Tetanus toxin C-fragment protects against excitotoxic spinal motoneuron degeneration in vivo

ABSTRACT The development of combined vaccines constitutes one of the priorities in modern vaccine research. One of the most successful combined vaccines in use is the diphtheria-pertussis-tetanus vaccine. However, concerns about the safety of the pertussis arm have led to decreased acceptance of the vaccine but also to the development of new, safer, and effective acellular vaccines against pertussis. Unfortunately, the production cost of these new vaccines is significantly higher than that of previous vaccines. Here, we explore the potential of live recombinant Mycobacterium bovis BCG producing the hybrid protein S1-TTC, which contains the S1 subunit of pertussis toxin fused to fragment C of tetanus toxin, as an alternative to the acellular vaccines. S1-TTC was produced in two different expression systems. In the first system its production was under the control of the 85A antigen promoter and signal peptide, and in the second system it was under the control of the hsp60promoter. Although expression of the hybrid antigen was obtained in both cases, only the second expression system yielded a recombinant BCG strain able to induce both a specific humoral immune response and a specific cellular immune response. The antibodies generated were directed against the TTC part and neutralized toxin activity in an in vivo challenge model, whereas interleukin-2 production was specific for both parts of the molecule. Since protection against tetanus is antibody mediated and protection against pertussis may be cell mediated, this constitutes a first promising step towards the development of a cost-effective, protective, and safe combined vaccine against pertussis, tetanus, and tuberculosis.

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Characterization of spinal motoneuron degeneration following different types of peripheral nerve injury in neonatal and adult mice

The Journal of Comparative Neurology ◽

10.1002/(sici)1096-9861(19980629)396:2<158::aid-cne2>3.0.co;2-# ◽

1998 ◽

Vol 396 (2) ◽

pp. 158-168 ◽

Cited By ~ 73

Author(s):

Linxi Li ◽

Lucien J. Houenou ◽

Wutian Wu ◽

Ming Lei ◽

David M. Prevette ◽

...

Keyword(s):

Peripheral Nerve ◽

Nerve Injury ◽

Peripheral Nerve Injury ◽

Spinal Motoneuron ◽

Adult Mice ◽

Different Types ◽

Motoneuron Degeneration

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Endobrevin/VAMP-8 Is the Primary v-SNARE for the Platelet Release Reaction

Molecular Biology of the Cell ◽

10.1091/mbc.e06-09-0785 ◽

2007 ◽

Vol 18 (1) ◽

pp. 24-33 ◽

Cited By ~ 110

Author(s):

Qiansheng Ren ◽

Holly Kalani Barber ◽

Garland L. Crawford ◽

Zubair A. Karim ◽

Chunxia Zhao ◽

...

Keyword(s):

Tetanus Toxin ◽

Vascular Inflammation ◽

Human Platelets ◽

In Vivo Model ◽

Attachment Protein ◽

Protein Receptors ◽

Platelet Release ◽

Dense Core Granules ◽

Platelet Exocytosis

Platelet secretion is critical to hemostasis. Release of granular cargo is mediated by soluble NSF attachment protein receptors (SNAREs), but despite consensus on t-SNAREs usage, it is unclear which Vesicle Associated Membrane Protein (VAMPs: synaptobrevin/VAMP-2, cellubrevin/VAMP-3, TI-VAMP/VAMP-7, and endobrevin/VAMP-8) is required. We demonstrate that VAMP-8 is required for release from dense core granules, alpha granules, and lysosomes. Platelets from VAMP-8−/−mice have a significant defect in agonist-induced secretion, though signaling, morphology, and cargo levels appear normal. In contrast, VAMP-2+/−, VAMP-3−/−, and VAMP-2+/−/VAMP-3−/−platelets showed no defect. Consistently, tetanus toxin had no effect on secretion from permeabilized mouse VAMP-3−/−platelets or human platelets, despite cleavage of VAMP-2 and/or -3. Tetanus toxin does block the residual release from permeabilized VAMP-8−/−platelets, suggesting a secondary role for VAMP-2 and/or -3. These data imply a ranked redundancy of v-SNARE usage in platelets and suggest that VAMP-8−/−mice will be a useful in vivo model to study platelet exocytosis in hemostasis and vascular inflammation.

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Hypoexcitability precedes denervation in the large fast-contracting motor units in two unrelated mouse models of ALS

eLife ◽

10.7554/elife.30955 ◽

2018 ◽

Vol 7 ◽

Cited By ~ 33

Author(s):

María de Lourdes Martínez-Silva ◽

Rebecca D Imhoff-Manuel ◽

Aarti Sharma ◽

CJ Heckman ◽

Neil A Shneider ◽

...

Keyword(s):

Motor Unit ◽

Mouse Models ◽

Neuromuscular Junctions ◽

Motor Units ◽

Loss Of Function ◽

Disease Markers ◽

Physiological Type ◽

Lateral Sclerosis ◽

Motoneuron Degeneration

Hyperexcitability has been suggested to contribute to motoneuron degeneration in amyotrophic lateral sclerosis (ALS). If this is so, and given that the physiological type of a motor unit determines the relative susceptibility of its motoneuron in ALS, then one would expect the most vulnerable motoneurons to display the strongest hyperexcitability prior to their degeneration, whereas the less vulnerable should display a moderate hyperexcitability, if any. We tested this hypothesis in vivo in two unrelated ALS mouse models by correlating the electrical properties of motoneurons with their physiological types, identified based on their motor unit contractile properties. We found that, far from being hyperexcitable, the most vulnerable motoneurons become unable to fire repetitively despite the fact that their neuromuscular junctions were still functional. Disease markers confirm that this loss of function is an early sign of degeneration. Our results indicate that intrinsic hyperexcitability is unlikely to be the cause of motoneuron degeneration.

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Fate of tetanus toxin bound to the surface of primary neurons in culture: evidence for rapid internalization.

The Journal of Cell Biology ◽

10.1083/jcb.100.5.1499 ◽

1985 ◽

Vol 100 (5) ◽

pp. 1499-1507 ◽

Cited By ~ 41

Author(s):

D R Critchley ◽

P G Nelson ◽

W H Habig ◽

P H Fishman

Keyword(s):

Cell Surface ◽

Tetanus Toxin ◽

Polyacrylamide Gel Electrophoresis ◽

Primary Cultures ◽

Protein A ◽

Sodium Dodecyl ◽

Neuronal Cell ◽

Subcellular Compartment ◽

Membrane Components

We examined the nature of the tetanus toxin receptor in primary cultures of mouse spinal cord by ligand blotting techniques. Membrane components were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled tetanus toxin. The toxin bound only to material at or near the dye front, which was lost when the cells were delipidated before electrophoresis. Gangliosides purified from the lipid extract were separated by thin-layer chromatography and the chromatogram was overlaid with 125I-toxin. The toxin bound to gangliosides corresponding to GD1b and GT1b. Similar results were obtained with brain membranes; thus, gangliosides rather than glycoproteins appear to be the toxin receptors both in vivo and in neuronal cell cultures. To follow the fate of tetanus toxin bound to cultured neurons, we developed an assay to measure cell-surface and internalized toxin. Cells were incubated with tetanus toxin at 0 degree C, washed, and sequentially exposed to antitoxin and 125I-labeled protein A. Using this assay, we found that much of the toxin initially bound to cell surface disappeared rapidly when the temperature was raised to 37 degrees C but not when the cells were kept at 0 degree C. Some of the toxin was internalized and could only be detected by our treating the cells with Triton X-100 before adding anti-toxin. Experiments with 125I-tetanus toxin showed that a substantial amount of the toxin bound at 0 degree C dissociated into the medium upon warming of the cells. Using immunofluorescence, we confirmed that some of the bound toxin was internalized within 15 min and accumulated in discrete structures. These structures did not appear to be lysosomes, as the cell-associated toxin had a long half-life and 90% of the radioactivity released into the medium was precipitated by trichloroacetic acid. The rapid internalization of tetanus toxin into a subcellular compartment where it escapes degradation may be important for its mechanism of action.

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