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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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SV2A/B KO neurons are resistant to TeNT.(A) Mouse spinal cord neurons with the following genotypes: WT, SV2B KO [SV2A (+/+) SV2B (−/−)], and SV2A/B KO [SV2A (−/−) SV2B (−/−)], were exposed to HCR/T (50 nM). HCR/T fluorescence in SV2A/B KO neurons was dramatically reduced as compared to WT and SV2B KO. (B) Quantification of HCR/T binding: fluorescence was reduced by 30% and 50% for SV2B KO and SV2A/B KO neurons, respectively. Error bars represent SD, WT n = 9, SV2B KO n = 11, SV2A/B KO n = 12, ***p≤0.001. (C) SV2B KO and SV2A/B KO cultures were exposed to TeNT (20 nM) and BoNT/A (10 nM). Cell lysates were subjected to immunoblot analysis and probed for syb II, SV2, SNAP-25, and actin. Syb II in SV2A/B KO neurons was largely protected from TeNT action until resensitized through lentiviral expression of SV2A or SV2B; arrow indicates the BoNT/A cleaved form of SNAP-25. (D) SV2B KO and SV2A/B KO spinal cord neurons from were assayed for susceptibility to TeNT. Syb II was cleaved by 5 nM TeNT in SV2B KO neurons, while syb II was protected from TeNT in SV2A/B KO neurons. SV2A/B KO neurons could be resensitized to TeNT, upon lentiviral expression of SV2A. (E) Three putative glycosylation sites in SV2A were removed by creating N to Q mutants (residues 498, 548 and 573) and were expressed in SV2A/B KO neurons along with WT SV2A. Syb II was cleaved by TeNT in neurons reinfected with WT SV2A as well as the three mutants. (F) WT and SV2B KO mice were injected with the indicated amounts of TeNT and their time-to-death was recorded. SV2B KO mice were more than five-times more resistant to TeNT as compared to their WT counterparts. (G) WT and SV2A/B KO neurons were cultured and treated with BoNT/F at the indicated concentrations. Cell lysates were probed for syb II and syp by immunoblot analysis. WT and SV2A/B KO neurons exhibited similar sensitivities to BoNT/F.
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ppat-1001207-g005: SV2A/B KO neurons are resistant to TeNT.(A) Mouse spinal cord neurons with the following genotypes: WT, SV2B KO [SV2A (+/+) SV2B (−/−)], and SV2A/B KO [SV2A (−/−) SV2B (−/−)], were exposed to HCR/T (50 nM). HCR/T fluorescence in SV2A/B KO neurons was dramatically reduced as compared to WT and SV2B KO. (B) Quantification of HCR/T binding: fluorescence was reduced by 30% and 50% for SV2B KO and SV2A/B KO neurons, respectively. Error bars represent SD, WT n = 9, SV2B KO n = 11, SV2A/B KO n = 12, ***p≤0.001. (C) SV2B KO and SV2A/B KO cultures were exposed to TeNT (20 nM) and BoNT/A (10 nM). Cell lysates were subjected to immunoblot analysis and probed for syb II, SV2, SNAP-25, and actin. Syb II in SV2A/B KO neurons was largely protected from TeNT action until resensitized through lentiviral expression of SV2A or SV2B; arrow indicates the BoNT/A cleaved form of SNAP-25. (D) SV2B KO and SV2A/B KO spinal cord neurons from were assayed for susceptibility to TeNT. Syb II was cleaved by 5 nM TeNT in SV2B KO neurons, while syb II was protected from TeNT in SV2A/B KO neurons. SV2A/B KO neurons could be resensitized to TeNT, upon lentiviral expression of SV2A. (E) Three putative glycosylation sites in SV2A were removed by creating N to Q mutants (residues 498, 548 and 573) and were expressed in SV2A/B KO neurons along with WT SV2A. Syb II was cleaved by TeNT in neurons reinfected with WT SV2A as well as the three mutants. (F) WT and SV2B KO mice were injected with the indicated amounts of TeNT and their time-to-death was recorded. SV2B KO mice were more than five-times more resistant to TeNT as compared to their WT counterparts. (G) WT and SV2A/B KO neurons were cultured and treated with BoNT/F at the indicated concentrations. Cell lysates were probed for syb II and syp by immunoblot analysis. WT and SV2A/B KO neurons exhibited similar sensitivities to BoNT/F.

Mentions: SV2 is a 12 transmembrane protein that is heavily glycosylated and is homologous to transmembrane transporters. SV2 exists in 3 isoforms - A, B, and C - and SV2 A and B, but not C, knock-out (KO) mice have been generated [51], [52]. We utilized dissociated spinal cord neurons from SV2 KO mice in order to directly determine whether SV2 was critical for the action of TeNT at a functional level. We observed significant reductions of HCR/T binding to inhibitory terminals in KO mice (Figure 5A). Compared to WT, there was a 30% reduction in HCR/T binding to SV2B KO spinal cord neurons and a 55% reduction of HCR/T binding to inhibitory terminals of SV2A/B KO neurons (Figure 5B). These results further demonstrate that SV2 plays a critical role in the binding of TeNT to inhibitory terminals in the spinal cord.


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)

SV2A/B KO neurons are resistant to TeNT.(A) Mouse spinal cord neurons with the following genotypes: WT, SV2B KO [SV2A (+/+) SV2B (−/−)], and SV2A/B KO [SV2A (−/−) SV2B (−/−)], were exposed to HCR/T (50 nM). HCR/T fluorescence in SV2A/B KO neurons was dramatically reduced as compared to WT and SV2B KO. (B) Quantification of HCR/T binding: fluorescence was reduced by 30% and 50% for SV2B KO and SV2A/B KO neurons, respectively. Error bars represent SD, WT n = 9, SV2B KO n = 11, SV2A/B KO n = 12, ***p≤0.001. (C) SV2B KO and SV2A/B KO cultures were exposed to TeNT (20 nM) and BoNT/A (10 nM). Cell lysates were subjected to immunoblot analysis and probed for syb II, SV2, SNAP-25, and actin. Syb II in SV2A/B KO neurons was largely protected from TeNT action until resensitized through lentiviral expression of SV2A or SV2B; arrow indicates the BoNT/A cleaved form of SNAP-25. (D) SV2B KO and SV2A/B KO spinal cord neurons from were assayed for susceptibility to TeNT. Syb II was cleaved by 5 nM TeNT in SV2B KO neurons, while syb II was protected from TeNT in SV2A/B KO neurons. SV2A/B KO neurons could be resensitized to TeNT, upon lentiviral expression of SV2A. (E) Three putative glycosylation sites in SV2A were removed by creating N to Q mutants (residues 498, 548 and 573) and were expressed in SV2A/B KO neurons along with WT SV2A. Syb II was cleaved by TeNT in neurons reinfected with WT SV2A as well as the three mutants. (F) WT and SV2B KO mice were injected with the indicated amounts of TeNT and their time-to-death was recorded. SV2B KO mice were more than five-times more resistant to TeNT as compared to their WT counterparts. (G) WT and SV2A/B KO neurons were cultured and treated with BoNT/F at the indicated concentrations. Cell lysates were probed for syb II and syp by immunoblot analysis. WT and SV2A/B KO neurons exhibited similar sensitivities to BoNT/F.
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Related In: Results  -  Collection

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ppat-1001207-g005: SV2A/B KO neurons are resistant to TeNT.(A) Mouse spinal cord neurons with the following genotypes: WT, SV2B KO [SV2A (+/+) SV2B (−/−)], and SV2A/B KO [SV2A (−/−) SV2B (−/−)], were exposed to HCR/T (50 nM). HCR/T fluorescence in SV2A/B KO neurons was dramatically reduced as compared to WT and SV2B KO. (B) Quantification of HCR/T binding: fluorescence was reduced by 30% and 50% for SV2B KO and SV2A/B KO neurons, respectively. Error bars represent SD, WT n = 9, SV2B KO n = 11, SV2A/B KO n = 12, ***p≤0.001. (C) SV2B KO and SV2A/B KO cultures were exposed to TeNT (20 nM) and BoNT/A (10 nM). Cell lysates were subjected to immunoblot analysis and probed for syb II, SV2, SNAP-25, and actin. Syb II in SV2A/B KO neurons was largely protected from TeNT action until resensitized through lentiviral expression of SV2A or SV2B; arrow indicates the BoNT/A cleaved form of SNAP-25. (D) SV2B KO and SV2A/B KO spinal cord neurons from were assayed for susceptibility to TeNT. Syb II was cleaved by 5 nM TeNT in SV2B KO neurons, while syb II was protected from TeNT in SV2A/B KO neurons. SV2A/B KO neurons could be resensitized to TeNT, upon lentiviral expression of SV2A. (E) Three putative glycosylation sites in SV2A were removed by creating N to Q mutants (residues 498, 548 and 573) and were expressed in SV2A/B KO neurons along with WT SV2A. Syb II was cleaved by TeNT in neurons reinfected with WT SV2A as well as the three mutants. (F) WT and SV2B KO mice were injected with the indicated amounts of TeNT and their time-to-death was recorded. SV2B KO mice were more than five-times more resistant to TeNT as compared to their WT counterparts. (G) WT and SV2A/B KO neurons were cultured and treated with BoNT/F at the indicated concentrations. Cell lysates were probed for syb II and syp by immunoblot analysis. WT and SV2A/B KO neurons exhibited similar sensitivities to BoNT/F.
Mentions: SV2 is a 12 transmembrane protein that is heavily glycosylated and is homologous to transmembrane transporters. SV2 exists in 3 isoforms - A, B, and C - and SV2 A and B, but not C, knock-out (KO) mice have been generated [51], [52]. We utilized dissociated spinal cord neurons from SV2 KO mice in order to directly determine whether SV2 was critical for the action of TeNT at a functional level. We observed significant reductions of HCR/T binding to inhibitory terminals in KO mice (Figure 5A). Compared to WT, there was a 30% reduction in HCR/T binding to SV2B KO spinal cord neurons and a 55% reduction of HCR/T binding to inhibitory terminals of SV2A/B KO neurons (Figure 5B). These results further demonstrate that SV2 plays a critical role in the binding of TeNT to inhibitory terminals in the spinal cord.

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