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P.D. Koeberle, M. Baehr; Intracellular Pathways Mediating the Neuroprotective Effects of GDNF and Neurturin in Axotomized Retinal Ganglion Cells . Invest. Ophthalmol. Vis. Sci. 2003;44(13):5220.
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Purpose: We have previously shown that GDNF and Neurturin (NTN) increase the survival of axotomized retinal ganglion cells (RGCs). We have also shown that GDNF increases the levels of the glial glutamate transporter GLAST-1, a possible neuroprotective mechanism. In the present study, we determined the intracellular signaling pathways that are required for GDNF and NTN neuroprotection in vivo, and the pathways that modulate GLAST-1 levels in vitro. Methods: Optic nerve transections were performed in adult rats. RGCs were retrogradely labeled with fluorogold. Animals received intraocular injections of a control solution (vehicle), GDNF (0.5 µg), NTN (0.5 µg), or GDNF or NTN in combination with the MAPK pathway inhibitor PD98059, or the PI3K pathway inhibitor LY294002, or the Src kinase inhibitor PP2. The effectiveness of inhibitors was assessed by Western Immunoblotting directed against phosphorylated isoforms of MAPK, Akt (downstream of PI3K), and Src. At 14 days postaxotomy, RGC densities were determined from flatmounted retinas. The effects of GDNF and NTN on GLAST-1 expression were determined in the rMC-1 retinal Müller cell line. rMC-1 cells were treated with GDNF or NTN (100 ng/ml), or GDNF or NTN in combination with PD98059, LY294002, or PP2. GLAST-1 and glutamine synthetase levels were measured by Western Immunoblotting at 12, 24, and 48 hours after treatment. Results: RGC densities (cells/mm2±SEM) in GDNF (1038±73) and NTN (987±74) treated retinas were significantly higher than control treated retinas (351±28). PI3K inhibition (500±42) abolished the neuroprotective effects of GDNF, while MAPK inhibition (872±61) or Src inhibition (672±35) produced less pronounced decreases in GDNF mediated protection. NTN dependent RGC survival was also attenuated by PI3K inhibition (507±43), however neither MAPK inhibition nor Src inhibition had any significant effect on RGC survival after NTN delivery. Both GDNF and NTN increased GLAST-1 expression in rMC-1 cells, without any effect on the levels of glutamine synthetase. PI3K or Src inhibition abolished the upregulation of GLAST-1 by GDNF. NTN mediated increases in GLAST-1 were attenuated only by coadministration with the PI3K inhibitor. Conclusions: Our results indicate that the survival promoting effects of GDNF are dependent on multiple intracellular signaling pathways including the MAPK, PI3K, and Src cascades. Conversely, Neurturin mediated survival appears to be exclusively dependent on PI3K activity. These results provide a molecular basis for independent neuroprotective signal transduction by GDNF and Neurturin.
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