Brain-derived neurotrophic factor facilitates in vivo internalization of tetanus neurotoxin C-terminal fragment fusion proteins in mature mouse motor nerve terminals.
In a previous study it was reported that fusion proteins composed of the atoxic C-terminal fragment of tetanus toxin (TTC) and green fluorescent protein or beta-galactosidase (GFP-TTC and beta-gal-TTC, respectively) rapidly cluster at motor nerve terminals of the mouse neuromuscular junction (NMJ). Because this traffic involves presynaptic activity, probably via the secretion of active molecules, we examined whether it is affected by brain-derived neurotrophic factor (BDNF). Quantitative confocal microscopy and a fluorimetric assay for beta-gal activity revealed that co-injecting BDNF and the fusion proteins significantly increased the kinetics and amount of the proteins' localization at the NMJ and their internalization by motor nerve terminals. The observed increases were independent of synaptic vesicle recycling because BDNF did not affect spontaneous quantal acetylcholine release. In addition, injecting anti-BDNF antibody shortly before injecting GFP-TTC, and before co-injecting GFP-TTC and BDNF, significantly reduced the fusion protein's localization at the NMJ. Co-injecting GFP-TTC with neurotrophin-4 (NT-4) or glial-derived neurotrophic factor (GDNF), but not with nerve growth factor, neurotrophin-3 or ciliary neurotrophic factor, also significantly increased the fusion protein's localization at the NMJ. Thus, TTC probes may use for their neuronal internalization endocytic pathways normally stimulated by BDNF, NT-4 and GDNF binding. Different tyrosine kinase receptors with similar signalling pathways are activated by BDNF/NT-4 and GDNF binding. Thus, activated components of these signalling pathways may be involved in the TTC probes' internalization, perhaps by facilitating localization of receptors of TTC in specific membrane microdomains or by recruiting various factors needed for internalization of TTC.
Roux
Sylvie
S
CNRS, Institut de Neurobiologie Alfred Fessard, FRC2118, Laboratoire de Neurobiologie Cellulaire et Moléculaire, UPR9040, 1 Avenue de la Terrasse, 91198 Gif sur Yvette, France.
Saint Cloment
Cécile
C
Curie
Thomas
T
Girard
Emmanuelle
E
Miana Mena
Francisco-Javier
FJ
Barbier
Julien
J
Osta
Rosario
R
Molgó
Jordi
J
Brûlet
Philippe
P
D000818:Animals; D000906:Antibodies / Q000494:pharmacology; D001370:Axonal Transport / Q000187:drug effects* / Q000502:physiology; D019208:Brain-Derived Neurotrophic Factor / Q000276:immunology / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D005260:Female; D005470:Fluorometry / Q000379:methods; D049452:Green Fluorescent Proteins / Q000378:metabolism; D051379:Mice; D018613:Microscopy, Confocal / Q000379:methods; D009046:Motor Neurons / Q000187:drug effects*; D016900:Neurofilament Proteins / Q000378:metabolism; D009469:Neuromuscular Junction / Q000166:cytology*; D010446:Peptide Fragments / Q000378:metabolism*; D021381:Protein Transport / Q000187:drug effects; D020813:Receptor, trkB / Q000378:metabolism; D012584:Sciatic Nerve / Q000187:drug effects / Q000502:physiology; D013744:Tetanus Toxin / Q000378:metabolism*; D013997:Time Factors; D001616:beta-Galactosidase / Q000378:metabolism
D000906:Antibodies; D019208:Brain-Derived Neurotrophic Factor; D016900:Neurofilament Proteins; D010446:Peptide Fragments; D013744:Tetanus Toxin; C040635:tetanus toxin fragment C; C051774:neurofilament protein H; D049452:Green Fluorescent Proteins; D020813:Receptor, trkB; D001616:beta-Galactosidase