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Andreas Ohlmann, Stephanie Leopold, Roswitha Seitz, Ernst Tamm; Norrin mediates neuroprotective effects on retinal ganglion cells via the induction of leukemia inhibitory factor. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3257. doi: https://doi.org/.
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To investigate if and how leukemia inhibitory factor (LIF) is involved in the neuroprotective effects of Norrin on retinal ganglion cell (RGC) survival following excitotoxic damage. Norrin is a secreted protein that activates the classical Wnt/β-catenin pathway via specific binding to Frizzled-4. In albino mice, Norrin protects RGC from excitotoxic damage and increases the retinal expression LIF and endothelin-2 (EDN2), as well as that of neuroprotective factors such as fibroblast growth factor-2 (FGF2) and ciliary neurotrophic factor (CNTF).
Recombinant human Norrin was isolated and purified from conditioned cell culture medium of HEK 293-EBNA cells. To induce excitotoxic RGC death, 3 µl NMDA [10 mM] were injected into the vitreous body of both hetero- and homozygous LIF-deficient mice in a C57/Black6 genetic background. The fellow eye received 3 µl of combined NMDA [10 mM] and Norrin [5 ng/µl]. To determine the degree of RGC damage, TUNEL labeling was performed on meridional sections 24 h after injection, and the number of labeled nuclei was quantified. The expression of mRNA for Lif, Edn2, and Fgf2 was investigated by quantitative real-time RT-PCR of treated retinae.
After injection of wild-type mice with combined NMDA/Norrin approximately 50% less TUNEL positive cells were observed in the RGC layer when compared to that of NMDA-treated littermates. The protective effect of Norrin was completely lost when homozygous LIF-deficient mice were treated with combined NMDA/Norrin. In addition, in LIF-deficient mice, NMDA induced a substantial increase in excitotoxic damage as 50% more apoptotic cells in the RGC layer were observed when compared to NMDA-injected wild-type littermates. By real-time RT-PCR for the expression of Lif and Edn2 mRNA in retinae of NMDA/Norrin-treated eyes, a significant increase was observed in wild-type mice when compared to eyes that received NMDA only. The Norrin-mediated effect was substantially reduced in heterozygous LIF-deficient mice. In contrast, only moderate changes were observed in the expression of Fgf2.
Norrin mediates its neuroprotective properties on retinal ganglion cells via an increased expression of Lif. In pigmented mice, the induction of LIF might involve an increased expression of Edn2 while Fgf2 plays no or only a minor role.
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