SV2 Mediates Entry of Tetanus Neurotoxin into Central Neurons

Felix L Yeh,Min Dong,Eric A Johnson,William H Tepp,Jun Yao,Guangyun Lin,Edwin R Chapman,Theresa Koehler

doi:10.1371/journal.ppat.1001207

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.

Highlights

The Clostridium genus of bacteria are responsible for the production of the clostridial neurotoxins (CNTs), which include both tetanus neurotoxin (TeNT) and seven botulinum neurotoxins (BoNT/A–G) [1]
The CNTs are 150 kDa proteins composed of a heavy chain (HC) and a light chain (LC) that are linked through a disulfide bond
In the current study we discovered that the ‘‘Trojan-horse’’, utilized by tetanus neurotoxin to enter central neurons, corresponds to recycling synaptic vesicles

Summary

Introduction

The Clostridium genus of bacteria are responsible for the production of the clostridial neurotoxins (CNTs), which include both tetanus neurotoxin (TeNT) and seven botulinum neurotoxins (BoNT/A–G) [1]. The LC cleaves one or more of three soluble N-ethylmaleimide-sensitive fusion protein receptor (SNARE) proteins: BoNT/A and E cleave the plasma membrane protein SNAP-25 (synaptosomal-associated protein of 25 kDa); BoNT/B, D, F, G and TeNT cleave the vesicle protein synaptobrevin (syb); and BoNT/C cleaves both SNAP-25 and syntaxin-1 [6,7,8,9,10]. Of sybNsyntaxinNSNAP-25 into parallel four-helix bundles is thought to pull the vesicle and plasma membranes together to drive membrane fusion [11]. Cleavage of these SNAREs by the CNTs either severs them from the membrane or disrupts their ability to assemble into stable/functional fusion complexes, thereby blocking synaptic vesicle (SVs) exocytosis and neurotransmitter release [1,12]

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Discussion

Conclusion