HETEROTRIMERIC G-PROTEIN ACTIVATION RAPIDLY INHIBITS OUTGROWTH OF OPTIC AXONS FROM ADULT AND EMBRYONIC MOUSE, AND GOLDFISH RETINAL EXPLANTS

Axons in the adult mammalian CNS normally do not regenerate following axotomy even though they retain the capacity for growth under certain experimental conditions. Although this implies that the regeneration o f adult axons is under regulative control, very little is known about the signaling pathways responsible for this regulation. This study exa mines the possibility that a G protein signaling system exists in adul t mouse optic fibers and that it functions to regulate axonal outgrowt h. To induce the growth of optic fibers, retinas from adult mouse were placed in organotypic culture under serum free conditions and allowed to regenerate onto a laminin substrate. Heterotrimeric G proteins wer e stimulated by adding mastoparan (MST) to the medium while monitoring growing fibers with time lapse microscopy. Mastoparan treatment produ ced rapid growth cone collapse and axonal retraction which persisted w hile MST was present. Prior addition of pertussis toxin (PTX), which i rreversibly inactivates the G proteins, G(o) and G(i), completely bloc ked the effect of MST, confirming that MST was acting through the PTX sensitive G proteins. Selective activation of G proteins in the growth cone by local application of MST with a micropipet was equally effect ive. For comparison, equivalent experiments were performed on embryoni c day 15 retinal explants and on retinal explants from adult goldfish, which normally regenerate in vivo. MST similarly inhibited these axon s and this effect was blocked by PTX. However, embryonic fibers were l ess reliably affected compared to goldfish or adult mouse, suggesting a developmentally regulated sensitivity. The presence of G-proteins in the mouse axons was further tested immunohistochemically using antibo dies against G(o)/G(i). Positive staining was detected in the growth c ones and shaft of adult and embryonic mouse optic fibers. These findin gs demonstrate that G protein activation inhibits axonal outgrowth and suggest that there may be a G protein signaling pathway that normally regulates this outgrowth. However, since this pathway appears to exis t in both axons that can regenerate and those that normally do not, th e presence of PTX-sensitive G proteins alone cannot account for regene rative failure. Regenerative failure may instead be explained as the s elective or increased activation of this pathway in the adult mammalia n CNS.