2 FACTORS SECRETED BY THE GOLDFISH OPTIC-NERVE INDUCE RETINAL GANGLION-CELLS TO REGENERATE AXONS IN CULTURE

Unlike mammals, lower vertebrates can regenerate an injured optic nerv e and other pathways of the CNS throughout life. We report here that i n dissociated cell culture, goldfish retinal ganglion cells regenerate their axons in response to two factors derived from the sheath cells of the optic nerve. Axogenesis factor 1 (AF-1) is a small peptide (700 -900 Da) that is inactivated by treatment with proteinase K but heat s table. A second factor, AF-2, is a polypeptide of ca 12 kDa. In the ab sence of these factors, dissociated retinal cells remained viable in s erum-free, defined media for at least a week but showed little outgrow th, as visualized using the vital dye 5,6-carboxyfluorescein diacetate (5,6-CFDA). The addition of AF-1 induced up to 25% of cells in cultur e to extend processes > 75 mu m in length by 6 d; AF-2 had a lesser bu t highly significant effect. To verify that neurite outgrowth was from retinal ganglion cells per se, we applied the lipophilic dye 4-Di-10- ASP to the optic tectum and allowed it to diffuse up the optic nerve f or several days before culturing the retina. A far greater percentage of cells containing the dye showed axonal outgrowth than was observed from the overall cell population, indicating that ganglion cells are s elective targets of the factors. The effects of AF-1 or AF-2 were not secondary to enhanced viability, since neither overall cell survival n or the number of retinal ganglion cells remaining in culture after 6 d was affected by the presence of the factors. The activity of AF-1 and AF-2 was not mimicked by several defined factors tested over a broad concentration range, for example, NGF, BDNF, NT-3, CNTF, taurine, reti noic acid, acidic or basic fibroblast growth factors. The concentratio n of AF-1 is considerably higher in CM than in optic nerve homogenates , suggesting that it is actively secreted; AF-2 has a similar concentr ation intra- and extracellularly. Insofar as AF-1 and AF-2 derive from cells of the optic nerve and act upon retinal ganglion cells, they ar e likely to be important in inducing optic nerve regeneration in vivo.