The Role of the Synaptic Protein SNAP-25 in the Potency of Botulinum Neurotoxin Type A*

James E. KellerDagger and Elaine A. Neale

From the Laboratory of Developmental Neurobiology, NICHD, National Institutes of Health, Bethesda, Maryland 20892

Received for publication, December 6, 2000, and in revised form, January 8, 2001



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Botulinum neurotoxin serotype A (BoNT/A) is distinguished from BoNT/E by longer duration of paralysis and greater potency. The proteolytic activity of BoNT/A in cultures of dissociated spinal cord neurons persists beyond 80 days, whereas BoNT/E activity persists for less than 1 day (Keller, J. E., Neale, E. A. Oyler, G., and Adler, M. (1999) FEBS Lett. 456, 137-142). This single quality of toxin activity can account for the differences observed in the duration of muscle block. In the present work we sought to understand the basis for the apparent greater potency of BoNT/A. BoNT/E cleaves a 26-amino acid fragment from the C terminus of the synaptic protein SNAP-25 whereas BoNT/A removes only nine residues creating a 197-amino acid fragment (P197) that is 95% the length of SNAP-25. We show that inhibition of neurotransmitter release by BoNT/E is equivalent to the damage caused to SNAP-25. However, synaptic blockade by BoNT/A is greater than the extent of SNAP-25 proteolysis. These findings can be explained if P197 produces an inhibitory effect on neurotransmitter release. A mathematical model of the experimentally determined relationship between SNAP-25 damage and blockade of neurotransmission supports this interpretation. Furthermore, neurotransmitter release following complete cleavage of SNAP-25 can be achieved by P197, but with about 5-fold less sensitivity to external Ca2+. In this case, vesicular release is restored by increasing intracellular Ca2+. These data demonstrate that P197 competes with intact SNAP-25, but is unable to initiate normal synaptic vesicle fusion in physiological concentrations of Ca2+.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Intoxication with botulinum neurotoxin (BoNT)1 in vivo leads to flaccid paralysis by blockade of acetylcholine release at the neuromuscular junction (1-3). The proteolytic activity of the toxin responsible for causing paralysis resides within the 50-kDa light chain domain (4, 5) and is directed against three synaptic proteins: synaptosomal-associated protein of 25-kDa (SNAP-25), vesicle-associated membrane protein (VAMP), or syntaxin (6-11). BoNT/A and -E both cleave SNAP-25 but at distinct sites (12). Interestingly, paralysis from BoNT/A lasts for many months whereas blockade caused by BoNT/E lasts for relatively brief periods (13, 14). Two hypotheses were proposed to account for this difference: (a) BoNT/A remains catalytically active for a longer interval than BoNT/E or, alternatively (b) the catalytic activity of both toxins is very short-term but the major SNAP-25 fragment generated by BoNT/A (P197) persists in nerve terminals and interferes with neurotransmitter release (15). The first hypothesis has been substantiated by direct demonstration of BoNT/A persistence in primary mouse spinal cord cultures, electroporated chromaffin cells, and mammalian neuromuscular junction preparations in vivo (16-18). Evidence for the second hypothesis is indirect. Overexpressed P197 in an insulinoma Hit-T15 cell line inhibits insulin secretion (19). BoNT/A action at the neuromuscular junction produces a strong immunogenic signal for P197 within nerve terminals, indicating that P197 resides for some time in the proper intracellular regions where it could exert an inhibitory effect on neurotransmission (20, 21). However, there has been no quantitative analysis to determine whether the concentrations of P197 generated by toxin activity are able to inhibit neurotransmitter release.

BoNT/A and -E are unique among the BoNTs in that, under certain experimental conditions, elevated Ca2+ can partially relieve a toxin-induced blockade (22-24). The effect of Ca2+ on reversing BoNT/A-induced paralysis has been reported for muscle preparations (25, 26), hippocampal brain slices (27, 28), synaptosomes (29), and chromaffin cells (31). The molecular relationship between SNAP-25 and Ca2+ is not well understood, however. The altered Ca2+ sensitivity observed in PC12 cells for either BoNT/A or -E (30) is consistent with earlier observations using neuromuscular or chromaffin cell preparations prior to the discovery of SNAP-25 as the toxin substrate (32-34). Furthermore, electrophysiological results indicate that the C terminus of SNAP-25, which is directly affected by BoNT/A and -E, contributes to the initiation of two distinct Ca2+-sensitive steps in the neurotransmitter release process (31).

SNAP-25 and P197 each bind to VAMP and syntaxin although the P197-containing complex is less stable to detergent solubilization than a complex containing SNAP-25 (35-37). Examining the possible inhibitory role of P197 within the framework of the SNARE hypothesis (38, 39) suggests that BoNT/A damage compromises the ability of the complex to facilitate vesicle fusion. Calcium induces a conformational transition of the trimeric SNARE complex from a cis (nonsecretory) to a trans orientation which triggers vesicle fusion and neurotransmitter release (40, 41). Given these two effects, SNAP-25 cleavage and reduced Ca2+ sensitivity, it is possible that BoNT/A action on SNAP-25 alters other interactions which affect neurotransmitter release. To this end, it was demonstrated that Ca2+ stimulates binding between SNAP-25 and P197 with synaptotagmin (30). Hence, there may be multiple roles for SNAP-25 and Ca2+ in the release process (34, 41-43).

Calcium ionophores have been used successfully to discriminate between the effects of voltage-gated Ca2+ channels and direct effects of elevated intracellular Ca2+ on BoNT/A action (27, 28, 44, 45). This approach has been extended to the present study to investigate the requirement for intracellular Ca2+ in overcoming the inhibitory action of BoNT/A in mouse spinal cord cultures. The findings demonstrate that a mathematical correlation exists between the generation of P197 by BoNT/A and inhibition of Ca2+-dependent neurotransmitter release, indicating that the amount of P197 attained within neurons will block exocytosis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- Monoclonal antibody to SNAP-25 (SMI-81) was obtained from Sternberger Monoclonals, Inc. (Lutherville, MD). Thapsigargin, ionomycin, monoclonal antibody to syntaxin, and alkaline phosphatase-labeled anti-mouse antibody were purchased from Sigma-Aldrich Chemical Co. All electrophoresis reagents were from Bio-Rad. Polyvinylidene difluoride membrane was obtained from Bio-Rad. Preparations of BoNT/A and -E toxin complex were from Wako Chemicals Inc. (Richmond, VA) with reported activities of 2.0 × 107 LD50 and 1.0 × 107 LD50 per mg of protein, respectively. BoNT/E (1 mg/ml) was activated (nicked) by incubating for 30 min at 37 °C with 0.3 mg/ml trypsin (type XI, bovine pancreas) in 30 mM HEPES, pH 6.75. Trypsin was subsequently inhibited by addition of 0.5 mg/ml trypsin inhibitor (type I-S, soybean) and incubation for 15 min at 20 °C (16). Toxins were aliquoted and stored at -20 °C; each experiment utilized a new aliquot of toxin to ensure uniform activity. Molar concentrations stated in the text were based upon masses of 500 and 300 kDa for BoNT/A and -E, respectively.

Spinal Cord Cultures-- Timed pregnant C57BL/6NCR mice were obtained from the Frederick Cancer Research and Development Center, Frederick, MD. Spinal cord cell cultures were prepared as described (46, 47). Briefly, spinal cords were removed from fetal mice at gestation day 13, dissociated with trypsin, and plated on VitrogenTM 100-coated dishes (Collagen Corp., Palo Alto, CA) at a density of 106 cells/dish. Cultures were maintained for 3 weeks at 37 °C in an atmosphere of 90% air, 10% CO2 before addition of toxins. Growth medium consisted of minimum essential medium (formula 82-0234AJ; Life Technologies, Inc., Bethesda, MD) supplemented with 5% heat-inactivated horse serum and a mixture of complex factors (48). Cultures were incubated with BoNT diluted with growth medium for times indicated in figure legends. BoNT-containing medium was removed by aspiration; cells were rinsed with toxin-free medium and prepared for Western blot analysis or neurotransmitter release.

Protein and Western Blot Analysis-- Protein was prepared by dissolving cells in 1% SDS with 1 mM EDTA and 1 mM EGTA. The slurry was transferred to 1.5-ml microcentrifuge tubes, incubated in a 95 °C water bath for 5 min to inactivate proteases, and then stored at -20 °C. Immediately prior to use samples were thawed, mixed with equal volumes of Tris-Tricine sample buffer (Bio-Rad), heated at 95 °C for 4 min, and then separated by SDS-polyacrylamide gel electrophoresis. Equal quantities of protein were loaded onto 16.5% acrylamide gels prepared by the method of Laemmli (49). Proteins were separated using 0.1 M Tris-Tricine, pH 8.3 (50), and then transferred to polyvinylidene difluoride membrane with a buffer containing 192 mM glycine, 25 mM Tris, pH 8.3, and 12% methanol. Membrane development was performed as previously described (16).

Neurotransmitter Release and Quantitation-- Potassium-stimulated Ca2+-dependent neurotransmitter release was determined as previously described (51). Cultures were labeled with [3H]glycine for 0.5 h at 35 °C and then washed with a series of low K+-containing buffers. Unless otherwise indicated, glycine release was stimulated by addition of 56 mM K+ and 2 mM Ca2+ to cultures; stimulation medium was collected after 5 min at 35 °C. Calcium-dependent release was determined by subtracting baseline radioactivity secreted from cultures in the absence of Ca2+, and expressed as a percentage of the total cellular radioactivity.

Regression Analysis and Curve Fitting-- Scanned images of Western blots were produced and edited utilizing NIH Image (National Institutes of Health, Bethesda, MD). Where indicated, images were digitally analyzed using IPLab Gel software (Scanalytics, Inc., Fairfax, VA). Nonlinear regression analysis of data was performed with Sigma Plot (SPSS Science, Inc., Chicago, IL). Error bars represent standard deviations of triplicate determinations unless otherwise indicated.

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Concentration-dependent Effects of BoNT/E on SNAP-25 Cleavage and Neurotransmitter Release-- BoNT/E treatment of spinal cord cell cultures results in cleavage of SNAP-25 in a concentration-dependent manner (Fig. 1A). Under the conditions of these experiments, the IC50 for BoNT/E cleavage of SNAP-25 is ~50 pM. Measurement of synaptic function indicates that K+-evoked glycine release is inhibited to the same extent as SNAP-25 cleavage (Fig. 1B). In control cultures, the extent of glycine release is related to Ca2+ concentrations in the external medium. Cultures depolarized in the presence of 0.15, 0.25, 2, and 10 mM Ca2+ release 4.3, 15.7, 26.5, and 25.8% of the total cellular radiolabel. Maximal release is achieved with 2 mM Ca2+ (Fig. 3). Varying external Ca2+ concentration over this range did not alter significantly the IC50 for the BoNT/E block of glycine release which varies from 49 to 55 pM for the conditions tested (Fig. 1B).


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Fig. 1.   Effects of BoNT/E on neurotransmitter release and SNAP-25 cleavage. A, Western blot showing cleavage of SNAP-25 at indicated concentrations of BoNT/E. Spinal cord cultures were incubated in toxin for 24 h at 37 °C. Cells were solubilized in sample buffer and 7 µg of total cellular protein was used for Western blotting with SNAP-25 and syntaxin antibodies. Cleavage of SNAP-25 increased in direct proportion to the toxin concentration. B, potassium-evoked glycine release was measured in the presence of 0.15 (black-square), 0.25 (), 2.0 (black-down-triangle ), or 10 mM (open circle ) Ca2+ in the depolarizing medium. SNAP-25 cleavage as determined by densitometric analysis of Western blots similar to that of A is also plotted (down-triangle). IC50 values for SNAP-25 proteolysis and glycine release were determined by regression analysis using the equation: Rres = Rmax/(1 + [toxin]/IC50)n. Rres represents the release determined at each dose of BoNT/E, [toxin]. Rmax is the maximal signal detected at zero toxin concentration. IC50 values ranged from 49 to 55 pM with a Hill coefficient (n) of 1.1 ± 0.1. C, model of the relationship of BoNT/E, SNAP-25, and K+-evoked release. In this depiction, SNAP-25 is cleaved by BoNT/E. Each incremental decrease in SNAP-25 results in an equivalent decrease in neurotransmitter release. A graphed representation of this modeled relationship generated the same overlapping pattern as the experimental data.

The model in Fig. 1C illustrates the effect of BoNT/E on the release process. In this model, SNAP-25 is shown to interact with elements (X) required for neurotransmitter release with an affinity represented by Kd. Potassium depolarization stimulates release in a Ca2+-dependent manner. Release is directly proportional to the amount of SNAP-25 present. As SNAP-25 is cleaved by increasing concentrations of BoNT/E, neurotransmitter release decreases to a similar extent. BoNT/E-cleaved SNAP-25 (P180) has been shown to interact with VAMP and syntaxin in vitro (36, 37). However, our data indicate that in this intact neuronal system, P180 does not interfere with K+-evoked release, possibly because P180 interacts with X very weakly relative to SNAP-25. Furthermore, increasing extracellular Ca2+ above physiological concentrations does not overcome the block (Fig. 3), similar to findings with neuromuscular preparations (14).

Concentration-dependent Effects of BoNT/A on SNAP-25 Cleavage and Neurotransmitter Release-- Unlike BoNT/E, dose-response data with BoNT/A produce nonoverlapping curves for SNAP-25 proteolysis and blockade of neurotransmitter release (Fig. 2B). SNAP-25 cleavage occurs with an approximate IC50 of 3 pM and glycine release is inhibited with IC50 values decreasing from 0.3 to 0.1 pM as Ca2+ concentrations decrease from 2 to 0.15 mM in the release medium (Fig. 2B). Increasing Ca2+ to 8 or 10 mM yielded results identical to that obtained with 2 mM Ca2+ (data not shown). At Ca2+ concentrations lower than 2 mM, a greater degree of inhibition occurs relative to SNAP-25 cleavage (Fig. 2B). At all Ca2+ concentrations tested, the slopes for neurotransmitter release were the same. Comparing these slopes with the slope for SNAP-25 yields a ratio of ~2:1.


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Fig. 2.   BoNT/A effects on neurotransmitter release and SNAP-25 cleavage. A, Western blot showing cleavage of SNAP-25 at the indicated concentrations of BoNT/A. Cultures were treated as described in the legend to Fig. 1. B, potassium-evoked glycine release was measured with 0.15 (black-square), 0.25 (down-triangle), or 2.0 mM () Ca2+. Level of SNAP-25 cleavage as determined by densitometric analysis of Western blots similar to that of A is also plotted (diamond ). Error bars represent standard deviations of at least three determinations. C, modeling SNAP-25 and P197 effects on neurotransmitter release. In this depiction, it is assumed that P197 interacts with the same cellular components as SNAP-25 and that this interaction yields an inhibitory complex, P197-X, that supports transmitter release only when internal Ca2+ exceeds normal activity-dependent concentrations (Ca2+'). D, graphed representation of modeled relationship. When interactions between SNAP-25 or P197 with X are predicted to have the same binding affinity (Kd = K<UP><SUB>d</SUB><SUP>′</SUP></UP>), the above graph is generated for neurotransmitter release () relative to SNAP-25 (diamond ). Slopes from the model curves demonstrate a 2:1 relationship similar to the experimental findings.

Fig. 2C depicts possible interactions between SNAP-25 and P197 and their combined effects on the release process. In this model P197 binds to elements X much the same as SNAP-25. Because elevated Ca2+ has been shown previously to reverse BoNT/A inhibition in brain slices (27), PC12 cells (29), and neuromuscular preparations (52), we hypothesize that neurotransmitter release through the P197-X pathway would require Ca2+ in excess of that achieved during normal neuronal stimulation. Elevated Ca2+ above normal levels is depicted with the term Ca2+'. This model allows us to predict the relationship between SNAP-25 cleavage and block of transmitter release. Assuming (a) that P197 interacts with elements X with the same or nearly the same affinity as SNAP-25, and (b) that at any given dose of BoNT/A, the molar sum of the remaining SNAP-25 and P197 will equal the amount of SNAP-25 in the absence of toxin, the profile in Fig. 2D is generated. According to this model, the ratio of the slope for neurotransmitter release relative to SNAP-25 is exactly 2:1. The data for neurotransmitter release measured with 2 mM Ca2+ (Fig. 2B) overlap the predicted result.

Extracellular Ca2+ Effects on Overcoming BoNT Intoxication-- Because at lower Ca2+ concentrations, BoNT/A-exposed cultures show a greater block in transmitter release (Fig. 2), we examined the effects of extracellular Ca2+ concentration on glycine release following exposure to a single, high dose of BoNT/A or -E. Both BoNTs at 1 nM cleave all detectable SNAP-25 after 24 h (Figs. 1A and 2A). [3H]Glycine release from control cultures (no toxin exposure) is Ca2+ dependent with an EC50 value for Ca2+ of 0.21 mM and a Hill coefficient of about 2 (Fig. 3). Glycine release from BoNT/A-treated cultures is similarly Ca2+ dependent with an EC50 value of 1.0 ± 0.1 mM and a plateau value that is 23% of control release (Fig. 3). Maximum release for both control and BoNT/A-treated cultures is attained when Ca2+ exceeds 2 mM (Fig. 3) and the slope of release is unaffected by toxin with an estimated Hill coefficient of 2.2 ± 0.3 (53, 54). Glycine release from BoNT/E-treated cultures is not detected at any concentration of Ca2+ tested (Fig. 3). This indicates that the rise of intracellular Ca2+ resulting from Ca2+-channel activation can restore some function to BoNT/A- but not to BoNT/E-treated neurons, consistent with results from Figs. 1 and 2.


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Fig. 3.   Calcium titration and K+-evoked glycine release. Spinal cord cultures were incubated in growth medium containing either BoNT/A or -E at 1 nM for 24 h at 37 °C, after which Ca2+-dependent release of glycine was assayed. Cultures not treated with toxin () demonstrated a standard titration curve relative to Ca2+ concentration with an EC50 value for Ca2+ of 0.21 ± 0.01 mM and a Hill coefficient of 1.9 ± 0.2. Maximal glycine release for each experiment ranged from 18 to 27% of the total cellular radioactivity and was set to 100%. BoNT/A-treated cultures (open circle ) demonstrated Ca2+ dependence with an EC50 value of 1.0 ± 0.1 mM and maximal release when Ca2+ exceeded 2 mM. The plateau value for this release was 23.2 ± 2.2% of the maximal release elicited from control cultures. BoNT/E-treated cultures (black-square) demonstrated no Ca2+ dependent release when measured in the presence of 1-8 mM Ca2+. Results were compiled from five (control) and four (BoNT/A-treated) experiments. Each symbol represents the average of duplicate determinations.

Intracellular Ca2+ Effects on Overcoming BoNT/A Intoxication-- To examine the effects of increased intracellular Ca2+ above levels attained from Ca2+-channel activation, glycine release was evoked by 56 mM K+ in the presence and absence of 2 mM Ca2+ and 5 µM ionomycin, a Ca2+-specific ionophore. Ionomycin partially restores glycine release after exposure to BoNT/A (Fig. 4). Treatment with BoNT/A reduces glycine release to ~23% of control release. Addition of ionomycin in combination with Ca2+ reduces the effect of BoNT/A, raising glycine release to 60% of control values. To determine the source of Ca2+ that contributes to this reversal, glycine release was assayed with ionomycin either in the absence of extracellular Ca2+, or after having treated cultures with thapsigargin to deplete intracellular Ca2+ stores (55, 56). The absence of any release with zero external Ca2+ with ionomycin and the failure of thapsigargin to affect the ionomycin-dependent release provide evidence that internal Ca2+ stores do not contribute significantly to ionomycin's reversal of the BoNT/A-induced block.


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Fig. 4.   The ability of ionomycin to reverse the BoNT/A block. Spinal cord cultures were treated with 2 nM BoNT/A for 29 h at 37 °C. Upon removal of the toxin some cultures were treated with 15 µM thapsigargin in minimal essential medium for 2.5 h. Glycine release was determined in the presence of 2 mM Ca2+ with release from control cultures (18.6% of total cellular radiolabel) set at 100% (first column). BoNT/A treatment reduced glycine release to 25% of controls (second column). Ionomycin at 5 µM added to release buffer containing 56 mM K+ and 2 mM Ca2+ did not enhance the total release from controls (data not shown) but increased release from BoNT/A-treated cultures to 60% (third column). Depletion of intracellular stores of Ca2+ with thapsigargin did not alter substantially the effect of ionomycin, indicating that ionomycin-induced recovery of release was independent of internal Ca2+ stores (fifth column). In contrast, reversal of the BoNT/A block did not occur in zero Ca2+ (fourth column), indicating a requirement for Ca2+ in the extracellular medium.

To expand upon this finding, cultures were treated with 1 nM BoNT/A or BoNT/E for 24 h and neurotransmitter release was measured in the presence of 10 µM ionomycin in 56 mM K+ with various external Ca2+ concentrations. Ionomycin produces a significant reversal of the BoNT/A block at all Ca2+ concentrations tested with complete recovery occurring near 2 mM Ca2+. Unlike K+/Ca2+ stimulation alone, stimulation in the presence of ionomycin partially alleviates BoNT/E-induced block with maximum glycine release at 53% of control cultures (Fig. 5). Within these experimental conditions, BoNT/A- and/E-treated neurons are about 4- and 7-fold less sensitive to Ca2+, respectively, than untreated neurons. Ionomycin has two measurable effects on reversing BoNT/E action: neurons release more glycine and demonstrate higher sensitivity to Ca2+. In the case of BoNT/A, ionomycin stimulates release of glycine to control values. However, ionomycin does not alter the EC50 value for Ca2+ which is 0.8 ± 0.1 mM (Fig. 5) compared with 1.0 ± 0.1 mM obtained for the partial release achieved in the absence of ionomycin (Fig. 3). Ionomycin at the tested concentration does not affect cultures that were not treated with toxin; neither the Ca2+ sensitivity nor the extent of glycine release was altered (Fig. 5).


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Fig. 5.   Relative sensitivity of neurotransmitter release to Ca2+ in the presence of ionomycin following exposure to BoNT. Cultures were treated with 1 nM BoNT/A (down-triangle) or/E (black-square) for 24 h. Potassium-stimulated Ca2+-dependent release of [3H]glycine was measured in the presence of 10 µM ionomycin and various concentrations of Ca2+ in release medium. Data are normalized to release obtained with 2 mM Ca2+ from toxin-free controls, which ranged from 19.1 to 23.4% of the total cellular radioactivity for each experiment. EC50 values for Ca2+ were obtained by regression fit of the equation R(release) = (Rmax × [Ca2+])/(EC50 + [Ca2+]) to the data. R(release) represents the fractional glycine release measured at any given concentration of Ca2+. Ionomycin did not alter the apparent EC50 value for control cultures () which is 0.20 ± 0.01 mM. EC50 values for BoNT/A- and E-treated cultures expressed relative to Rmax from controls are 0.8 ± 0.1 mM and 1.4 ± 0.2 mM, respectively. Error bars represent S.D. values of three separate determinations.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In this study, we examined the relationship between blockade of neurotransmitter release and cleavage of SNAP-25 caused by BoNT/A and -E to aid in understanding how BoNT/A seems to derive greater potency than BoNT/E (14, 22, 26, 32). Both BoNTs induce a concentration-dependent block in synaptic vesicle exocytosis and proteolysis of SNAP-25. However, it appears likely that in addition to disabling SNAP-25, BoNT/A action has a secondary inhibitory effect. The nonoverlapping pattern of SNAP-25 cleavage and block of K+-evoked transmitter release in this latter case points to the formation of an inhibitory complex containing a cleavage fragment produced by BoNT/A. Most simply, BoNT/A action on SNAP-25 produces a dual effect: a reduction in functional SNAP-25 and production of a fragment that antagonizes synaptic vesicle fusion.

The identity of this fragment could be either the 197-amino acid fragment, P197, or the smaller 9-residue fragment cleaved from the C terminus. C-terminal peptides of SNAP-25 disrupt synaptic vesicle trafficking in permeabilized chromaffin cells (57, 58). Peptides with sequences corresponding to the C-terminal 26 and 20 amino acids inhibited neurosecretion with IC50 values of ~0.25 and 20 µM, respectively (58, 59). A 12-residue peptide of the C terminus failed to inhibit release to any significant extent (60). The correlation of shorter peptide length with lower potency suggests that the 9-residue cleavage fragment generated by BoNT/A does not interfere with neurotransmitter release.

An inhibitory role of P197 was demonstrated by transfection of P197 into an Hit-T15 cell line and subsequent inhibition of insulin secretion (19). Additionally, P197 has been shown to interact directly with the SNARE proteins VAMP and syntaxin in a manner similar to SNAP-25 (35, 37). Based on the estimated half-life for BoNT/A being 3 days (61), detection of P197 in intoxicated motor nerve terminals in vivo generated the hypothesis that P197 was degraded slowly relative to SNAP-25 and could, therefore, interfere with the normal vesicular pathway for neurotransmitter release for weeks or months (20, 21). Two studies since then have provided direct evidence that BoNT/A catalytic activity persists within neurons with a half-life far greater than 3 days (16, 18). Information on the degradation rate of P197, although indirect at present, suggests that it occurs at a rate similar to SNAP-25 (16). The prolonged duration of the catalytic action of BoNT/A does not, however, invalidate the role of SNAP-25 cleavage products in the extent of paralysis. For example, the persistence of BoNT/A activity would provide a constant production of P197 to account for its continued presence in intoxicated nerve terminals (20, 21). Furthermore, because nerve stimulation accelerates the onset of paralysis (23, 62, 63), toxin probably accumulates within nerve terminals where production of P197 would occur in the proximity of vesicular activity. Additive effects on muscle twitch from the production of P197 and loss of SNAP-25 could also explain why muscle paralysis occurs prior to detectable loss of SNAP-25 by immunohistochemical methods (64).

The model presented in Fig. 2C predicts that cleavage of x% of the SNAP-25 population would result in a 2x% block of neurotransmitter release. This is based on simple competition of P197 and SNAP-25 for components of the fusion complex and the relationship is depicted graphically in Fig. 2D. Otto et al. (72) reported that cleavage of approximately half of SNAP-25 in synaptosomal preparations caused nearly a complete block of Ca2+-dependent glutamate secretion, in agreement with our model and experimental observations. In contrast to the sigmoidal curve observed for BoNT/E (Fig. 1B), the linear relationship of SNAP-25 cleavage relative to BoNT/A concentration may reflect the preferential anchoring of BoNT/A (20) but not BoNT/E within nerve terminals. SNAP-25 cleavage, therefore, may represent both BoNT/A concentration and movement of SNAP-25 within proximity of the toxin, for example, SNAP-25 must be transported to the toxin. The model does not require that SNAP-25-X is necessarily composed of VAMP and syntaxin; however, the current SNARE hypothesis (38, 39) would support the involvement of these or other proteins (30, 65, 66).

BoNT/E-treated spinal cord cultures developed a block in neurotransmitter release no greater than the extent of SNAP-25 cleavage. The direct relationship between SNAP-25 cleavage and neurotransmitter blockade indicates that, in intact neurons, SNAP-25 fragments created by BoNT/E do not interfere with the release process. Neurotransmitter release from BoNT/E-treated spinal cord neurons is approximately 7-fold less sensitive to Ca2+ than release from untreated neurons when ionomycin is used, although in the absence of ionomycin release does not occur. The ionomycin/Ca2+ combination could not produce a full recovery, however. Similar observations were obtained with other neuronal preparations (28, 45) and are consistent with observations in vivo that the minimal amount of the K+ channel blocker 3,4-diaminopyridine that stimulates a full recovery from BoNT/A does not elicit recovery from BoNT/E (14).

Unlike BoNT/E-treated neurons, glycine release from BoNT/A-treated neurons could be elicited without ionomycin. This release was Ca2+-dependent with an EC50 value for external Ca2+ of 1.0 mM, approximately 5-fold higher than controls (0.21 mM). At the lower concentrations of Ca2+ tested, BoNT/A completely blocked K+-stimulated release and at higher Ca2+ concentrations, maximal release was ~23% of the release from control cultures. The effect of Ca2+ on the release process is consistent with studies using intact chromaffin cells and hippocampal neurons (31, 53, 67, 68); i.e. a pool of neurotransmitter can be released after complete cleavage of SNAP-25 although the concentration of Ca2+ required for this to occur is higher than physiological Ca2+ levels. Release that occurs despite treatment with BoNT/A may reflect the weaker ability of P197 relative to SNAP-25 to facilitate Ca2+ requiring step(s) given the extent of Ca2+ influx that occurs under normal physiological conditions.

To further investigate the possible P197-release pathway (Fig. 2C), we employed ionomycin to determine whether elevated cytosolic Ca2+ would fully reverse the effects of BoNT/A (24, 29, 69). Ionomycin improved the efficiency of vesicular release in BoNT/A-treated neurons. The apparent EC50 for Ca2+ in the presence and absence of ionomycin was statistically unaffected, 0.8 ± 0.1 mM compared with 1.0 ± 0.1 mM, respectively. Lack of an effect on Ca2+ sensitivity and promotion of a full recovery of transmitter release suggests that ionomycin facilitates the hypothesized P197 release process by raising Ca2+ above physiologically attainable levels. An alternative explanation is that the action of an undetectable population of uncleaved SNAP-25 could account for the observed synaptic release. If this were the case, however, and SNAP-25 were solely responsible, then BoNT/A treatment would reduce the plateau values for transmitter release but the observed EC50 value for Ca2+ would approximate the value for neurons not treated with toxin, i.e. 0.2 mM.

The lower Ca2+ sensitivity following BoNT/A exposure may be due to disruption of several fusion processes. P197, for example, may hinder vesicle trafficking in nerve terminals. BoNT/A and tetanus neurotoxin cause significant accumulation of synaptic vesicles at active zones (63, 70). These vesicles, unable to fuse with the presynaptic membrane, might produce a physical obstruction for the recruitment of new synaptic vesicles to dock and fuse upon Ca2+-dependent stimulation (31, 63, 71). Hence, we show that an intact neuronal system treated with BoNT/A requires more Ca2+ to attain maximal release. This effect, in part, may be due to changes in the dynamics of synaptic vesicle movement in and near release sites caused by one or more Ca2+-related changes from SNAP-25 cleavage (30).

Primary spinal cord cultures were used to provide the first proof that catalytic activity of BoNT/A but not BoNT/E ensues for many months following intoxication (16). This finding is consistent with in vivo observations that BoNT/A but not BoNT/E causes long-term paralysis (13, 18). In the present work, Ca2+ stimulates release of neurotransmitter with a Hill coefficient of approximately 2 in the absence of toxin, which is consistent with electrophysiological estimates (54, 67). Calcium stimulates release following exposure to BoNT/A and to a lesser extent, BoNT/E, each observation being similar to in vivo experiments with local injections of either toxin into the extensor digitorum longus muscle of rats (14, 22, 23). Our data demonstrate that the amount of P197 generated by BoNT/A is sufficient to block neuronal activity in a concentration-dependent manner. Furthermore, the ability of P197 to inhibit release is based upon its ability to compete against uncleaved SNAP-25 in the release mechanism. In support of this latter assertion, electroporation of anti-toxin antibodies into BoNT/A-treated, chromaffin cells accelerates the recovery of secretion by nearly 10-fold (17), indicating that inactivation of BoNT/A allows newly synthesized SNAP-25 to displace P197. This demonstrates that the effect of P197 is brief without BoNT/A activity indicating P197 itself has a brief lifetime relative to BoNT/A. Integrating these observations into a unified model we suggest that paralysis produced by BoNT/A, (a) is due to persistent catalytic activity of this toxin within nerve terminals and (b) is caused by loss of functional SNAP-25 and concomitant generation of P197 that competes with the remaining intact SNAP-25 to exacerbate the block.

    ACKNOWLEDGEMENTS

We are grateful to Drs. Robert Sheridan, Michael Adler, Sharad Deshpande, and George Oyler for comments during the preparation of this manuscript. We also thank Dr. Robert Phair for assistance in interpreting the models depicted in the current work; J. Caitlin Sticco for help in preparing and analyzing Western blot data and, most certainly, we thank Karen Bateman for preparation and maintenance of the spinal cord cell cultures and help with preparing the present paper.

    FOOTNOTES

* The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Dagger To whom correspondence should be addressed. Tel.: 301-496-6419; Fax: 301-496-9939; E-mail: jekeller@codon.nih.gov.

Published, JBC Papers in Press, January 23, 2001, DOI 10.1074/jbc.M010992200

    ABBREVIATIONS

The abbreviations used are: BoNT, botulinum neurotoxin; SNAP-25, synaptosomal-associated protein of 25 kDa; P197, BoNT/A-truncated SNAP-25; P180, BoNT/E-truncated SNAP-25; VAMP, vesicle-associated membrane protein; Tricine, N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]glycine.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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