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Fragment C of Tetanus Toxin: New Insights into Its Neuronal Signaling Pathway

DOI: 10.3390/ijms13066883

Keywords: clathrin-mediated pathway, dynamin, fragment C, tetanus toxin, neurotrophin, Trk receptors

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When Clostridium tetani was discovered and identified as a Gram-positive anaerobic bacterium of the genus Clostridium, the possibility of turning its toxin into a valuable biological carrier to ameliorate neurodegenerative processes was inconceivable. However, the non-toxic carboxy-terminal fragment of the tetanus toxin heavy chain (fragment C) can be retrogradely transported to the central nervous system; therefore, fragment C has been used as a valuable biological carrier of neurotrophic factors to ameliorate neurodegenerative processes. More recently, the neuroprotective properties of fragment C have also been described in vitro and in vivo, involving the activation of Akt kinase and extracellular signal-regulated kinase (ERK) signaling cascades through neurotrophin tyrosine kinase (Trk) receptors. Although the precise mechanism of the molecular internalization of fragment C in neuronal cells remains unknown, fragment C could be internalized and translocated into the neuronal cytosol through a clathrin-mediated pathway dependent on proteins, such as dynamin and AP-2. In this review, the origins, molecular properties and possible signaling pathways of fragment C are reviewed to understand the biochemical characteristics of its intracellular and synaptic transport.


[1]  Johnson, J.L.; Francis, G. Taxonomy of the clostridia: Ribosomal ribonucleic acid homologies among the species. J. Gen. Microbiol 1975, 88, 229–244.
[2]  Johnson, E.A. Clostridial toxins as therapeutic agents: Benefits of natures’s most toxic proteins. Annu. Rev. Microbiol 1999, 53, 551–575.
[3]  Anderson, J.F.; Leake, J.P. A method of producing tetanus toxin. J. Med. Res 1915, 33, 239–241.
[4]  Noguchi, H. The nature of the antitetanic action of eosin. J. Exp. Med 1907, 9, 281–290.
[5]  Flexner, S.; Noguchi, H. The effect of eosin upon tetanus toxin and upon tetanus in rats and guinea-pigs. J. Exp. Med 1906, 8, 1–7.
[6]  Cowie, D.M.; Greenthal, R.M. Studies on the nature of the action of non-specific protein in disease processes. III. Non-specific proteins and soluble toxin (diphtheria-tetanus). J. Med. Res 1922, 43, 21–28.
[7]  Sherrington, C.S. On reciprocal innervation of antagonistic muscles. VIIIth note. Proc. R. Soc. B 1905, 76, 269–297.
[8]  Acheson, G.H.; Oscar, M.D.; Ratnoff, D.; Schoenbach, E.B. The localized action on the spinal cord of intramuscularly injected tetanus toxin. J. Exp. Med 1942, 75, 465–480.
[9]  Brooks, V.B.; Curtis, D.R.; Eccles, J.C. The action of tetanus toxin on the inhibition of motor neurons. J. Physiol 1957, 135, 655–672.
[10]  Firor, W.M.; Lamont, A. The apparent alteration of tetanus toxin within the spinal cord of dogs. Ann. Surg 1938, 108, 941–957.
[11]  Martini, E.; Torda, C.; Zironi, A. The effect of tetanus toxin on the choline esterase activity of the muscles of rats. J. Physiol 1939, 96, 168–171.
[12]  Harvey, A.M. The peripheral action of tetanus toxin. J. Physiol 1939, 96, 348–365.
[13]  Manwaring, W.H. Types of tetanus toxin. Cal. West. Med 1943, 59, 306–307.
[14]  Ipsen, J. The effect of environmental temperature on the reaction of mice to tetanus toxin. J. Immunol 1951, 66, 687–694.
[15]  Wright, E.A. The effect of the injection of tetanus toxin into the central nervous system of rabbits. J. Immunol 1953, 71, 41–44.
[16]  Roaf, M.D.; Sherrington, C.S. Experiments in examination of the locked jaw induced by tetanus toxin. J. Physiol 1906, 34, 315–331.
[17]  Wassermann, A.; Takaki, T. über Tetanusantitoxische Eigenschaften des normalen Centralnervensystems. Berl. Klin. Wochenschr 1898, 35, 5–6.
[18]  Landsteiner, K.; Botteri, A. über Verbindungen von Tetanustoxin mit Lipoiden IV. Zbl. Bakt. Orig 1906, 42, 562.
[19]  Van Heyningen, W.E. The fixation of tetanus toxin by nervous tissue. J. Gen. Microbiol 1959, 20, 291–300.
[20]  Van Heyningen, W.E. Chemical assay of the tetanus toxin receptor in nervous tissue. J. Gen. Microbiol 1959, 20, 301–309.
[21]  Van Heyningen, W.E.; Miller, P.A. The fixation of tetanus toxin by ganglioside. J. Gen. Microbiol 1961, 24, 107–119.
[22]  Van Heyningen, W.E. Binding of ganglioside by the chains of tetanus toxin. FEBS Lett 1976, 68, 5–7.
[23]  Sugiyama, H. Clostridium botulinum neurotoxin. Microbiol. Rev 1980, 44, 419–448.
[24]  Johnson, E.A. Clostridial toxins as therapeutic agents: Benefits of nature’s most toxic proteins. Ann. Rev. Microbiol 1999, 53, 551–575.
[25]  Pellizari, R.; Rossetto, O.; Schiavo, G.; Montecucco, C. Tetanus and botulinum neurotoxins: Mechanism of action and therapeutic uses. Philos. Trans. R. Soc. Lond. B 1999, 354, 259–268.
[26]  Montal, M. Botulinum neurotoxin. Annu. Rev. Biochem 2010, 79, 591–617.
[27]  Habermann, E.; Dreyer, F. Clostridial neurotoxins: Handling and action at the cellular and molecular level. Curr. Top. Microbiol. Immunol 1986, 129, 93–179.
[28]  Chen, S.; Karalewitz, A.P.A.; Barbieri, J.T. Insights into the different catalytic activities of Clostridium neurotoxins. Biochemistry 2012, 51, 3941–3947.
[29]  Neubauer, V.; Helting, T.B. Structure of tetanus toxin: The arrangement of papain digestion products within the heavy chain-light chain framework of extracellular toxin. Biochim. Biophys. Acta 1981, 27, 141–148.
[30]  Deinhardt, K.; Berninghausen, O.; Willison, H.J.; Hopkins, C.R.; Schiavo, G. Tetanus toxin is internalized by a sequential clathrin-dependent mechanism initiated within lipid microdomains and independent of epsin (eosin?) 1. J. Cell Biol 2006, 174, 459–471.
[31]  Mochida, S. Protein-protein interactions in neurotransmitter release. Neurosci. Res 2000, 36, 175–182.
[32]  Humeau, Y.; Doussau, F.; Grant, N.J.; Poulain, B. How botulinum and tetanus neurotoxins block neurotransmitter release. Biochimie 2000, 82, 427–446.
[33]  Ungar, D.; Hughson, F.M. SNARE protein structure and function. Annu. Rev. Cell Dev. Biol 2003, 19, 493–517.
[34]  Boquet, P.; Duflot, E.; Hauttecoeur, B. Low pH induces a hydrophobic domain in the tetanus toxin molecule. Eur. J. Biochem 1984, 144, 339–344.
[35]  Simpson, L.L.; Hoch, D.H. Neuropharmacological characterization of fragment B from tetanus toxin. J. Pharmacol. Exp. Ther 1985, 232, 223–227.
[36]  Menestrina, G.; Forti, S.; Gambale, F. Interaction of tetanus toxin with lipid vesicles. Effects of pH, surface charge and transmembrane potential on the kinetics of channel formation. Biophys. J 1989, 55, 393–405.
[37]  Calappi, E.; Masserini, M.; Schiavo, G.; Montecucco, C.; Tettamanti, G. Lipid interaction of tetanus toxin. A calorimetric and fluorescence spectroscopy study. FEBS 1992, 309, 107–110.
[38]  Habermann, E.; Albus, U. Interaction between tetanus toxin and rabbit kidney: A comparison with rat brain preparations. J. Neurochem 1986, 46, 1219–1226.
[39]  Lazarovici, P.; Yanai, P.; Llavín, E. Molecular interactions between micellar polysialogangliosides and affinity-purified tetanotoxins in aqueous solution. J. Biol. Chem 1987, 262, 2645–2651.
[40]  Emsley, P.; Fotinou, C.; Black, I.; Fairweather, N.F.; Charles, I.G.; Watts, C.; Hewitt, E.; Isaacs, N.W. The structures of the HC fragment of tetanus toxin with carbohydrate subunit complexes provide insight into ganglioside binding. J. Biol. Chem 2000, 275, 8889–8894.
[41]  Sinha, K.; Box, M.; Lalli, G.; Schiavo, G.; Schneider, H.; Groves, M.; Siligardi, G.; Fairweather, N. Analysis of mutants of tetanus toxin HC fragment: Ganglioside binding, cell binding and retrograde axonal transport properties. Mol. Microbiol 2000, 37, 1041–1051.
[42]  Louch, H.A.; Buczko, E.S.; Woody, M.A.; Venable, R.M.; Vann, W.F. Identification of a binding site for ganglioside on the receptor binding domain of tetanus toxin. Biochemistry 2002, 41, 13644–13652.
[43]  Conway, P.M.C.; Whittal, R.M.; Baldwin, M.A.; Burlingame, A.L.; Balhorn, R. Electrospray mass spectrometry of NeuAc oligomers associated with the C fragment of the tetanus toxin. J. Am. Soc. Mass Spectrom 2006, 17, 967–976.
[44]  Siade, A.L.; Schoeniger, J.S.; Sasaki, D.Y.; Yip, C.M. In situ canning probe microscopy studies of tetanus toxin-membrane interacions. Biophys. J 2006, 91, 4565–4574.
[45]  Helting, T.B.; Zwisler, O.; Wiegandt, H. Structure of tetanus toxin. II. Toxin binding to ganglioside. J. Biol. Chem 1977, 252, 194–198.
[46]  Sutton, J.M.; Chow-Worn, O.; Spaven, L.; Silman, N.J.; Hallis, B.; Shone, C.C. Tyrosyne-1290 of tetanus neurotoxin plays a key role in its binding to gangliosides and functional binding to neurons. FEBS Lett 2001, 493, 45–49.
[47]  The 1.61 angstrom structure of the tetanus toxin ganglioside binding region: Solved by MAD and Mir phase combination. Available online: , accessed on 31 May 2012.
[48]  Rummel, A.; Bade, S.; Alves, J.; Bigalke, H.; Binz, T. Two carbohydrate binding sites in the HCC-domain of tetanus neurotoxin are required for toxicity. J. Mol. Biol 2003, 326, 835–847.
[49]  Jayaraman, S.; Eswaramoorthy, S.; Kumaran, D.; Swaminathan, S. Common binding site for disialyllactose and tri-peptide in C-fragment of tetanus neurotoxin. Proteins Struct. Funct. Bioinform 2005, 61, 288–295.
[50]  Cosman, M.; Lightstone, F.C.; Krishnan, V.V.; Zeller, L.; Prieto, M.C.; Roe, D.C.; Balhorn, R. Identification of novel small molecules that bind to two different sites on the surface of tetanus toxin C fragment. Chem. Res. Toxicol 2002, 15, 1218–1228.
[51]  Fotinou, C.; Emsley, P.; Black, I.; Ando, H.; Ishida, H.; Kiso, M.; Sinha, K.A.; Fairweather, N.F.; Isaacs, W. The crystal structure of the tetanus-toxin HC fragment complexed with a synthetic GT1b analogue suggests cross-linking between ganglioside receptors and the toxin. J. Biol. Chem 2001, 276, 32274–32281.
[52]  Jayaraman, S.; Swaramoorthy, S.; Kumaran, D.; Swaminathan, S. Common binging site for disialyllactose and tri-peptide in C-fragment of tetanus neurotoxin. Proteins 2005, 61, 288–295.
[53]  Chen, C.; Fu, Z.; Kim, J.-J.P.; Barbieri, J.T.; Baldwin, M.R. Gangliosides as high affinity receptors for tetanus neurotoxin. J. Biol. Chem 2009, 284, 26569–26577.
[54]  Herreros, J.; Lalli, G.; Montecucco, C.; Schiavo, G. Tetanus toxin fragment C binds to a protein present in neuronal cell lines and motorneurons. J. Neurochem 2000, 74, 1941–1950.
[55]  Herreros, J.; Ng, T.; Schiavo, G. Lipid rafts act as specialized domains for tetanus toxin binding and internalization into neurons. Mol. Biol. Cell 2001, 12, 2947–2960.
[56]  Deinhardt, K.; Salinas, K.; Verastigui, C.; Watson, R.; Worth, D.; Hanrahan, S.; Bucci, C.; Schiavo, G. Rab5 and Rab7 control endocytic sorting along the axonal retrograde transport pathway. Neuron 2006, 52, 293–305.
[57]  Evinger, C.; Erichsen, J.T. Transsynaptic retrograde transport of fragment C of tetanus toxin demonstrated by immunohistochemical localization. Brain Res 1986, 380, 383–388.
[58]  Manning, K.A.; Erichsen, J.T.; Evinger, C. Retrograde transneuronal transport properties of fragment C of tetanus toxin. Neuroscience 1990, 34, 251–263.
[59]  Fishman, P.S.; Carrigan, D.R. Retrograde transneuronal transfer of the fragment C of tetanus toxin. Brain Res 1987, 406, 275–279.
[60]  Coen, L.; Osta, R.; Maury, M.; Br?let, P. Construction of hybrid proteins that migrate retrogradely and transynaptically into the central nervous system. Proc. Natl. Acad. Sci. USA 1997, 94, 9400–9405.
[61]  Miana-Mena, F.J.; Mu?oz, M.J.; Ciriza, J.; Soria, J.; Br?let, P.; Zaragoza, P.; Osta, R. Fragment C tetanus toxin: A putative activity-dependent neuroanatomical tracer. Acta Neurobiol. Exp 2003, 63, 211–218.
[62]  Miana-Mena, F.J.; Mu?oz, M.J.; Roux, S.; Ciriza, J.; Zaragoza, P.; Br?let, P.; Osta, R. A non-viral vector for targeting gene therapy to motoneurons in the CNS. Neurodegener. Dis 2004, 1, 101–108.
[63]  Miana-Mena, F.J.; Roux, S.; Benichou, J.C.; Osta, R.; Br?let, P. Neuronal activity-dependent membrane traffic at the neuromuscular junction. Proc. Natl. Acad. Sci. USA 2002, 99, 3234–3239.
[64]  Barati, S.; Chegini, F.; Hurtado, P.; Rush, R.A. Hybrid tetanus toxin C fragment-diphtheria toxin translocation domain allows specific gene transfer into PC12 cells. Exp. Neurol 2002, 177, 75–87.
[65]  Oliveira, H.; Fernandez, R.; Pires, L.R.; Martins, M.C.L.; Sim?es, S.; Barbosa, M.A.; Pêgo, A.P. Targeted gene delivery into peripheral sensorial neurons mediated by self-assembled vectors composed of poly (ethylene imine) and tetanus toxin fragment C. J. Control Release 2010, 143, 350–358.
[66]  Larsen, K.E.; Benn, S.C.; Ay, I.; Chian, R.J.; Celia, S.A.; Remington, M.P.; Bejarano, M.; Liu, M.; Ross, J.; Carmillo, P.; et al. A glial cell line-derived neurotrophic factor (GDNF): Tetanus toxin fragment C protein conjugate improves delivery of GDNF to spinal cord motor neurons in mice. Brain Res 2006, 1120, 1–12.
[67]  Ciriza, J.; Moreno-Igoa, M.; Calvo, A.C.; Yagüe, G.; Palacio, J.; Miana-Mena, F.J.; Mu?oz, M.J.; Zaragoza, P.; Br?let, P.; Osta, R. A genetic fusion GDNF-C fragment of tetanus toxin prolongs survival in a symptomatic mouse ALS model. Restor. Neurol. Neurosci 2008, 26, 459–465.
[68]  Moreno-Igoa, M.; Calvo, A.C.; Ciriza, J.; Mu?oz, M.J.; Zaragoza, P.; Osta, R. Non-viral gene delivery of the GDNF, either alone or fused to the C-fragment of tetanus toxin protein, prolongs survival in a mouse ALS model. Restor. Neurol. Neurosci 2012, 30, 69–80.
[69]  Calvo, A.C.; Moreno-Igoa, M.; Mancuso, R.; Manzano, R.; Oliván, S.; Munoz, M.J.; Penas, C.; Zaragoza, P.; Navarro, X.; Osta, R. Lack of a synergistic effect of a non-viral ALS gene therapy based on BDNF and a TTC fusion molecule. Orphanet J. Rare Dis 2011, doi:10.1186/1750-1172-6-10.
[70]  Bordet, T.; Castelnau-Ptakhine, L.; Fauchereau, F.; Friocourt, G.; Kahn, A.; Haase, G. Neuronal targeting of cardiotrophin-1 by coupling with tetanus toxin C fragment. Mol. Cell. Neurosci 2001, 17, 842–854.
[71]  Carlton, E.; Teng, Q.; Federici, T.; Yang, J.; Riley, J.; Boulis, N.M. Fusion of the tetanus toxin C fragment binding domain and Bcl-XL for protection of peripheral-nerve neurons. Neurosurgery 2008, 63, 1175–1184.
[72]  Lalli, G.; Schiavo, G. Analysis of retrograde transport in motor neurons reveals common endocytic carriers for tetanus toxin and neurotrophin receptor p75NTR. J. Cell Biol 2002, 156, 233–239.
[73]  Ibánez, C.F.; Simi, A. p75 neurotrophin receptor signaling in nervous system injury and degeneration: Paradox and opportunity. Trends Neurosci 2012, doi:10.1016/j.tins.2012.03.007.
[74]  Roux, S.; Saint Cloment, C.; Curie, T.; Girard, E.; Mena, F.J.; Barbier, J.; Osta, R.; Molgó, J.; Br?let, P. Brain-derived neurotrophic factor facilitates in vivo internalization of tetanus neurotoxin C-terminal fragment fusion proteins in mature mouse motor nerve terminals. Eur. J. Neurosci 2006, 24, 1546–1554.
[75]  Skeldal, S.; Matusica, D.; Nykjaer, A.; Coulson, E.J. Proteolytic processing of the p75 neurotrophin receptor: A prerequisite for signalling? Neuronal life, growth and death signalling are crucially regulated by intra-membrane proteolysis and trafficking of p75(NTR). Bioessays 2011, 33, 614–625.
[76]  Skaper, S.D. The biology of neurotrophins, signalling pathways, and functional peptide mimetics of neurotrophins and their receptors. CNS Neurol. Disord. Drug Targets 2008, 7, 46–62.
[77]  Deinhardt, K.; Reversi, A.; Berninghausen, O.; Hopkins, C.R.; Schiavo, G. Neurotrophins redirect p75NTR from a clathrin-independent to a clathrin-dependent endocytic pathway coupled to axonal transport. Traffic 2007, 8, 1736–1749.
[78]  Wang, K.C.; Kim, J.A.; Sivasankaran, R.; Segal, R.; He, Z. p75 interacts with the Nogo receptor as a co-receptor for Nogo, MAG and OMgp. Nature 2002, 420, 74–78.
[79]  Twiss, J.L.; Chang, J.H.; Schanen, N.C. Pathophysiological mechanisms for actions of the neurotrophins. Brain Pathol 2006, 16, 320–332.
[80]  Aguilera, J.; Lopez, L.A.; Yavin, E. Tetanus toxin-induced protein kinase C activation and elevated serotonin levels in the perinatal rat brain. FEBS 1990, 263, 61–65.
[81]  Gil, C.; Ruiz-Meana, M.; álava, M.; Yavin, E.; Aguilera, J. Tetanus toxin enhances protein kinase C activity translocation and increases polyphosphoinositide hydrolysis in rat cerebral cortex preparations. J. Neurochem 1998, 70, 1636–1643.
[82]  Inserte, J.; Najib, A.; Pelliccioni, P.; Gil, C.; Aguilera, J. Inhibition by tetanus toxin of sodium-dependent, high-affinity [3H]5-hydroxitryptamine uptake in rat synaptosomes. Biochem. Pharmacol 1999, 57, 111–120.
[83]  Gil, C.; Chaib, I.; Pelliccioni, P.; Aguilera, J. Activation of signal transduction pathways involving TrkA, PLCγ-1, PKC isoforms and ERK-1/2 by tetanus toxin. FEBS Lett 2000, 481, 177–182.
[84]  Pelliccioni, P.; Gil, C.; Najib, A.; Sarri, E.; Picatoste, F.; Aguilera, J. Tetanus toxin modulates serotonin transport in rat-brain neuronal cultures. J. Mol. Neurosci 2001, 17, 303–310.
[85]  Gil, C.; Chaib-Oukadour, I.; Blasi, J.; Aguilera, J. HC fragment (C-terminal portion of the heavy chain) of tetanus toxin activates protein kinase C isoforms and phosphoproteins involved in signal transduction. Biochem. J 2001, 356, 97–103.
[86]  Gil, C.; Chaib-Oukadour, I.; Aguilera, J. C-terminal fragment of tetanus toxin heavy chain activates Akt and MEK/ERK signalling pathways in a Trk receptor-dependent manner in cultured cortical neurons. Biochem. J 2003, 373, 613–620.
[87]  Chaib-Oukadour, I.; Gil, C.; Aguilera, J. The C-terminal domain of heavy chain of tetanus toxin rescues cerebellar granule neurons from apoptotic death: Involvement of phosphatidylinositol 3-kinase and mitogen-activated protein kinase pathways. J. Neurochem 2004, 90, 1227–1236.
[88]  Mendieta, L.; Venegas, B.; Moreno, N.; Patricio, A.; Martínez, I.; Aguilera, J.; Limón, I.D. The carboxyl-terminal domain of the heavy chain of tetanus toxin prevents dopaminergic degeneration and improves motor behaviour in rats with striatal MPP+-lesions. Neurosci. Res 2009, 65, 98–106.
[89]  Chaib-Oukadour, I.; Gil, C.; Rodríguez-álvarez, J.; Ortega, A.; Aguilera, J. Tetanus toxin HC fragment reduces neuronal MPP+ toxicity. Mol. Cell. Neurosci 2009, 41, 297–303.
[90]  Thorne, R.G.; Frey, W.H. Delivery of neurotrophic factors to the central nervous system: Pharmacokinetic considerations. Clin. Pharmacokinet 2001, 40, 907–946.
[91]  Borasio, G.D.; Robberecht, W.; Leigh, P.N.; Emile, J.; Guiloff, R.J.; Jerusalem, F.; Silani, V.; Vos, P.E.; Wokke, J.H.; Dobbins, T. A placebo-controlled trial of insulin-like growth factor-I in amyotrophic lateral sclerosis. European ALS/IGF-I Study Group. Neurology 1998, 51, 583–586.
[92]  Check, E. Harmful potential of viral vectors fuels doubts over gene therapy. Nature 2003, 423, 573–574.
[93]  Moreno-Igoa, M.; Calvo, A.C.; Penas, C.; Manzano, R.; Oliván, S.; Mu?oz, M.J.; Mancuso, R.; Zaragoza, P.; Aguilera, J.; Navarro, X.; et al. Fragment C of tetanus toxin, more than a carrier. Novel perspectives in non-viral ALS gene therapy. J. Mol. Med 2010, 88, 297–308.


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