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Adanna Udeh, Galina Dvoriantchikova, Tal Carmy, Dmitry V Ivanov, Abigail S Hackam; Wnt-induced neurite growth in retinal ganglion cells (RGCs) is regulated by Ripk1. Invest. Ophthalmol. Vis. Sci. 2018;59(9):5493.
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© ARVO (1962-2015); The Authors (2016-present)
Wingless-type (Wnt) signaling plays important roles in regulating neuronal survival and axonal regeneration in the adult CNS after injury. Previous studies using axonal injury in various model systems showed that Wnt ligands activate signaling pathways in both non-neuronal cells and neurons during axonal growth. However, the cellular mediators of the regenerative effect of Wnt in the retina are unclear. Additionally, the molecular mechanisms of Wnt3a-mediated RGC axonal growth remain unknown. A commonly used model of axon regeneration is neurite growth in cultured RGCs. In this study, we tested the hypothesis that Wnt signaling induces RGC neurite growth through the activation of intrinsic signaling pathways.
Mouse primary RGC cultures were obtained at PN12, and incubated with increasing concentrations of Wnt3a ligand to induce Wnt signaling. Additionally, RGCs were treated with Wnt inhibitor Dkk1, Wnt3a+Dkk1, PBS (vehicle control) or the receptor-interacting serine/threonine-protein kinase 1 (Ripk1) inhibitor Necrostatin-1. RGC neurite growth was quantified by measuring length and number of the beta-tubulin-positive neurites. Ripk1 and Ripk3 expression was measured using qPCR.
Wnt3a induced significant dose-dependent increases in average neurite length (up to 134.8 um + 5.8) compared to controls (35.5 um + 12.6) and increased the number of neurites per cell (up to 5.1+ 0.45) compared to controls (1.9 + 0.73, n=3-6, p<0.05). Dkk1 blocked Wnt3a-induced neurite growth (n=3, p<0.05). Furthermore, significant reductions of Ripk1 and Ripk3 transcripts were associated with Wnt-dependent neurite growth (n=4, p<0.05), and inhibiting Ripk1 signaling lead to increased neurite numbers per cell (50% increase, n=4 p<0.05), but not increased neurite length.
Wnt3a induced neurite growth within RGCs in a dose-dependent manner by activating the canonical Wnt signaling pathway, indicating that RGCs are direct targets of Wnt-induced axonal growth. Furthermore, we demonstrated a novel association between Wnt signaling and Rip kinases in neurite formation.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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