Characterization of New Cell Permeable C3-like Proteins That Inactivate Rho and Stimulate Neurite Outgrowth on Inhibitory Substrates

Abstract

The activation state of Rho is an important determinant of axon growth and regeneration in neurons. Axons can extend neurites on growth inhibitory substrates when Rho is inactivated by C3-ADP-ribosyltransferase (C3). We found by Rho-GTP pull-down assay that inhibitory substrates activate Rho. To inactivate Rho, scrapeloading of C3 was necessary because it does not freely enter cells. To overcome the poor permeability of C3, we made and characterized five new recombinant C3-like chimeric proteins designed to cross the cell membrane by receptor-independent mechanisms. These proteins were constructed by the addition of short transport peptides to the carboxyl-terminal of C3 and tested using a bioassay measuring neurite outgrowth of PC-12 cells plated on growth inhibitory substrates. All five constructs stimulated neurite outgrowth but with different dose-response profiles. Biochemical properties of the chimeric proteins were examined using C3-05, the most effective construct tested. Gel shift assays showed that C3-05 retained the ability to ADP-ribosylate Rho. Western blots and immunocytochemistry were used to verify the presence of C3 inside treated cells. C3-05 was also effective at promoting neurite outgrowth in primary neuronal cultures, as well as causing the disassembly of actin stress fibers and focal adhesions complexes in fibroblasts. These studies demonstrate that the new C3-like proteins are effective in delivering biologically active C3 into different cell types, thereby, inactivating Rho.

Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS

“…Most studies have focused on two situations in which this occurs -neonatal spinal neurons, which often regenerate to produce complete functional recovery 91,92 , and regeneration of the results in regeneration 62 . It remains to be seen whether the effects of the new generation of C3 molecules, which are more membrane-permeable, can be similarly confined and effective 72 .…”

Rho Kinase Inhibition Enhances Axonal Regeneration in the Injured CNS

“…The relative contribution of the neuroprotective effect of C3 and of long-distance axonal regeneration to functional recovery is unclear. Use of cell-permeable C3-like proteins (Winton et al, 2002) and a slow release protocol in vivo would help to define the contribution of these cellular responses.…”

C3 exoenzymes, novel insights into structure and action of Rho-ADP-ribosylating toxins

“…Several studies have shown that the Cterminal fusion of short transport peptide sequences enhances the cellular uptake of C3 (Sauzeau et al 2001;Park et al 2003;Winton et al 2002;Sahai and Olson 2006). In many studies, the C3 gene was introduced into eukaryotic target cells by transient and stable transfection using plasmids or by viral infection (Fujisawa et al 1998;Hill et al 1995;Caron and Hall 1998;Henning et al 1997;Meacci et al 1999;Genot et al 1996).…”