Tetanus Toxin Is Transported in a Novel Neuronal Compartment Characterized by a Specialized pH Regulation

Bohnert, Stephanie; Schiavo, Giampietro

doi:10.1074/jbc.m506750200

Cited by 89 publications

(83 citation statements)

References 44 publications

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“…The retrograde movement of HCR/T is dependent on Rab5 and Rab7 and occurs in Rab7 vesicles which are also positive for low-affinity neurotrophin receptor, TrkB, and brain-derived neutrotrophic factor (BDNF) (14). These vesicles are of neutral pH and vATPase excluded (13). Little is known concerning the mechanism by which TeNT is transcytosed, but TeNT intoxication of inhibitory interneurons requires entry into a vesicle, which will undergo acidification, making endosomes an attractive mechanism of entry.…”

Section: Discussionmentioning

confidence: 99%

“…TeNT can bind a glycophosphatidylinositol (GPI)-anchored protein (8) or SV2 (9), but the significance of these interactions has not been defined (10) or reproduced (11), respectively. TeNT enters motor neurons upon endocytosis (12) and traffics through motor neurons associated with Rab7-enriched endosomes that are of neutral pH (13,14). Retrograde trafficking proceeds from the axon to the soma, where TeNT transcytoses from the motor neuron into an inhibitory neuron of the central nervous system (CNS).…”

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confidence: 99%

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Entry of a Recombinant, Full-Length, Atoxic Tetanus Neurotoxin into Neuro-2a Cells

et al. 2014

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Tetanus neurotoxin (TeNT) and botulinum neurotoxin (BoNT) are clostridial neurotoxins (CNTs) that are the most toxic proteins for humans (1). TeNT and BoNT share ϳ35% identity and ϳ65% similarity and overall structure-function properties (2). BoNT intoxication results in flaccid paralysis through the inhibition of acetylcholine release by motor neurons, while TeNT intoxication yields a spastic paralysis due to inhibition of glycine release by inhibitory neurons (3). TeNT and BoNT are expressed as ϳ150-kDa single-chain proteins that are cleaved to form dichain proteins linked by a disulfide bond (2). The N-terminal 50-kDa light chain (LC) is a zinc-metalloprotease that cleaves neuronspecific soluble NSF attachment protein (SNAP) receptor (SNARE) proteins (4). TeNT and BoNT serotype B cleave the same residue within vesicle-associated membrane protein 2 (VAMP2), a SNARE protein of synaptic vesicles (SVs). The C-terminal 100-kDa heavy chain (HC) contains two structurally distinct domains with separate functions. The translocation domain (HCT) facilitates LC translocation from the SV lumen into the cell cytosol, and the receptor binding domain (HCR) binds dual host receptors.BoNT/A binds a ganglioside and synaptic vesicle protein 2 (SV2) and enters neurons upon SV recycling from the plasma membrane (5). Upon SV acidification within the periphery of motor neurons, the HCT undergoes a pH-dependent conformational change and inserts into the SV membrane, forming a channel that allows the LC to escape into the cytosol. Within the periphery of the motor neuron, the LC cleaves SNARE proteins, resulting in loss of stimulatory signaling between neurons and muscles, yielding flaccid paralysis. The LC of BoNT/A localizes to the plasma membrane to target synaptosomal-associated protein 25 (SNAP25) for cleavage within neurons (6).TeNT binds two gangliosides as functional receptors (7). TeNT can bind a glycophosphatidylinositol (GPI)-anchored protein (8) or SV2 (9), but the significance of these interactions has not been defined (10) or reproduced (11), respectively. TeNT enters motor neurons upon endocytosis (12) and traffics through motor neurons associated with Rab7-enriched endosomes that are of neutral pH (13,14). Retrograde trafficking proceeds from the axon to the soma, where TeNT transcytoses from the motor neuron into an inhibitory neuron of the central nervous system (CNS). Upon vesicle acidification, the LC is translocated into the cytosol and cleaves VAMP2. The block in signaling between the inhibitory neurons and motor neurons leads to the spastic paralysis characteristic of tetanus. The molecular mechanism responsible for the unique entry of BoNT and TeNT is not clearly understood.The modular structural domains of the CNTs have permitted the study of individual domains to assess protein structure-function in vitro; the LC is a functional SNARE protease (15), the HCT inserts into lipid bilayers and forms a channel (16), and the HCR binds host receptors (17). The tetanus HCR (HCR/T) can retrograde traffic in both cultu...

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Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

Entry of a Recombinant, Full-Length, Atoxic Tetanus Neurotoxin into Neuro-2a Cells

et al. 2014

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“…Five or six days after being plated on glass bottom dishes (MatTek Corporation) (3,12), spinal cord MNs were incubated with 7 ϫ 10 5 PFU/well of Alexa Fluor 555-labeled strain Sabin 1 at 35.5°C or with 6 ϫ 10 7 PFU/well of Alexa Fluor 488-labeled strain Mahoney at 37°C for 15 min; then they were washed and imaged by confocal laser scanning microscopy (Carl Zeiss MicroImaging). For the competition assay, MNs were incubated with or without 4.7 ϫ 10 8 PFU/well of unlabeled PV or an anti-hPVR MAb at 37°C for 5 min; labeled PV and 667 g/ml of tetramethyl rhodamine (TMR)-conjugated dextran (molecular weight, 3,000; Invitrogen) was then added, and MNs were incubated at 37°C for 15 min and then washed and observed by confocal laser scanning microscopy.…”

Section: Virusesmentioning

confidence: 99%

“…All mice were 6 to 10 weeks of age. For the preparation of MNs in culture (3,12), embryonic day 14 (E14) SpragueDawley rat embryos or E13 IQI-PVRTg21 or C57BL/6-PVRTg21 mouse embryos were used. Mice and rats were treated according to the guidelines for the care and use of laboratory animals of the University of Tokyo.…”

Section: Virusesmentioning

confidence: 99%

Receptor-Dependent and -Independent Axonal Retrograde Transport of Poliovirus in Motor Neurons

Ohka

Sakai

Bohnert

et al. 2009

J Virol

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Poliovirus (PV), when injected intramuscularly into the calf, is incorporated into the sciatic nerve and causes an initial paralysis of the inoculated limb in transgenic (Tg) mice carrying the human PV receptor (hPVR/ CD155) gene. We have previously demonstrated that a fast retrograde axonal transport process is required for PV dissemination through the sciatic nerves of hPVR-Tg mice and that intramuscularly inoculated PV causes paralytic disease in an hPVR-dependent manner. Here we showed that hPVR-independent axonal transport of PV was observed in hPVR-Tg and non-Tg mice, indicating that several different pathways for PV axonal transport exist in these mice. Using primary motor neurons (MNs) isolated from these mice or rats, we demonstrated that the axonal transport of PV requires several kinetically different motor machineries and that fast transport relies on a system involving cytoplasmic dynein. Unexpectedly, the hPVR-independent axonal transport of PV was not observed in cultured MNs. Thus, PV transport machineries in cultured MNs and in vivo differ in their hPVR requirements. These results suggest that the axonal trafficking of PV is carried out by several distinct pathways and that MNs in culture and in the sciatic nerve in situ are intrinsically different in the uptake and axonal transport of PV.In humans, paralytic poliomyelitis results from the invasion of the central nervous system by circulating poliovirus (PV), probably via the blood-brain barrier. This conclusion is supported by the finding that circulating PV after intravenous inoculation in mice appears to cross the blood-brain barrier at a high rate in a human PV receptor (hPVR/CD155)-independent manner (44). After the virus enters the central nervous system, it replicates in neurons, especially in motor neurons (MNs), inducing the cell death that causes paralytic poliomyelitis. Along with this route of dissemination, a neuron-specific pathway has been reported in humans (31), monkeys (18), and PV-sensitive transgenic (Tg) mice carrying the hPVR gene (34,37). This neuron-specific pathway appears to be important in causing "provocation poliomyelitis," which is triggered by injuries after PV ingestion (11). Using differentiated PC12 cells and a PV-sensitive Tg mouse line, we have shown that intramuscularly inoculated PV is taken up by endocytosis at synapses.hPVR is a member of the immunoglobulin (Ig) superfamily, with three linked extracellular Ig-like domains, followed by a membrane-spanning domain and a cytoplasmic domain. Two membrane-bound forms (␣ and ␦) and two secreted forms (␤ and ␥) of hPVR derived by alternative splicing are likely to be expressed in human cells (23). Membrane-bound hPVRs are considered to play important roles in the early steps of infection, such as the binding of the virus to the cell surface, its entry into the cell, and the uncoating of the virus. The N-terminal Ig-like domain harbors the sites for PV binding, and antihPVR monoclonal antibodies (MAbs) directed against this region block PV infection (9,24,39).hPVR ...

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“…In addition, tetanus neurotoxin (TeNT) shares structural homology with the BoNTs and cleaves VAMP-2 at the same site as BoNT/B. Despite this similarity, TeNT elicits spastic paralysis due to the ability to retrograde traffic within the peripheral neurons to target interneurons (6,(13)(14)(15).…”

mentioning

confidence: 99%

Substrate Recognition of VAMP-2 by Botulinum Neurotoxin B and Tetanus Neurotoxin

Chen

Hall

Barbieri

2008

Journal of Biological Chemistry

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Botulinum neurotoxin (BoNT; serotypes A-G) and tetanus neurotoxin elicit flaccid and spastic paralysis, respectively. These neurotoxins are zinc proteases that cleave SNARE proteins to inhibit synaptic vesicle fusion to the plasma membrane. Although BoNT/B and tetanus neurotoxin (TeNT) cleave VAMP-2 at the same scissile bond, their mechanism(s) of VAMP-2 recognition is not clear. Mapping experiments showed that residues 60 -87 of VAMP-2 were sufficient for efficient cleavage by BoNT/B and that residues 40 -87 of VAMP-2 were sufficient for efficient TeNT cleavage. Alanine-scanning mutagenesis and kinetic analysis identified three regions within VAMP-2 that were recognized by BoNT/B and TeNT: residues adjacent to the site of scissile bond cleavage (cleavage region) and residues located within N-terminal and C-terminal regions relative to the cleavage region. Analysis of residues within the cleavage region showed that mutations at the P7, P4, P2, and P1 residues of VAMP-2 had the greatest inhibition of LC/B cleavage (>32-fold), whereas mutations at P7, P4, P1, and P2 residues of VAMP-2 had the greatest inhibition of LC/TeNT cleavage (>64-fold). Residues within the cleavage region influenced catalysis, whereas residues N-terminal and C-terminal to the cleavage region influenced binding affinity. Thus, BoNT/B and TeNT possess similar organization but have unique residues to recognize and cleave VAMP-2. These studies provide new insights into how the clostridial neurotoxins recognize their substrates.The botulinum neurotoxins (BoNTs) 2 are the most potent protein toxins for humans (1). BoNTs have been used for malicious purposes (2) but also relieve numerous human neurological afflictions, such as blepharospasm, in addition to being used for cosmetic enhancement (3-5). Recent studies provide a more detailed understanding of BoNT action, which should provide insight for developing novel products and therapies to treat human suffering.BoNTs are zinc proteases that cleave SNARE proteins to block synaptic vesicle fusion and neurotransmitter release to elicit flaccid paralysis. BoNTs are 150-kDa dichain proteins with AB structure-function properties; the N terminus encodes the zinc protease domain (light chain (LC)), and the C terminus encodes a translocation domain and a receptor binding domain (6, 7). The seven serotypes of BoNTs, termed A-G, are based upon antisera neutralization properties (8). Each BoNT serotype cleaves one of three neuronal SNARE proteins: SNAP-25, VAMP-2, or syntaxin 1a, except for BoNT/C, which cleaves both SNAP-25 and syntaxin 1a. BoNT/A and BoNT/E cleave SNAP-25 at different sites, whereas BoNT/B, BoNT/D, BoNT/F, and BoNT/G cleave VAMP-2 at distinct sites (9 -12). In addition, tetanus neurotoxin (TeNT) shares structural homology with the BoNTs and cleaves VAMP-2 at the same site as BoNT/B. Despite this similarity, TeNT elicits spastic paralysis due to the ability to retrograde traffic within the peripheral neurons to target interneurons (6, 13-15).Unlike other zinc proteases, BoNTs and TeNT rec...

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Tetanus Toxin Is Transported in a Novel Neuronal Compartment Characterized by a Specialized pH Regulation

Cited by 89 publications

References 44 publications

Entry of a Recombinant, Full-Length, Atoxic Tetanus Neurotoxin into Neuro-2a Cells

Entry of a Recombinant, Full-Length, Atoxic Tetanus Neurotoxin into Neuro-2a Cells

Receptor-Dependent and -Independent Axonal Retrograde Transport of Poliovirus in Motor Neurons

Substrate Recognition of VAMP-2 by Botulinum Neurotoxin B and Tetanus Neurotoxin

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