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Tetanus toxin and botulinum toxin a utilize unique mechanisms to enter neurons of the central nervous system.

ABSTRACT: Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most toxic proteins for humans. While BoNTs cause flaccid paralysis, TeNT causes spastic paralysis. Characterized BoNT serotypes enter neurons upon binding dual receptors, a ganglioside and a neuron-specific protein, either synaptic vesicle protein 2 (SV2) or synaptotagmin, while TeNT enters upon binding gangliosides as dual receptors. Recently, TeNT was reported to enter central nervous system (CNS) neurons upon synaptic vesicle cycling that was mediated by the direct binding to SV2, implying that TeNT and BoNT utilize common mechanisms to enter CNS neurons. This prompted an assessment of TeNT entry into CNS neurons, using the prototypic BoNT serotype A as a reference for SV2-mediated entry into synaptic vesicles, analyzing the heavy-chain receptor binding domain (HCR) of each toxin. Synaptic vesicle cycling stimulated the entry of HCR/A into neurons, while HCR/T entered neurons with similar levels of efficiency in depolarized and nondepolarized neurons. ImageJ analysis identified two populations of cell-associated HCR/T in synaptic vesicle cycling neurons, a major population which segregated from HCR/A and a minor population which colocalized with HCR/A. HCR/T did not inhibit HCR/A entry into neurons in competition experiments and did not bind SV2, the protein receptor for BoNT/A. Intoxication experiments showed that TeNT efficiently cleaved VAMP2 in depolarized neurons and neurons blocked for synaptic vesicle cycling. These experiments demonstrate that TeNT enters neurons by two pathways, one independent of stimulated synaptic vesicle cycling and one by synaptic vesicles independent of SV2, showing that TeNT and BoNT/A enter neurons by unique mechanisms.

SUBMITTER: Blum FC

PROVIDER: S-EPMC3347426 | biostudies-literature | 2012 May

REPOSITORIES: biostudies-literature

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Tetanus toxin and botulinum toxin a utilize unique mechanisms to enter neurons of the central nervous system.

Blum Faith C FC Chen Chen C Kroken Abby R AR Barbieri Joseph T JT

Infection and immunity 20120305 5

Botulinum neurotoxins (BoNTs) and tetanus neurotoxin (TeNT) are the most toxic proteins for humans. While BoNTs cause flaccid paralysis, TeNT causes spastic paralysis. Characterized BoNT serotypes enter neurons upon binding dual receptors, a ganglioside and a neuron-specific protein, either synaptic vesicle protein 2 (SV2) or synaptotagmin, while TeNT enters upon binding gangliosides as dual receptors. Recently, TeNT was reported to enter central nervous system (CNS) neurons upon synaptic vesicl ...[more]

PMID: 22392932

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Project description:Background: Synaptic transmission is required for functional maturation of the CNS and to induce gene transcription involved in neuronal plasticity. Our aim is to identify genes that are transcriptionally regulated by synaptic transmission during development. cDNA microarrays will be used to compare gene expression in normal embryos and embryos with no synaptic transmission. Using the Gal4 UAS system, the tetanus toxin light fragment (TET) will be expressed throughout the nervous system. The effects of TET are well characterised: the synaptic protein n-Synaptobrevin is cleaved, blocking evokes vesicle fusion in TET expressing neurons and as a result synaptic transmission is blocked. Embryos expressing an inactive, mutant version of TET (iTET) show no detectable phenotype. Thus a comparison of gene expression in iTET expressing embryos and TET expressing embryos focuses our screen cleanly and specifically on genes that are regulated by synaptic transmission. At the end of larval life, during metamorphosis, there is a second phase of nervous system development with the same requirement for neural circuit differentiation and maturation as in the embryo. We will compare gene expression in TET expressing and iTET expressing pupae to identify genes which are regulated by synaptic transmission in this second phase of nervous system development. We will focus our analysis on genes which are similarly regulated in both systems. Plan: elav Gal4 (expressed in all the neurons) for the embryos and Heat Shock Gal4 (which allows a temporally controlled expression) for the pupae are used to drive UAS TET and UAS iTET throughout the nervous system. Crosses are set up with flies homozygous for Gal4 or UAS transgenes, so that all the progeny have the same genotype. Total RNA is extracted from 500 carefully staged embryos that have been collected at the time of hatching. Pupae are heat shocked at 24 hours after puparium formation, and heads are then collected 5 days after puparium formation. TET expressing pupae are paralysed but morphologically normal whereas iTET expressing pupae emerge as normal adults. Total RNA will be extracted from 250 heads of each genotype. Probes made from TET and iTET expressing embryos extracts will be hybridised together to the chip and so will be probes made from TET and iTET expressing pupae extracts. Genes regulated in both systems will be compared together.

Dataset Information

Tetanus toxin and botulinum toxin a utilize unique mechanisms to enter neurons of the central nervous system.

Publications

Tetanus toxin and botulinum toxin a utilize unique mechanisms to enter neurons of the central nervous system.

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