A 29-amino acid fragment of Clostridium botulinum C3 protein enhances neuronal outgrowth, connectivity, and reinnervation.
Small GTPases of the Rho family play versatile roles in the formation and development of axons and dendrites, effects often studied by the Rho-inactivating C3 transferase (C3bot) from Clostridium botulinum. Recently, we reported that transferase-deficient C3bot also exerted axonotrophic activity. Using overlapping peptides from the C3bot sequence, we identified a small peptide of 29 amino acids (covering residues 154-182) from the C-terminal region of C3bot that promotes both axonal and dendritic growth, as well as branching of hippocampal neurons, at submicromolar concentrations. Several C3bot constructs, including the short peptide, enhanced the number of axonal segments from mid- to higher-order segments. C3bot(154-182) also increased the number of synaptophysin-expressing terminals, up-regulated various synaptic proteins, and functionally increased the glutamate uptake. Staining against the vesicular glutamate and GABA transporters further revealed that the effect was attributable to a higher number of glutamatergic and GABAergic inputs on proximal dendrites of enhanced green fluorescent protein (EGFP)-transfected neurons. Using organotypical slice cultures, we also detected trophic effects of C3bot(154-182) on length and density of outgrowing fibers from the entorhinal cortex that were comparable to the effects elicited by full-length C3bot. In addition, an enhanced reinnervation was observed in a hippocampal-entorhinal lesion model. In summary, the neurotrophic effect of C3bot is executed by a C-terminal peptide fragment covering aa 154-182 of C3; it triggers dendritic and axonal growth and branching as well as increased synaptic connectivity. In contrast to full-length C3, this C3 peptide selectively acts on neurons but not on glial cells.
Höltje
Markus
M
Centrum für Anatomie, AG Funktionelle Zellbiologie, Charité-Universitätsmedizin Berlin, Berlin, Germany. markus.hoeltje@charite.de
Djalali
Susann
S
Hofmann
Fred
F
Münster-Wandowski
Agnieszka
A
Hendrix
Sven
S
Boato
Francesco
F
Dreger
Stefanie C
SC
Grosse
Gisela
G
Henneberger
Christian
C
Grantyn
Rosemarie
R
Just
Ingo
I
Ahnert-Hilger
Gudrun
G
D036002:ADP Ribose Transferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D000596:Amino Acids / Q000737:chemistry / Q000494:pharmacology*; D000818:Animals; D001369:Axons / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D015415:Biomarkers / Q000378:metabolism; D001905:Botulinum Toxins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D002478:Cells, Cultured; D003014:Clostridium botulinum / Q000235:genetics / Q000378:metabolism; D018920:Coculture Techniques; D003712:Dendrites / Q000187:drug effects / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004622:Embryo, Mammalian; D005982:Glutathione Transferase / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006624:Hippocampus / Q000166:cytology / Q000196:embryology; D007150:Immunohistochemistry; D051379:Mice; D008815:Mice, Inbred Strains; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D008970:Molecular Weight; D016900:Neurofilament Proteins / Q000737:chemistry / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D013997:Time Factors
D000596:Amino Acids; D015415:Biomarkers; D008869:Microtubule-Associated Proteins; D016900:Neurofilament Proteins; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins; D036002:ADP Ribose Transferases; C063007:exoenzyme C3, Clostridium botulinum; D005982:Glutathione Transferase; D001905:Botulinum Toxins