We have shown that application of basic fibroblast growth factor (FGF-2) to axotomized optic nerve promotes the survival of frog retinal ganglion cells (RGCs). In the present study we used western blotting and immunocytochemistry to investigate the effects of this FGF-2 treatment upon the activation of the extracellular signal-regulated kinase (ERK) pathway, the amounts and distribution of Bcl-2 family proteins, and the activation of caspase-3. Axotomy alone temporarily increased ERK activation; FGF-2 treatment to the nerve prolonged this activation. This effect was blocked by U0126, a selective ERK kinase (MEK) inhibitor. Axotomy caused a decrease in Bcl-2 and a small increase in Bcl-x L . FGF-2 treatment caused an ERK-dependent increase in Bcl-2 and an ERK-independent increase in Bcl-x L . The pro-apoptotic Bax was increased by axotomy; FGF-2 treatment greatly decreased Bax levels, an effect that was inhibited by U0126. Axotomy induced the cleavage of caspase-3; FGF-2 treatment blocked this effect in an ERKdependent manner. Finally, intraocular application of the MEK inhibitor caused a large reduction in the survival-promoting effect that FGF-2 application to the nerve stump had on RGCs. Our results suggest that FGF-2 acts, at least in part, via the ERK pathway to prevent apoptosis of axotomized RGCs not only by increasing amounts of antiapoptotic proteins, but also by a striking reduction in the levels of apoptotic effectors themselves.

In this study we used immunocytochemistry to investigate the distribution of brain-derived neurotrophic factor (BDNF) and its receptor tyrosine kinase (trkB) in retina and optic tectum of the frog Rana pipiens during regeneration after axotomy. We also measured changes in BDNF mRNA in retina and tectum. Retrograde labeling was used to identify retinal ganglion cells (RGCs) prior to quantification of the BDNF immunoreactivity. In control animals, BDNF was found in the majority of RGCs and displaced amacrine cells and in some cells in the inner nuclear layer (INL). After axotomy, BDNF immunoreactivity was reduced in RGCs but increased in the INL. BDNF mRNA levels in the retina remained high before and after axotomy. Three months after axotomy, after reconnection to the target, the staining intensity of many of the surviving RGCs had partially recovered. In the control tectum, BDNF staining was present in ependymoglial cells and in neurons throughout layers 4, 6, 8, and 9. After axotomy, BDNF staining in tectal neurons became more intense, even though mRNA synthesis was transiently down-regulated. In control retinas, trkB receptor immunostaining was present in most RGCs; no significant changes were observed after axotomy. In control tectum, trkB was detected only in ependymoglial cells. After axotomy, many neuronal cell bodies were transiently labeled. Our data are consistent with the hypothesis that a considerable fraction of the BDNF normally present in RGCs is acquired from their targets in the tectum. However, there are also intraretinal sources of BDNF that could contribute to the survival of RGCs.

Application of basic fibroblast growth factor (FGF-2) to the optic nerve after axotomy promotes the survival of retinal ganglion cells (RGCs) in the frog, Rana pipiens, and results in a rapid up-regulation of BDNF and TrkB synthesis by the RGCs. Here we investigate whether this upregulation is maintained in the long term, and whether it is required for FGF-2's survival effect. At 6 weeks after axotomy and FGF-2 treatment we found more RGCs immunopositive for BDNF protein and higher intensity of BDNF and TrkB immunostaining, accompanied by increases in BDNF and TrkB mRNA in RGCs. Application of fluorescently-labeled siRNA targeted against BDNF to the cut RGC axons showed that it was transported to the cell bodies. Axonal siRNA treatment eliminated the increases in BDNF immunostaining and mRNA that were induced by FGF-2, and had no effect on TrkB mRNA. This reduction in BDNF synthesis by siRNA greatly reduced the long term survival effect of FGF-2 on RGCs. This, taken together with previous results, suggests that, while FGF-2 may initially activate survival pathways via ERK signaling, its main long-term survival effects are mediated via its upregulation of BDNF synthesis by the RGCs.

Retinoic acid (RA) is important during development, in neuronal plasticity, and also in peripheral nervous system regeneration. Here we use the frog visual system as a model to investigate the changes in RA signaling that take place after axonal injury to the central nervous system. Immunocytochemistry was used to localize different components of RA signaling within sections of the retina and optic tectum, namely, the synthetic enzyme retinaldehyde dehydrogenase (RALDH), the RA binding proteins CRABPI and II, the retinoic acid receptors RARα, β and (, and finally the catabolic enzyme CYP26A1. The levels of these proteins were quantified in extracts of retina and tectum using Western blotting. Animals were studied at 1 week, 3 weeks and 6 weeks after optic nerve transection. At the latter time point the RGC axons were re-entering the optic tectum. All the components of RA signaling were present at low to moderate levels in retinas and tecta of control, unoperated animals. In retina, soon after optic nerve injury there was a large increase in RALDH, some increase in the CRABPs, and a large increase in RGC RARβ and ( expression. These increases continued as the RGC axons were regenerating, with the addition of later RARα expression at 6 weeks. At no stage did CYP26A1 expression significantly change. In the tectum the levels of RALDH increased after axotomy and during regrowth of axons (3 weeks), then decreased at 6 weeks, at which time the levels of CYP26A1 increased. Axotomy did not cause an immediate increase in tectal RAR levels but RARα and RARβ increased after 3 weeks and RAR( only after 6 weeks. These results are consistent with RA signaling playing an important role in the survival and regeneration of frog RGCs.

After lesions to the mammalian optic nerve, the great majority of retinal ganglion cells (RGCs) die before their axons have even had a chance to regenerate. Frog RGCs, on the other hand, suffer only an approximately 50% cell loss, and we have previously investigated the mechanisms by which the application of growth factors can increase their survival rate. Retinoic acid (RA) is a vitamin A-derived lipophilic molecule that plays major roles during development of the nervous system. The RA signaling pathway is also present in parts of the adult nervous system, and components of it are upregulated after injury in peripheral nerves but not in the CNS. Here we investigate whether RA signaling affects long-term RGC survival at 6 weeks after axotomy. Intraocular injection of all-trans retinoic acid (ATRA), the retinoic acid receptor (RAR) type-α agonist AM80, the RARβ agonist CD2314, or the RARγ agonist CD1530, returned axotomized RGC numbers to almost normal levels. On the other hand, inhibition of RA synthesis with disulfiram, or of RAR receptors with the pan-RAR antagonist Ro-41-5253, or the RARβ antagonist LE135E, greatly reduced the survival of the axotomized neurons. Axotomy elicited a strong activation of the MAPK, STAT3 and AKT pathways; this activation was prevented by disulfiram or by RAR antagonists. Finally, addition of exogenous ATRA stimulated the activation of the first two of these pathways. Future experiments will investigate whether these strong survival-promoting effects of RA are mediated via the upregulation of neurotrophins.