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SV2 mediates entry of tetanus neurotoxin into central neurons.

Yeh FL, Dong M, Yao J, Tepp WH, Lin G, Johnson EA, Chapman ER - PLoS Pathog. (2010)

Bottom Line: Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons.Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals.Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Howard Hughes Medical Institute, University of Wisconsin, Madison, Wisconsin, USA.

ABSTRACT
Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons - due to preferential loss of inhibitory transmission - that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.

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TeNT requires actively recycling synaptic vesicles to enter neurons and cleave synaptobrevin II.(A) Cultured hippocampal neurons were incubated with full-length TeNT (5 nM) in non-depolarizing (TTX) or depolarizing (high K+) buffer for 3 minutes, washed, then returned to media and incubator for 4 hours. Cells were processed for immunostaining; the synaptobrevin II (syb II, Cl 69.1) signal was significantly reduced under depolarizing conditions. (B) Quantification of syb II intensity at excitatory or inhibitory terminals normalized to VGLUT1 or vGAT intensities, respectively. Error bars represent SD, n = 9, ***p≤0.001. (C) Cultured spinal cord neurons were incubated with TeNT holotoxin (500 pM) in TTX or high K+ buffers for 3 minutes, washed, then returned to media and incubated for 4 hours. (D) Quantification of syb II to vGAT fluorescence intensity ratios. Error bars represent SD, n = 9, ***p≤0.001.
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ppat-1001207-g002: TeNT requires actively recycling synaptic vesicles to enter neurons and cleave synaptobrevin II.(A) Cultured hippocampal neurons were incubated with full-length TeNT (5 nM) in non-depolarizing (TTX) or depolarizing (high K+) buffer for 3 minutes, washed, then returned to media and incubator for 4 hours. Cells were processed for immunostaining; the synaptobrevin II (syb II, Cl 69.1) signal was significantly reduced under depolarizing conditions. (B) Quantification of syb II intensity at excitatory or inhibitory terminals normalized to VGLUT1 or vGAT intensities, respectively. Error bars represent SD, n = 9, ***p≤0.001. (C) Cultured spinal cord neurons were incubated with TeNT holotoxin (500 pM) in TTX or high K+ buffers for 3 minutes, washed, then returned to media and incubated for 4 hours. (D) Quantification of syb II to vGAT fluorescence intensity ratios. Error bars represent SD, n = 9, ***p≤0.001.

Mentions: The previous experiments strongly suggest that TeNT binds to a receptor that is a resident of SVs, so we next determined whether this interaction results in functional entry of the toxin. To test this idea, we determined whether the ability of TeNT holotoxin to enter neurons and cleave syb II also depended on SV recycling. We employed a monoclonal antibody raised against syb II (Cl. 69.1) that cannot recognize the enzymatically cleaved form of the protein. We began by comparing the ability of TeNT to cleave syb II under TTX (which blocks action potentials to inhibit SV recycling) or high potassium (which depolarizes neurons to drive SV recycling) conditions. We observed that syb II fluorescence was markedly reduced when hippocampal neurons were depolarized with potassium to drive SV recycling (Figure 2A); this occurred at both excitatory and inhibitory nerve terminals (Figure 2B). Syb II immunofluorescence was reduced in excitatory terminals by 39% in the TTX condition and was further reduced by 65% of under depolarizing conditions, as compared to control. Inhibitory terminals exhibited no significant reduction of syb II immunofluorescence under TTX conditions, but a 53% reduction was observed in neurons that had been depolarized (Figure 2B).


SV2 mediates entry of tetanus neurotoxin into central neurons.

Yeh FL, Dong M, Yao J, Tepp WH, Lin G, Johnson EA, Chapman ER - PLoS Pathog. (2010)

TeNT requires actively recycling synaptic vesicles to enter neurons and cleave synaptobrevin II.(A) Cultured hippocampal neurons were incubated with full-length TeNT (5 nM) in non-depolarizing (TTX) or depolarizing (high K+) buffer for 3 minutes, washed, then returned to media and incubator for 4 hours. Cells were processed for immunostaining; the synaptobrevin II (syb II, Cl 69.1) signal was significantly reduced under depolarizing conditions. (B) Quantification of syb II intensity at excitatory or inhibitory terminals normalized to VGLUT1 or vGAT intensities, respectively. Error bars represent SD, n = 9, ***p≤0.001. (C) Cultured spinal cord neurons were incubated with TeNT holotoxin (500 pM) in TTX or high K+ buffers for 3 minutes, washed, then returned to media and incubated for 4 hours. (D) Quantification of syb II to vGAT fluorescence intensity ratios. Error bars represent SD, n = 9, ***p≤0.001.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2991259&req=5

ppat-1001207-g002: TeNT requires actively recycling synaptic vesicles to enter neurons and cleave synaptobrevin II.(A) Cultured hippocampal neurons were incubated with full-length TeNT (5 nM) in non-depolarizing (TTX) or depolarizing (high K+) buffer for 3 minutes, washed, then returned to media and incubator for 4 hours. Cells were processed for immunostaining; the synaptobrevin II (syb II, Cl 69.1) signal was significantly reduced under depolarizing conditions. (B) Quantification of syb II intensity at excitatory or inhibitory terminals normalized to VGLUT1 or vGAT intensities, respectively. Error bars represent SD, n = 9, ***p≤0.001. (C) Cultured spinal cord neurons were incubated with TeNT holotoxin (500 pM) in TTX or high K+ buffers for 3 minutes, washed, then returned to media and incubated for 4 hours. (D) Quantification of syb II to vGAT fluorescence intensity ratios. Error bars represent SD, n = 9, ***p≤0.001.
Mentions: The previous experiments strongly suggest that TeNT binds to a receptor that is a resident of SVs, so we next determined whether this interaction results in functional entry of the toxin. To test this idea, we determined whether the ability of TeNT holotoxin to enter neurons and cleave syb II also depended on SV recycling. We employed a monoclonal antibody raised against syb II (Cl. 69.1) that cannot recognize the enzymatically cleaved form of the protein. We began by comparing the ability of TeNT to cleave syb II under TTX (which blocks action potentials to inhibit SV recycling) or high potassium (which depolarizes neurons to drive SV recycling) conditions. We observed that syb II fluorescence was markedly reduced when hippocampal neurons were depolarized with potassium to drive SV recycling (Figure 2A); this occurred at both excitatory and inhibitory nerve terminals (Figure 2B). Syb II immunofluorescence was reduced in excitatory terminals by 39% in the TTX condition and was further reduced by 65% of under depolarizing conditions, as compared to control. Inhibitory terminals exhibited no significant reduction of syb II immunofluorescence under TTX conditions, but a 53% reduction was observed in neurons that had been depolarized (Figure 2B).

Bottom Line: Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons.Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals.Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms.

View Article: PubMed Central - PubMed

Affiliation: Department of Physiology, Howard Hughes Medical Institute, University of Wisconsin, Madison, Wisconsin, USA.

ABSTRACT
Tetanus neurotoxin causes the disease tetanus, which is characterized by rigid paralysis. The toxin acts by inhibiting the release of neurotransmitters from inhibitory neurons in the spinal cord that innervate motor neurons and is unique among the clostridial neurotoxins due to its ability to shuttle from the periphery to the central nervous system. Tetanus neurotoxin is thought to interact with a high affinity receptor complex that is composed of lipid and protein components; however, the identity of the protein receptor remains elusive. In the current study, we demonstrate that toxin binding, to dissociated hippocampal and spinal cord neurons, is greatly enhanced by driving synaptic vesicle exocytosis. Moreover, tetanus neurotoxin entry and subsequent cleavage of synaptobrevin II, the substrate for this toxin, was also dependent on synaptic vesicle recycling. Next, we identified the potential synaptic vesicle binding protein for the toxin and found that it corresponded to SV2; tetanus neurotoxin was unable to cleave synaptobrevin II in SV2 knockout neurons. Toxin entry into knockout neurons was rescued by infecting with viruses that express SV2A or SV2B. Tetanus toxin elicited the hyper excitability in dissociated spinal cord neurons - due to preferential loss of inhibitory transmission - that is characteristic of the disease. Surprisingly, in dissociated cortical cultures, low concentrations of the toxin preferentially acted on excitatory neurons. Further examination of the distribution of SV2A and SV2B in both spinal cord and cortical neurons revealed that SV2B is to a large extent localized to excitatory terminals, while SV2A is localized to inhibitory terminals. Therefore, the distinct effects of tetanus toxin on cortical and spinal cord neurons are not due to differential expression of SV2 isoforms. In summary, the findings reported here indicate that SV2A and SV2B mediate binding and entry of tetanus neurotoxin into central neurons.

Show MeSH
Related in: MedlinePlus