Journal of Neuroscience, Vol 15, 5514-5525, Copyright © 1995 by Society for Neuroscience
Two factors secreted by the goldfish optic nerve induce retinal ganglion cells to regenerate axons in culture
JM Schwalb, NM Boulis, MF Gu, J Winickoff, PS Jackson, N Irwin and LI Benowitz
Department of Neurosurgery, Children's Hospital, Boston, Massachusetts 02115, USA.
Unlike mammals, lower vertebrates can regenerate an injured optic nerve and
other pathways of the CNS throughout life. We report here that in
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 stable. A second
factor, AF-2, is a polypeptide of ca 12 kDa. In the absence of these
factors, dissociated retinal cells remained viable in serum-free, defined
media for at least a week but showed little outgrowth, 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 culture to extend processes > 75
microns in length by 6 d; AF-2 had a lesser but 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 for 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 selective targets of the factors. The effects of
AF-1 or AF-2 were not secondary to enhanced viability, since neither
overall cell survival nor 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,
retinoic acid, acidic or basic fibroblast growth factors. The concentration
of AF-1 is considerably higher in CM than in optic nerve homogenates,
suggesting that it is actively secreted; AF-2 has a similar concentration
intra- and extracellularly. Insofar as AF-1 and AF-2 derive from cells of
the optic nerve and act upon retinal ganglion cells, they are likely to be
important in inducing optic nerve regeneration in vivo.
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