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A 29-amino acid fragment of Clostridium botulinum C3 protein enhances neuronal outgrowth, connectivity, and reinnervation

Markus Höltje^*,1, Susann Djalali^*, Fred Hofmann, Agnieszka Münster-Wandowski^*, Sven Hendrix^,||, Francesco Boato, Stefanie C. Dreger, Gisela Große^*, Christian Henneberger, Rosemarie Grantyn, Ingo Just and Gudrun Ahnert-Hilger^*

^* Centrum für Anatomie, AG Funktionelle Zellbiologie,

Centrum für Anatomie, Institut für Zell- und Neurobiologie, and

Johannes Müller Institut für Neurophysiologie, AG Entwicklungsphysiologie, Charité-Universitätsmedizin Berlin, Berlin, Germany;

Institut für Toxikologie der Medizinischen Hochschule Hannover, Hannover, Germany; and

^|| Department of Morphology and Biomedical Research Institute (BIOMED), Hasselt University, Hasselt, Belgium

¹Correspondence: Centrum für Anatomie, Institut für Integrative Neuroanatomie, Charité-Universitätsmedizin Berlin, Philippstr. 12, D-10115 Berlin, Germany. E-mail: markus.hoeltje{at}charite.de

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, M., Djalali, S., Hofmann, F., Münster-Wandowski, A., Hendrix, S., Boato, F., Dreger, S. C., Große, G., Henneberger, C., Grantyn, R., Just, I., Ahnert-Hilger, G. A 29-amino acid fragment of Clostridium botulinum C3 protein enhances neuronal outgrowth, connectivity, and reinnervation.

Key Words: regeneration • hippocampus • axon