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Apoptotic cell death of photoreceptors is the final event leading to blindness in the heterogeneous group of inherited retinal degenerations. GDNF (glial cell-line-derived neurotrophic factor) was found to rescue photoreceptor function and survival very effectively in an animal model of retinal degeneration (M. Frasson, S. Picaud, T. Leveillard, M. Simonutti, S. Mohand-Said, H. Dreyfus, D. Hicks, and J. Sahel, Investig. Ophthalmol. Vis. Sci. 40:2724-2734, 1999). However, the cellular mechanism of GDNF action remained unresolved. We show here that in porcine retina, GDNF receptors GFRalpha-1 and RET are expressed on retinal Mueller glial cells (RMG) but not on photoreceptors. Additionally, RMG express the receptors for the GDNF family members artemin and neurturin (GFRalpha-2 and GFRalpha-3). We further investigated GDNF-, artemin-, and neurturin-induced signaling in isolated primary RMG and demonstrate three intracellular cascades, which are activated in vitro: MEK/ERK, stress-activated protein kinase (SAPK), and PKB/AKT pathways with different kinetics in dependence on stimulating GFL. We correlate the findings to intact porcine retina, where GDNF induces phosphorylation of ERK in the perinuclear region of RMG located in the inner nuclear layer. GDNF signaling resulted in transcriptional upregulation of FGF-2, which in turn was found to support photoreceptor survival in an in vitro assay. We provide here a detailed model of GDNF-induced signaling in mammalian retina and propose that the GDNF-induced rescue effect on mutated photoreceptors is an indirect effect mediated by retinal Mueller glial cells.

Purpose: To map the expression and distribution of FGFR and potential FGFR-related signaling molecules within rat and human retina., Methods: Sections of postnatal 5 day old and adult rat, and aged human retina, and cell cultures prepared from selected cell populations of young rat retina, were immunolabeled with specific antisera to FGFR (FGFR-1, -2, -3, and -4) or candidate signaling molecules [phospholipase Cg1 (PLCg1), son of sevenless 1 and 2 (SOS1, SOS2), extracellular signal-regulated kinase 1 and 2 (ERK1/2), protein tyrosine phosphatase (SH-PTP2) and SH2-containing protein (Shc)], and with multiple retinal cell-type specific antibodies. Controls were conducted using primary antisera pre-adsorbed with the corresponding immunizing peptide., Results: All FGFR antisera showed strong labeling of inner retina [inner nuclear layer, inner plexiform layer and ganglion cell layer (INL, IPL and GCL respectively)] in rat and human retina, although there were distinct differences in individual patterns. FGFR-3 was particularly intense in ganglion cell bodies and dendrites, and was absent from photoreceptors and bipolar cells in vitro. FGFR-1 and FGFR-4 also labeled the outer nuclear layer (ONL), more intensely in adult than in young tissue, and FGFR-4 was especially prominent within inner segments. FGFR-2 and -3 were only weakly expressed in the ONL, but FGFR-2 showed specific labeling of cone outer segments in human retina. Candidate FGFR-signaling molecules also showed generally higher expression in the inner than outer retina in the different samples. Shc immunolabeling was apparent in the GCL and nascent photoreceptor outer segments in young and adult retina. SOS1 expression was much more intense than SOS2 in the ONL, although the latter showed selective intense staining of a sub-population in the INL and GCL. These ex vivo data were confirmed in cultures prepared from young rat retina. Pure photoreceptor cultures exhibited strong expression of FGFR-1 and -4, and faint expression of FGFR-2 and -3. In mixed inner retinal cultures, anti-FGFR-1 labeled neurons and Müller glia with equal intensity, while the other FGFR antisera showed preferential staining of neurons. FGFR-3 was strongly expressed by ganglion and amacrine cells but not by other types. Signaling molecules showed widespread expression, but of variable intensity, in all cells. All control experiments using corresponding peptide pre-adsorption led to complete removal of immunostaining., Conclusions: Rat and human retinal cells showed a largely similar, widespread expression of multiple FGFR and candidate FGFR-related signaling molecules. Distinct differences in development, species, cell- and sub-cell type distribution were apparent, suggesting that specific FGFR/FGF ligands and transduction pathways may operate in different cells.