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Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal.

Neale EA, Bowers LM, Jia M, Bateman KE, Williamson LC - J. Cell Biol. (1999)

Bottom Line: Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis.We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis.In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. eneale@codon.nih.gov

ABSTRACT
The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval.

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FM1-43 staining (a–h) and destaining (i–k) with K+ depolarization. (a–c) Control cultures. (a) 56 mM KCl; 2 mM CaCl2. The fluorescent image is strikingly similar to Fig. 1b and Fig. 4a. The labeled structures are individual active synaptic terminals containing a number of synaptic vesicles whose membranes are labeled with the fluorescent dye as a result of vesicle membrane recycling during the stimulation interval. (b) 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. Uptake of FM1-43 is not detectable. (c) 3 mM KCl and 2 mM CaCl2. Some uptake of FM1-43 occurs as a result of spontaneous network activity. (d) TeNT (10 ng/ml for 22 h), 56 mM KCl, and 2 mM CaCl2. (e) BoNT C (100 ng/ml for 22 h), 56 mM KCl, and 2 mM CaCl2. When synaptic vesicle exocytosis is blocked by TeNT or BoNT C, uptake of FM1-43 is decreased substantially. (f–h) BoNT A (200 ng/ml; i.e., >20 times that required to block neurotransmitter release). (f) 56 mM KCl and 2 mM CaCl2. Although intensity is reduced from controls, FM1-43 labeling is unequivocal in cultures blocked by BoNT A. (g) 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. As in control cultures, uptake of FM1-43 requires Ca2+. (h) 3 mM KCl and 2 mM CaCl2. There is essentially no labeling of toxin-blocked cultures without K+ depolarization. (i and j) Control cultures loaded with FM1-43 in 56 mM KCl and 2 mM CaCl2, as in a. (i) Loaded culture subsequently depolarized with 56 mM KCl and 2 mM CaCl2. There is substantial loss of FM1-43 from synaptic terminals as a result of exocytosis of labeled vesicles (compare with a). (j) Loaded culture subsequently depolarized with 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. The absence of Ca2+ prevents synaptic vesicle exocytosis and the destaining of labeled terminals. (k) BoNT A (200 ng/ml for 22 h)–blocked culture loaded with FM1-43 in 56 mM KCl and 2 mM CaCl2, as in f, and subsequently incubated with 56 mM KCl and 2 mM CaCl2 in an attempt to destain. In contrast to control cultures, it is not possible to destain BoNT A–blocked cultures, providing further evidence that vesicle exocytosis cannot be stimulated. Bar, 25 μm.
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Figure 6: FM1-43 staining (a–h) and destaining (i–k) with K+ depolarization. (a–c) Control cultures. (a) 56 mM KCl; 2 mM CaCl2. The fluorescent image is strikingly similar to Fig. 1b and Fig. 4a. The labeled structures are individual active synaptic terminals containing a number of synaptic vesicles whose membranes are labeled with the fluorescent dye as a result of vesicle membrane recycling during the stimulation interval. (b) 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. Uptake of FM1-43 is not detectable. (c) 3 mM KCl and 2 mM CaCl2. Some uptake of FM1-43 occurs as a result of spontaneous network activity. (d) TeNT (10 ng/ml for 22 h), 56 mM KCl, and 2 mM CaCl2. (e) BoNT C (100 ng/ml for 22 h), 56 mM KCl, and 2 mM CaCl2. When synaptic vesicle exocytosis is blocked by TeNT or BoNT C, uptake of FM1-43 is decreased substantially. (f–h) BoNT A (200 ng/ml; i.e., >20 times that required to block neurotransmitter release). (f) 56 mM KCl and 2 mM CaCl2. Although intensity is reduced from controls, FM1-43 labeling is unequivocal in cultures blocked by BoNT A. (g) 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. As in control cultures, uptake of FM1-43 requires Ca2+. (h) 3 mM KCl and 2 mM CaCl2. There is essentially no labeling of toxin-blocked cultures without K+ depolarization. (i and j) Control cultures loaded with FM1-43 in 56 mM KCl and 2 mM CaCl2, as in a. (i) Loaded culture subsequently depolarized with 56 mM KCl and 2 mM CaCl2. There is substantial loss of FM1-43 from synaptic terminals as a result of exocytosis of labeled vesicles (compare with a). (j) Loaded culture subsequently depolarized with 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. The absence of Ca2+ prevents synaptic vesicle exocytosis and the destaining of labeled terminals. (k) BoNT A (200 ng/ml for 22 h)–blocked culture loaded with FM1-43 in 56 mM KCl and 2 mM CaCl2, as in f, and subsequently incubated with 56 mM KCl and 2 mM CaCl2 in an attempt to destain. In contrast to control cultures, it is not possible to destain BoNT A–blocked cultures, providing further evidence that vesicle exocytosis cannot be stimulated. Bar, 25 μm.

Mentions: To demonstrate further the toxin-induced electrical quiescence, we used the styryl dye FM1-43 for the optical detection of synaptic activity (Betz and Bewick 1992). Depolarization of control cultures in the presence of FM1-43 results in a pattern of fluorescence consonant with the labeling of synaptic terminals (Fig. 6 a). Uptake of FM1-43 is not observed with depolarization in the absence of Ca2+ (Fig. 6 b) and is markedly reduced without depolarization (Fig. 6 c). As expected, cultures treated for 22 h with 10 ng/ml TeNT (Fig. 6 d), 10–100 ng/ml BoNT C (Fig. 6 e) (Williamson et al. 1996), or BoNTs B or D (10 ng/ml; not shown), fail to release neurotransmitter with K+ depolarization, and also fail to take up FM1-43. In marked contrast, cultures blocked with 10 ng/ml of BoNT A, which exhibit no K+-evoked release of neurotransmitter (Williamson et al. 1996) show intense K+-stimulated uptake of FM1-43. Increasing the concentration of BoNT A to 200 ng/ml (Fig. 6 f) or 300 ng/ml (not shown) failed to abolish FM1-43 uptake. As in controls, FM1-43 loading of BoNT A–treated cultures requires both extracellular Ca2+ (Fig. 6 g) and elevated K+ (Fig. 6 h). These results indicate that BoNT A blockade of neurotransmitter release does not prevent vesicle membrane reuptake by endocytosis, and that membrane retrieval is dependent on Ca2+.


Botulinum neurotoxin A blocks synaptic vesicle exocytosis but not endocytosis at the nerve terminal.

Neale EA, Bowers LM, Jia M, Bateman KE, Williamson LC - J. Cell Biol. (1999)

FM1-43 staining (a–h) and destaining (i–k) with K+ depolarization. (a–c) Control cultures. (a) 56 mM KCl; 2 mM CaCl2. The fluorescent image is strikingly similar to Fig. 1b and Fig. 4a. The labeled structures are individual active synaptic terminals containing a number of synaptic vesicles whose membranes are labeled with the fluorescent dye as a result of vesicle membrane recycling during the stimulation interval. (b) 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. Uptake of FM1-43 is not detectable. (c) 3 mM KCl and 2 mM CaCl2. Some uptake of FM1-43 occurs as a result of spontaneous network activity. (d) TeNT (10 ng/ml for 22 h), 56 mM KCl, and 2 mM CaCl2. (e) BoNT C (100 ng/ml for 22 h), 56 mM KCl, and 2 mM CaCl2. When synaptic vesicle exocytosis is blocked by TeNT or BoNT C, uptake of FM1-43 is decreased substantially. (f–h) BoNT A (200 ng/ml; i.e., >20 times that required to block neurotransmitter release). (f) 56 mM KCl and 2 mM CaCl2. Although intensity is reduced from controls, FM1-43 labeling is unequivocal in cultures blocked by BoNT A. (g) 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. As in control cultures, uptake of FM1-43 requires Ca2+. (h) 3 mM KCl and 2 mM CaCl2. There is essentially no labeling of toxin-blocked cultures without K+ depolarization. (i and j) Control cultures loaded with FM1-43 in 56 mM KCl and 2 mM CaCl2, as in a. (i) Loaded culture subsequently depolarized with 56 mM KCl and 2 mM CaCl2. There is substantial loss of FM1-43 from synaptic terminals as a result of exocytosis of labeled vesicles (compare with a). (j) Loaded culture subsequently depolarized with 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. The absence of Ca2+ prevents synaptic vesicle exocytosis and the destaining of labeled terminals. (k) BoNT A (200 ng/ml for 22 h)–blocked culture loaded with FM1-43 in 56 mM KCl and 2 mM CaCl2, as in f, and subsequently incubated with 56 mM KCl and 2 mM CaCl2 in an attempt to destain. In contrast to control cultures, it is not possible to destain BoNT A–blocked cultures, providing further evidence that vesicle exocytosis cannot be stimulated. Bar, 25 μm.
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Figure 6: FM1-43 staining (a–h) and destaining (i–k) with K+ depolarization. (a–c) Control cultures. (a) 56 mM KCl; 2 mM CaCl2. The fluorescent image is strikingly similar to Fig. 1b and Fig. 4a. The labeled structures are individual active synaptic terminals containing a number of synaptic vesicles whose membranes are labeled with the fluorescent dye as a result of vesicle membrane recycling during the stimulation interval. (b) 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. Uptake of FM1-43 is not detectable. (c) 3 mM KCl and 2 mM CaCl2. Some uptake of FM1-43 occurs as a result of spontaneous network activity. (d) TeNT (10 ng/ml for 22 h), 56 mM KCl, and 2 mM CaCl2. (e) BoNT C (100 ng/ml for 22 h), 56 mM KCl, and 2 mM CaCl2. When synaptic vesicle exocytosis is blocked by TeNT or BoNT C, uptake of FM1-43 is decreased substantially. (f–h) BoNT A (200 ng/ml; i.e., >20 times that required to block neurotransmitter release). (f) 56 mM KCl and 2 mM CaCl2. Although intensity is reduced from controls, FM1-43 labeling is unequivocal in cultures blocked by BoNT A. (g) 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. As in control cultures, uptake of FM1-43 requires Ca2+. (h) 3 mM KCl and 2 mM CaCl2. There is essentially no labeling of toxin-blocked cultures without K+ depolarization. (i and j) Control cultures loaded with FM1-43 in 56 mM KCl and 2 mM CaCl2, as in a. (i) Loaded culture subsequently depolarized with 56 mM KCl and 2 mM CaCl2. There is substantial loss of FM1-43 from synaptic terminals as a result of exocytosis of labeled vesicles (compare with a). (j) Loaded culture subsequently depolarized with 56 mM KCl, 0 mM CaCl2, and 0.5 mM EGTA. The absence of Ca2+ prevents synaptic vesicle exocytosis and the destaining of labeled terminals. (k) BoNT A (200 ng/ml for 22 h)–blocked culture loaded with FM1-43 in 56 mM KCl and 2 mM CaCl2, as in f, and subsequently incubated with 56 mM KCl and 2 mM CaCl2 in an attempt to destain. In contrast to control cultures, it is not possible to destain BoNT A–blocked cultures, providing further evidence that vesicle exocytosis cannot be stimulated. Bar, 25 μm.
Mentions: To demonstrate further the toxin-induced electrical quiescence, we used the styryl dye FM1-43 for the optical detection of synaptic activity (Betz and Bewick 1992). Depolarization of control cultures in the presence of FM1-43 results in a pattern of fluorescence consonant with the labeling of synaptic terminals (Fig. 6 a). Uptake of FM1-43 is not observed with depolarization in the absence of Ca2+ (Fig. 6 b) and is markedly reduced without depolarization (Fig. 6 c). As expected, cultures treated for 22 h with 10 ng/ml TeNT (Fig. 6 d), 10–100 ng/ml BoNT C (Fig. 6 e) (Williamson et al. 1996), or BoNTs B or D (10 ng/ml; not shown), fail to release neurotransmitter with K+ depolarization, and also fail to take up FM1-43. In marked contrast, cultures blocked with 10 ng/ml of BoNT A, which exhibit no K+-evoked release of neurotransmitter (Williamson et al. 1996) show intense K+-stimulated uptake of FM1-43. Increasing the concentration of BoNT A to 200 ng/ml (Fig. 6 f) or 300 ng/ml (not shown) failed to abolish FM1-43 uptake. As in controls, FM1-43 loading of BoNT A–treated cultures requires both extracellular Ca2+ (Fig. 6 g) and elevated K+ (Fig. 6 h). These results indicate that BoNT A blockade of neurotransmitter release does not prevent vesicle membrane reuptake by endocytosis, and that membrane retrieval is dependent on Ca2+.

Bottom Line: Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis.We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis.In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins.

View Article: PubMed Central - PubMed

Affiliation: Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20892, USA. eneale@codon.nih.gov

ABSTRACT
The supply of synaptic vesicles in the nerve terminal is maintained by a temporally linked balance of exo- and endocytosis. Tetanus and botulinum neurotoxins block neurotransmitter release by the enzymatic cleavage of proteins identified as critical for synaptic vesicle exocytosis. We show here that botulinum neurotoxin A is unique in that the toxin-induced block in exocytosis does not arrest vesicle membrane endocytosis. In the murine spinal cord, cell cultures exposed to botulinum neurotoxin A, neither K(+)-evoked neurotransmitter release nor synaptic currents can be detected, twice the ordinary number of synaptic vesicles are docked at the synaptic active zone, and its protein substrate is cleaved, which is similar to observations with tetanus and other botulinal neurotoxins. In marked contrast, K(+) depolarization, in the presence of Ca(2+), triggers the endocytosis of the vesicle membrane in botulinum neurotoxin A-blocked cultures as evidenced by FM1-43 staining of synaptic terminals and uptake of HRP into synaptic vesicles. These experiments are the first demonstration that botulinum neurotoxin A uncouples vesicle exo- from endocytosis, and provide evidence that Ca(2+) is required for synaptic vesicle membrane retrieval.

Show MeSH
Related in: MedlinePlus