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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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Fine structure of the active zone. Active zones are marked by vertical bars, and docked synaptic vesicles (within 10 nm of the presynaptic membrane), with black squares. (a–c) Resting cultures. a is from a spontaneously active, control culture; b is from a culture in which neurotransmitter release was blocked by TeNT; and c is from a culture blocked by BoNT A. The number of synaptic vesicles docked at the active zone is increased in toxin-treated terminals. Note clathrin organized into baskets (arrowheads) in b. In this experiment, TeNT (100 ng/ml) and BoNT A (1.0 μg/ml) were added for 16 h. (d–f) K+-stimulated active zones. In a stimulated control culture (d), fewer synaptic vesicles appear docked (marked with squares) than in spontaneously active cultures. Note the increased occurrence of clathrin-coated vesicles (arrows). In contrast, when TeNT (100 ng/ml)– or BoNT A (List, 200 ng/ml)–blocked cultures are stimulated with K+ (e and f), the number of docked vesicles remains high. Arrowheads mark empty clathrin baskets. Bar, 0.2 nm.
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Figure 5: Fine structure of the active zone. Active zones are marked by vertical bars, and docked synaptic vesicles (within 10 nm of the presynaptic membrane), with black squares. (a–c) Resting cultures. a is from a spontaneously active, control culture; b is from a culture in which neurotransmitter release was blocked by TeNT; and c is from a culture blocked by BoNT A. The number of synaptic vesicles docked at the active zone is increased in toxin-treated terminals. Note clathrin organized into baskets (arrowheads) in b. In this experiment, TeNT (100 ng/ml) and BoNT A (1.0 μg/ml) were added for 16 h. (d–f) K+-stimulated active zones. In a stimulated control culture (d), fewer synaptic vesicles appear docked (marked with squares) than in spontaneously active cultures. Note the increased occurrence of clathrin-coated vesicles (arrows). In contrast, when TeNT (100 ng/ml)– or BoNT A (List, 200 ng/ml)–blocked cultures are stimulated with K+ (e and f), the number of docked vesicles remains high. Arrowheads mark empty clathrin baskets. Bar, 0.2 nm.

Mentions: Purified TeNT (2 × 107 mouse lethal doses/mg of protein) was a gift from Dr. William Habig (Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD). Except as noted, purified BoNT A was purchased from List Biological Laboratories, Inc. and BoNT C was from the Centre for Applied Microbiology and Research (5.2 × 107 and 1.0 × 107 mouse LD50/mg protein, respectively). BoNT A (9.4 × 106 mouse LD50/mg protein) from Calbiochem-Novabiochem Corp. was used for the experiments presented in Fig. 5, a–c, and Fig. 8. BoNT A provided by Drs. Eric Johnson and Michael Goodenough (University of Wisconsin, Madison, WI) was used for the experiments in Fig. 4 and Fig. 5. Concentration of BoNT A was adjusted based on the IC50 for blockade of glycine release; the effects of each preparation on exo- and endocytosis were tested and found to be identical. BoNT B (1.26 × 106 LD50/mg protein) was purchased from Calbiochem-Novabiochem Corp. [3H]Glycine (sp act 12.2 Ci/mmol) was purchased from Amersham Corp. Affinity-purified rabbit polyclonal antisynapsin 1a was obtained from Chemicon International, Inc. Affinity-purified rabbit polyclonal antibodies against amino acids 1–32 of the variable domain of vesicle-associated membrane protein (VAMP) 1 and against the COOH-terminal 12 amino acids of synaptosomal-associated protein of 25 kD (SNAP-25) (Rossetto et al. 1996) were a gift of Dr. Cesare Montecucco (University of Padova, Padova, Italy). FM1-43 was obtained from Molecular Probes and HRP Type VI from Sigma Chemical Co.


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)

Fine structure of the active zone. Active zones are marked by vertical bars, and docked synaptic vesicles (within 10 nm of the presynaptic membrane), with black squares. (a–c) Resting cultures. a is from a spontaneously active, control culture; b is from a culture in which neurotransmitter release was blocked by TeNT; and c is from a culture blocked by BoNT A. The number of synaptic vesicles docked at the active zone is increased in toxin-treated terminals. Note clathrin organized into baskets (arrowheads) in b. In this experiment, TeNT (100 ng/ml) and BoNT A (1.0 μg/ml) were added for 16 h. (d–f) K+-stimulated active zones. In a stimulated control culture (d), fewer synaptic vesicles appear docked (marked with squares) than in spontaneously active cultures. Note the increased occurrence of clathrin-coated vesicles (arrows). In contrast, when TeNT (100 ng/ml)– or BoNT A (List, 200 ng/ml)–blocked cultures are stimulated with K+ (e and f), the number of docked vesicles remains high. Arrowheads mark empty clathrin baskets. Bar, 0.2 nm.
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Related In: Results  -  Collection

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Figure 5: Fine structure of the active zone. Active zones are marked by vertical bars, and docked synaptic vesicles (within 10 nm of the presynaptic membrane), with black squares. (a–c) Resting cultures. a is from a spontaneously active, control culture; b is from a culture in which neurotransmitter release was blocked by TeNT; and c is from a culture blocked by BoNT A. The number of synaptic vesicles docked at the active zone is increased in toxin-treated terminals. Note clathrin organized into baskets (arrowheads) in b. In this experiment, TeNT (100 ng/ml) and BoNT A (1.0 μg/ml) were added for 16 h. (d–f) K+-stimulated active zones. In a stimulated control culture (d), fewer synaptic vesicles appear docked (marked with squares) than in spontaneously active cultures. Note the increased occurrence of clathrin-coated vesicles (arrows). In contrast, when TeNT (100 ng/ml)– or BoNT A (List, 200 ng/ml)–blocked cultures are stimulated with K+ (e and f), the number of docked vesicles remains high. Arrowheads mark empty clathrin baskets. Bar, 0.2 nm.
Mentions: Purified TeNT (2 × 107 mouse lethal doses/mg of protein) was a gift from Dr. William Habig (Center for Biologics Evaluation and Research, Food and Drug Administration, Bethesda, MD). Except as noted, purified BoNT A was purchased from List Biological Laboratories, Inc. and BoNT C was from the Centre for Applied Microbiology and Research (5.2 × 107 and 1.0 × 107 mouse LD50/mg protein, respectively). BoNT A (9.4 × 106 mouse LD50/mg protein) from Calbiochem-Novabiochem Corp. was used for the experiments presented in Fig. 5, a–c, and Fig. 8. BoNT A provided by Drs. Eric Johnson and Michael Goodenough (University of Wisconsin, Madison, WI) was used for the experiments in Fig. 4 and Fig. 5. Concentration of BoNT A was adjusted based on the IC50 for blockade of glycine release; the effects of each preparation on exo- and endocytosis were tested and found to be identical. BoNT B (1.26 × 106 LD50/mg protein) was purchased from Calbiochem-Novabiochem Corp. [3H]Glycine (sp act 12.2 Ci/mmol) was purchased from Amersham Corp. Affinity-purified rabbit polyclonal antisynapsin 1a was obtained from Chemicon International, Inc. Affinity-purified rabbit polyclonal antibodies against amino acids 1–32 of the variable domain of vesicle-associated membrane protein (VAMP) 1 and against the COOH-terminal 12 amino acids of synaptosomal-associated protein of 25 kD (SNAP-25) (Rossetto et al. 1996) were a gift of Dr. Cesare Montecucco (University of Padova, Padova, Italy). FM1-43 was obtained from Molecular Probes and HRP Type VI from Sigma Chemical Co.

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