated that phosphorylation of SNAP-25 may play an essential role in the inhibitory regulation of morphine on SNARE complex formation. These results are consistent with the morphine-induced inhibition of SNAP-25 phosphorylation and SNARE complex formation in synaptosomes and suggested that the regulation of SNAP-25 by phosphorylation may be involved in PKC-mediated exocytotic process during opiate abuse.

The molecular mechanism of PKC-mediated vesicle exocytosis remains an open question. It has been shown that PKC mediates secretory vesicle translocation to the plasma membrane (18, 47). Lonart and Südhof (48) also propose a working model that SNARE complex assembly prior to fusion pore opening is up-regulated by PKC and then sets the readily pool of synaptic vesicles. Our unpublished data2 showed that SNARE complex formation was up-regulated after PMA treatment, and PMA-stimulated up-regulation of complex formation was inhibited by PKC blocker, indicating that the PKC pathway participated, at least in part, in SNARE complex formation. However, how the SNARE complex and the related step of membrane fusion are regulated by the PKC pathway is still elusive.

Noticeably, PKC can mediate the localization of SNAP-25 in PC12 cells (13, 14). Further electrophysiological study shows that PKC-dependent phosphorylation of SNAP-25 at Ser187 potentiates the vesicle recruitment (17), which indicates a critical role of SNAP-25 phosphorylation in the refilling of vesicle pools rather than in the fusion of vesicles from the readily releasable pool (17, 49). With biochemical methods, the present study demonstrated that SNAP-25 phosphorylation at Ser187 participated in the modulation of SNARE complex formation, which may lead to several considerations about the phosphorylation-mediated regulation of SNARE complex and its possible effect on vesicle recruitment. On the one hand, previous biochemical study demonstrates that phosphorylation of SNAP-25 accelerated the dissociation of SNAP-25 from syntaxin in vitro (15). It is possible that phosphorylation of SNAP-25 speeds up disassembly of nonproductive binary complexes, involving two molecules of syntaxin and one SNAP-25, thereby favoring the formation of productive ternary complexes by replacing one syntaxin with synaptobrevin (50). This possibility was also mentioned in the previous study (17). On the other hand, SNARE complex formation regulated by SNAP-25 phosphorylation may function as fusion machinery localized in the membrane of synaptic vesicles participating in vesicle refilling and translocation to the plasma membrane (51), because SNARE complex can be assembled and disassembled in vesicle membrane (27) and governs the probability of synaptic vesicles (52). Although the evidence to support these possibilities is not adequate so far, these hypotheses merit future investigation of the mechanism of opiate addiction.

In summary, the present study revealed a decrease of SNAP-25 phosphorylation in chronically morphine-treated hippocampal synaptosomes, which is involved in consequent down-regulation of SNARE complex formation. The alteration in SNAP-25 phosphorylation and SNARE complex formation may serve as a potential molecular mechanism in the modulation of neurotransmission and synaptic plasticity under opiate abuse, which may be associated with the development of aberrant learning and memory induced by abused drug.


   FOOTNOTES
 
* This work was supported by the Ministry of Science and Technology (Grants G1999053907, G2000077800, and 2003CB515405), the Chinese Academy of Sciences (Grants KSCX1-SW-11 and KSCX2-SW-204), the National Natural Science Foundation of China (Grants 30021003 and 30321002), the Shanghai Science and Technology Committee (Grants 03DZ19213 and 018014015), and the K. C. Wong Education Foundation of Hong Kong. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

§ Both authors contributed equally to this work. Back

Present address: University of Texas Southwestern Medical Center, Dallas, TX 75235 Back

** To whom correspondence should be addressed. Tel.: 86-21-5492-1371; Fax: 86-21-5492-0078, E-mail: gpei{at}sibs.ac.cn.

1 The abbreviations used are: PKA, cAMP-dependent protein kinase; PKC, protein kinase C; CaMKII, Ca2+/calmodulin-dependent protein kinase II; SNARE, soluble N-ethylmaleimide-sensitive factor attachment protein receptor; SNAP-25, synaptosomal-associated protein of 25kDa; synaptobrevin, vesicle-associated SNARE synaptobrevin 2; 2-DE, two-dimensional electrophoresis; MALDI-TOF-MS, matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry; EGFP, enhanced green fluorescent protein; WT, wild-type; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid. Back

2 N.-J. Xu, Y.-X. Yu, J.-M. Zhu, H. Liu, L. Shen, R. Zeng, X. Zhang, and G. Pei, unpublished data. Back


   ACKNOWLEDGMENTS
 
We thank Dr. Masakazu Kataoka for providing EGFP-SNAP-25, EGFP-SNAP-25E, and EGFP-SNAP-25A and Dr. Masami Takahashi for anti-phosphorylated SNAP-25 antibody. We also thank Drs. Lan Bao, Mark Henkemeyer, and Jiu-Hong Kang for discussions and critical comments on the manuscript and Ya-Lan Wu, Sun-Mei Xin, Pei-Hua Wu, Guo-Bin Bao, Lin Teng, Wen-Bo Zhang, Peng Xia, Jian-Ping Mao, Ji-Song Guan, Dan Xu, and Shi-Jian Ding for their technical assistance.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Nestler, E. J., and Aghajanian, G. K. (1997) Science 278, 58–63[Abstract/Free Full Text]
  2. Robbins, T. W., and Everitt, B. J. (1999) Nature 398, 567–570[CrossRef][Medline] [Order article via Infotrieve]
  3. Williams, J. T., Christie, M. J., and Manzoni, O. (2001) Physiol. Rev. 81, 299–343[Abstract/Free Full Text]
  4. Haas, H. L., and Ryall, R. W. (1980) J. Physiol. 308, 315–330[Abstract/Free Full Text]
  5. Mansouri, F. A., Motamedi, F., and Fathollahi, Y. (1999) Brain Res. 815, 419–423[CrossRef][Medline] [Order article via Infotrieve]
  6. Mansouri, F. A., Motamedi, F., Fathollahi, Y., Atapour, N., and Semnanian, S. (1997) Brain Res. 769, 119–124[CrossRef][Medline] [Order article via Infotrieve]
  7. Pu, L., Bao, G. B., Xu, N. J., Ma, L., and Pei, G. (2002) J. Neurosci. 22, 1914–1921[Abstract/Free Full Text]
  8. Xu, N. J., Bao, L., Fan, H. P., Bao, G. B., Pu, L., Lu, Y. J., Wu, C. F., Zhang, X., and Pei, G. (2003) J. Neurosci. 23, 4775–4784[Abstract/Free Full Text]
  9. Law, P. Y., Wong, Y. H., and Loh, H. H. (2000) Annu. Rev. Pharmacol. Toxicol. 40, 389–430[CrossRef][Medline] [Order article via Infotrieve]
  10. Sudhof, T. C. (1995) Nature 375, 645–653[CrossRef][Medline] [Order article via Infotrieve]
  11. Sollner, T., Bennett, M. K., Whiteheart, S. W., Scheller, R. H., and Rothman, J. E. (1993) Cell 75, 409–418[CrossRef][Medline] [Order article via Infotrieve]
  12. Lin, R. C., and Scheller, R. H. (2000) Annu. Rev. Cell Dev. Biol. 16, 19–49[CrossRef][Medline] [Order article via Infotrieve]
  13. Iwasaki, S., Kataoka, M., Sekiguchi, M., Shimazaki, Y., Sato, K., and Takahashi, M. (2000) J Biochem. (Tokyo) 128, 407–414[Abstract/Free Full Text]
  14. Kataoka, M., Kuwahara, R., Iwasaki, S., Shoji-Kasai, Y., and Takahashi, M. (2000) J. Neurochem. 74, 2058–2066[CrossRef][Medline] [Order article via Infotrieve]
  15. Shimazaki, Y., Nishiki, T., Omori, A., Sekiguchi, M., Kamata, Y., Kozaki, S., and Takahashi, M. (1996) J. Biol. Chem. 271, 14548–14553[Abstract/Free Full Text]
  16. Risinger, C., and Bennett, M. K. (1999) J. Neurochem. 72, 614–624[CrossRef][Medline] [Order article via Infotrieve]
  17. Nagy, G., Matti, U., Nehring, R. B., Binz, T., Rettig, J., Neher, E., and Sorensen, J. B. (2002) J. Neurosci. 22, 9278–9286[Abstract/Free Full Text]
  18. Shoji-Kasai, Y., Itakura, M., Kataoka, M., Yamamori, S., and Takahashi, M. (2002) Eur. J. Neurosci. 15, 1390–1394[CrossRef][Medline] [Order article via Infotrieve]
  19. Tian, J. H., Das, S., and Sheng, Z. H. (2003) J. Biol. Chem. 278, 26265–26274[Abstract/Free Full Text]
  20. Ledent, C., Valverde, O., Cossu, G., Petitet, F., Aubert, J. F., Beslot, F., Bohme, G. A., Imperato, A., Pedrazzini, T., Roques, B. P., Vassart, G., Fratta, W., and Parmentier, M. (1999) Science 283, 401–404[Abstract/Free Full Text]
  21. Ortiz, J., Harris, H. W., Guitart, X., Terwilliger, R. Z., Haycock, J. W., and Nestler, E. J. (1995) J. Neurosci. 15, 1285–1297[Abstract]
  22. Pozzo-Miller, L. D., Gottschalk, W., Zhang, L., McDermott, K., Du, J., Gopalakrishnan, R., Oho, C., Sheng, Z. H., and Lu, B. (1999) J. Neurosci. 19, 4972–4983[Abstract/Free Full Text]
  23. Bradford, M. M. (1976) Anal. Biochem. 72, 248–254[CrossRef][Medline] [Order article via Infotrieve]
  24. Genoud, S., Pralong, W., Riederer, B. M., Eder, L., Catsicas, S., and Muller, D. (1999) J. Neurochem. 72, 1699–1706[CrossRef][Medline] [Order article via Infotrieve]
  25. Araki, N., Morimasa, T., Sakai, T., Tokuoh, H., Yunoue, S., Kamo, M., Miyazaki, K., Abe, K., Saya, H., and Tsugita, A. (2000) Electrophoresis 21, 1880–1889[CrossRef][Medline] [Order article via Infotrieve]
  26. Itakura, M., Misawa, H., Sekiguchi, M., Takahashi, S., and Takahashi, M. (1999) Biochem. Biophys. Res. Commun. 265, 691–696[CrossRef][Medline] [Order article via Infotrieve]
  27. Otto, H., Hanson, P. I., and Jahn, R. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 6197–6201[Abstract/Free Full Text]
  28. Lou, L. G., and Pei, G. (1997) Biochem. Biophys. Res. Commun. 236, 626–629[Medline] [Order article via Infotrieve]
  29. Oyler, G. A., Higgins, G. A., Hart, R. A., Battenberg, E., Billingsley, M., Bloom, F. E., and Wilson, M. C. (1989) J. Cell Biol. 109, 3039–3052[Abstract/Free Full Text]
  30. Sorensen, J. B., Nagy, G., Varoqueaux, F., Nehring, R. B., Brose, N., Wilson, M. C., and Neher, E. (2003) Cell 114, 75–86[CrossRef][Medline] [Order article via Infotrieve]
  31. Ventayol, P., Busquets, X., and Garcia-Sevilla, J. A. (1997) Naunyn-Schmiedeberg's Arch. Pharmacol. 355, 491–500[CrossRef][Medline] [Order article via Infotrieve]
  32. Escriba, P. V., and Garcia-Sevilla, J. A. (1999) Br. J. Pharmacol. 127, 343–348[CrossRef][Medline] [Order article via Infotrieve]
  33. Hayashi, T., McMahon, H., Yamasaki, S., Binz, T., Hata, Y., Sudhof, T. C., and Niemann, H. (1994) EMBO J. 13, 5051–5061[Medline] [Order article via Infotrieve]
  34. Yoshikawa, M., Nakayama, H., Ueno, S., Hirano, M., Hatanaka, H., and Furuya, H. (2000) Brain Res. 859, 217–223[CrossRef][Medline] [Order article via Infotrieve]
  35. Niu, S., Kuo, C. H., Gan, Y., Nishikawa, E., Sadakata, T., Ichikawa, H., and Miki, N. (2000) Jpn. J. Pharmacol. 84, 412–417[CrossRef][Medline] [Order article via Infotrieve]
  36. Abood, M. E., and Tao, Q. (1995) J. Pharmacol. Exp. Ther. 274, 1566–1573[Abstract/Free Full Text]
  37. Bao, L., Jin, S. X., Zhang, C., Wang, L. H., Xu, Z. Z., Zhang, F. X., Wang, L. C., Ning, F. S., Cai, H. J., Guan, J. S., Xiao, H. S., Xu, Z. Q., He, C., Hokfelt, T., Zhou, Z., and Zhang, X. (2003) Neuron 37, 121–133[CrossRef][Medline] [Order article via Infotrieve]
  38. Nestler, E. J. (2001) Nat. Rev. Neurosci. 2, 119–128[CrossRef][Medline] [Order article via Infotrieve]
  39. Nestler, E. J. (2001) Science 292, 2266–2267[Free Full Text]
  40. Lu, W., Man, H., Ju, W., Trimble, W. S., MacDonald, J. F., and Wang, Y. T. (2001) Neuron 29, 243–254[CrossRef][Medline] [Order article via Infotrieve]
  41. Renger, J. J., Egles, C., and Liu, G. (2001) Neuron 29, 469–484[CrossRef][Medline] [Order article via Infotrieve]
  42. Fundytus, M. E., and Coderre, T. J. (1996) Eur. J. Pharmacol. 300, 173–181[CrossRef][Medline] [Order article via Infotrieve]
  43. Cerezo, M., Laorden, M. L., and Milanes, M. V. (2002) Eur. J. Pharmacol. 452, 57–66[CrossRef][Medline] [Order article via Infotrieve]
  44. Koticha, D. K., McCarthy, E. E., and Baldini, G. (2002) J. Cell Sci. 115, 3341–3351[Abstract/Free Full Text]
  45. Hanson, P. I., Heuser, J. E., and Jahn, R. (1997) Curr. Opin. Neurobiol. 7, 310–315[CrossRef][Medline] [Order article via Infotrieve]
  46. Sutton, R. B., Fasshauer, D., Jahn, R., and Brunger, A. T. (1998) Nature 395, 347–353[CrossRef][Medline] [Order article via Infotrieve]
  47. Tsuboi, T., Kikuta, T., Warashina, A., and Terakawa, S. (2001) Biochem. Biophys. Res. Commun. 282, 621–628[CrossRef][Medline] [Order article via Infotrieve]
  48. Lonart, G., and Sudhof, T. C. (2000) J. Biol. Chem. 275, 27703–27707[Abstract/Free Full Text]
  49. Finley, M. F., Scheller, R. H., and Madison, D. V. (2003) Neuropharmacology 45, 857–862[CrossRef][Medline] [Order article via Infotrieve]
  50. Margittai, M., Fasshauer, D., Pabst, S., Jahn, R., and Langen, R. (2001) J. Biol. Chem. 276, 13169–13177[Abstract/Free Full Text]
  51. Duman, J. G., and Forte, J. G. (2003) Am. J. Physiol. 285, C237–C249
  52. Finley, M. F., Patel, S. M., Madison, D. V., and Scheller, R. H. (2002) J. Neurosci. 22, 1266–1272[Abstract/Free Full Text]

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