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Ganeswara Rao Musada, Galina Dvoriantchikova, Ciara Myer, Dmitry V Ivanov, Sanjoy K Bhattacharya, Abigail Hackam; Characterizing The Effect Of Extrinsic Wnt/β-Catenin Signaling In Muller Glia On Retinal Ganglion Cell (RGC) Neurite Growth. Invest. Ophthalmol. Vis. Sci. 2020;61(7):697.
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© ARVO (1962-2015); The Authors (2016-present)
We previously demonstrated that Wnt signaling induction by Wnt3a leads to signaling activation in Muller glia (MG) and Retinal Ganglion Cell (RGC) and significantly enhances RGC axon regeneration after optic nerve crush in the mouse. This study was to investigate the effect of Wnt signaling in MG on RGC neurite growth in primary cultures, to identify Wnt pathway components expressed by MG and RGCs and to characterize the secretome of MG when Wnt signaling is induced and inhibited.
Indirect co-cultures of primary MG with RGC using transwells were performed after induction or inhibition of Wnt signaling specifically in MG using adenoviral constructs containing genes for the Wnt pathway activator β-catenin-S33A, Wnt inhibitor β-eng or the control GFP, under the GFAP promoter. Wnt ligand and Frizzled (Fzd) receptor expression by primary MG and RGC respectively were identified by PCR and sequencing. The secretome of Muller glia after induction or inhibition of Wnt signaling was characterized using protein profiling of conditioned media by Q Exactive mass spectrometry.
In indirect co-cultures, activation of Wnt signaling in MG using the constitutively active β-catenin-S33A significantly increased RGC average neurite length (ANL) (101±18 µm; mean±SD) and average branch site number (ABS) (3±0.4) compared to MG incubated with GFP control (ANL =44±3 µm; p=0.0006, ABS =1.3±0.3; p=0.005) or the Wnt inhibitor β-eng (ANL =34±2 µm; p=0.0002, ABS =1.1±0.4; p=0.002 ). We identified expression of Wnt2, Wnt4, Wnt5a, Wnt5b, Wnt9a, Wnt10b and Wnt16 in MG and Fzd-1, 3, 4, 5 and 7 expression in RGC. The MG secretome after activation of Wnt signaling had distinct and more numerous proteins involved in regulation of axonogenesis, axon projection development, cell growth, axon guidance, cell adhesion proteins, proteins involved in MAPK signaling and TGF- β signaling, compared with the secretome of Wnt inhibited cells.
This study demonstrated a significant inducing effect of extrinsic Wnt/β-catenin signaling in MG on RGC neurite growth and neurite complexity. Additionally, we showed highly redundant expression of Wnt signaling components in MG and RGC. Furthermore, the proteomic analysis provides new knowledge about potential neurite growth promoting molecules in the MG secretome, and identified new Wnt-dependent target proteins that may mediate neurite growth.
This is a 2020 ARVO Annual Meeting abstract.
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