Fibroblast growth factor 2 applied to the optic nerve after axotomy increases Bcl-2 and decreases Bax in ganglion cells by activating the extracellular signal-regulated kinase signaling pathway Wilson Rı ´os-Mun ˜oz*, Ileana Soto*, Mildred V. Duprey-Dı ´az, Jonathan Blagburn and Rosa E. Blanco Institute of Neurobiology, Departments of Anatomy and Physiology, University of Puerto Rico Medical Sciences Campus, Old San Juan, Puerto Rico Abstract 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 pro- teins, 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 treat- ment greatly decreased Bax levels, an effect that was inhibited by U0126. Axotomy induced the cleavage of ca- spase-3; FGF-2 treatment blocked this effect in an ERK- dependent manner. Finally, intraocular application of the MEK inhibitor caused a large reduction in the survival-pro- moting 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 axo- tomized RGCs not only by increasing amounts of anti- apoptotic proteins, but also by a striking reduction in the levels of apoptotic effectors themselves. Keywords: apoptosis, axotomy, frog, growth factor, retinal ganglion cells, survival. J. Neurochem. (2005) 93, 1422–1433. The CNS of mammals has little or no regenerative capacity compared with that of lower vertebrates. One of the reasons for this is that injured neurons die; for example, adult mammals lose more than 90% of retinal ganglion cells (RGCs) after axotomy (Watanabe et al. 1997; Chen and Weber 2001). Lower vertebrates, such as amphibians and fish, are better able to regenerate axons after axotomy or other trauma to the CNS (Scalia et al. 1985; Blanco et al. 2000; Dawson and Meyer 2001). In the frog visual system, approximately 40–70% of RGCs die after axotomy of the optic nerve, but the surviving cells regenerate and recovery of vision can occur (Scalia et al. 1985; Singman and Scalia 1991; Dunlop et al. 1992). In our laboratory we have shown that the application of basic fibroblast growth factor (FGF-2) to the frog optic nerve after axotomy enhances the survival of a large proportion of the RGCs that would otherwise die (Blanco et al. 2000). Fibroblast growth factors (FGFs) are known to play a role in cell growth, differentiation, migration, morphogenesis and survival during the development of the CNS. The FGFs make up a large family of at least 23 polypeptides, 10 of which have been identified in the brain, and exert their actions by activating cell surface receptors, FGFR1–5 (Goldfarb 1996, 2001; Sleeman et al. 2001; Reuss et al. 2003). FGFR1–4 are members of the tyrosine kinase family and trigger signal Received December 22, 2004; revised manuscript received February 3, 2005; accepted February 3, 2005. Address correspondence and reprint requests to Dr R. E. Blanco, Insti- tute of Neurobiology, 201 Boulevard del Valle, San Juan, Puerto Rico 00901. E-mail: rblanco@neurobio.upr.clu.edu *These authors contributed equally to this work. Abbreviations used: DMSO, dimethyl sulfoxide; ERK, extracellular signal-regulated kinase; FGF, fibroblast growth factor; FGF-2, basic fibroblast growth factor; GAPDH, glyceraldehyde-3-phosphate dehy- drogenase; GCL, ganglion cell layer; INL, inner nuclear layer; IPL, inner plexiform layer; MEK, ERK kinase; PBS, phosphate-buffered saline; p-ERK, phosphorylated ERK; RGC, retinal ganglion cell; TDA, tetra- methylrhodamine dextran amine. Journal of Neurochemistry , 2005, 93, 1422–1433 doi:10.1111/j.1471-4159.2005.03129.x 1422 Ó 2005 International Society for Neurochemistry, J. Neurochem. (2005) 93, 1422–1433