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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 associates with SV2.(A) Biotinylated BoNT/B, E and TeNT were pre-incubated with neutravidin beads and then mixed with brain detergent extract. Beads were washed and bound material was subjected to SDS-PAGE and immunoblot analysis. BoNT/B and BoNT/E both associated with their receptors, synaptotagmin 1 (syt1, Cl 69.1) and SV2 (pan-SV2), respectively. TeNT showed a strong association with SV2, but not with other synaptic vesicle proteins such as syt1 and synaptoporin (porin). (B) BoNT/E (20 nM) was analyzed for binding to hippocampal neurons in the presence and absence of 2 µM HCR/T. Binding of BoNT/E to neurons was substantially reduced by HCR/T, indicating competition for binding to SV2. (C) Hippocampal neurons were treated with 200 pM BoNT/E in the presence and absence of 20 nM HCR/T. The arrow indicates the BoNT/E cleaved form of SNAP-25. Immunoblot analysis revealed that the presence of HCR/T resulted in the reduced cleavage of SNAP-25 by BoNT/E, again indicating competition for binding to the same receptor.
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ppat-1001207-g004: TeNT associates with SV2.(A) Biotinylated BoNT/B, E and TeNT were pre-incubated with neutravidin beads and then mixed with brain detergent extract. Beads were washed and bound material was subjected to SDS-PAGE and immunoblot analysis. BoNT/B and BoNT/E both associated with their receptors, synaptotagmin 1 (syt1, Cl 69.1) and SV2 (pan-SV2), respectively. TeNT showed a strong association with SV2, but not with other synaptic vesicle proteins such as syt1 and synaptoporin (porin). (B) BoNT/E (20 nM) was analyzed for binding to hippocampal neurons in the presence and absence of 2 µM HCR/T. Binding of BoNT/E to neurons was substantially reduced by HCR/T, indicating competition for binding to SV2. (C) Hippocampal neurons were treated with 200 pM BoNT/E in the presence and absence of 20 nM HCR/T. The arrow indicates the BoNT/E cleaved form of SNAP-25. Immunoblot analysis revealed that the presence of HCR/T resulted in the reduced cleavage of SNAP-25 by BoNT/E, again indicating competition for binding to the same receptor.

Mentions: To identify the potential SV binding partner for TeNT, we biotinylated this toxin, as well as BoNT/B, BoNT/E (as controls), and bound them to neutravidin beads, through biotin-avidin interactions. After incubating the toxin-linked beads with brain detergent extracts (BDE), we screened for bound SV proteins. Previously it has been shown that the receptor for BoNT/B is synaptotagmin (syt) I/II and the receptor for BoNT/E is SV2A/B [29], [30]. Consistent with previous reports, we detected that BoNT/B associated with syt I and BoNT/E with SV2, but surprisingly, we observed that TeNT also strongly associated with SV2 (Figure 4A). To further confirm that TeNT associated with SV2, we used HCR/T to see if it could compete with BoNT/E for binding to its native receptor, SV2, on hippocampal neurons. A 100-fold molar excess of HCR/T, compared to BoNT/E, markedly reduced BoNT/E binding to nerve terminals in this preparation (Figure 4B). Moreover, competition of HCR/T with BoNT/E in hippocampal neurons reduced the extent of cleavage of SNAP-25 (Figure 4C). These data are in agreement with previous studies on the NMJ that also demonstrate HCR/T can antagonize BoNT/E entry [49], [50]. The ability of the receptor-binding domain of TeNT to reduce BoNT/E binding and entry into hippocampal neurons - through competition for binding SV2 - further supports the idea that TeNT utilizes SV2 as its receptor protein.


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 associates with SV2.(A) Biotinylated BoNT/B, E and TeNT were pre-incubated with neutravidin beads and then mixed with brain detergent extract. Beads were washed and bound material was subjected to SDS-PAGE and immunoblot analysis. BoNT/B and BoNT/E both associated with their receptors, synaptotagmin 1 (syt1, Cl 69.1) and SV2 (pan-SV2), respectively. TeNT showed a strong association with SV2, but not with other synaptic vesicle proteins such as syt1 and synaptoporin (porin). (B) BoNT/E (20 nM) was analyzed for binding to hippocampal neurons in the presence and absence of 2 µM HCR/T. Binding of BoNT/E to neurons was substantially reduced by HCR/T, indicating competition for binding to SV2. (C) Hippocampal neurons were treated with 200 pM BoNT/E in the presence and absence of 20 nM HCR/T. The arrow indicates the BoNT/E cleaved form of SNAP-25. Immunoblot analysis revealed that the presence of HCR/T resulted in the reduced cleavage of SNAP-25 by BoNT/E, again indicating competition for binding to the same receptor.
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ppat-1001207-g004: TeNT associates with SV2.(A) Biotinylated BoNT/B, E and TeNT were pre-incubated with neutravidin beads and then mixed with brain detergent extract. Beads were washed and bound material was subjected to SDS-PAGE and immunoblot analysis. BoNT/B and BoNT/E both associated with their receptors, synaptotagmin 1 (syt1, Cl 69.1) and SV2 (pan-SV2), respectively. TeNT showed a strong association with SV2, but not with other synaptic vesicle proteins such as syt1 and synaptoporin (porin). (B) BoNT/E (20 nM) was analyzed for binding to hippocampal neurons in the presence and absence of 2 µM HCR/T. Binding of BoNT/E to neurons was substantially reduced by HCR/T, indicating competition for binding to SV2. (C) Hippocampal neurons were treated with 200 pM BoNT/E in the presence and absence of 20 nM HCR/T. The arrow indicates the BoNT/E cleaved form of SNAP-25. Immunoblot analysis revealed that the presence of HCR/T resulted in the reduced cleavage of SNAP-25 by BoNT/E, again indicating competition for binding to the same receptor.
Mentions: To identify the potential SV binding partner for TeNT, we biotinylated this toxin, as well as BoNT/B, BoNT/E (as controls), and bound them to neutravidin beads, through biotin-avidin interactions. After incubating the toxin-linked beads with brain detergent extracts (BDE), we screened for bound SV proteins. Previously it has been shown that the receptor for BoNT/B is synaptotagmin (syt) I/II and the receptor for BoNT/E is SV2A/B [29], [30]. Consistent with previous reports, we detected that BoNT/B associated with syt I and BoNT/E with SV2, but surprisingly, we observed that TeNT also strongly associated with SV2 (Figure 4A). To further confirm that TeNT associated with SV2, we used HCR/T to see if it could compete with BoNT/E for binding to its native receptor, SV2, on hippocampal neurons. A 100-fold molar excess of HCR/T, compared to BoNT/E, markedly reduced BoNT/E binding to nerve terminals in this preparation (Figure 4B). Moreover, competition of HCR/T with BoNT/E in hippocampal neurons reduced the extent of cleavage of SNAP-25 (Figure 4C). These data are in agreement with previous studies on the NMJ that also demonstrate HCR/T can antagonize BoNT/E entry [49], [50]. The ability of the receptor-binding domain of TeNT to reduce BoNT/E binding and entry into hippocampal neurons - through competition for binding SV2 - further supports the idea that TeNT utilizes SV2 as its receptor protein.

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