Abstract
Purpose: :
Identifying survival factors for retinal ganglion cell (RGC) is an important strategy in the development of novel therapies for glaucoma. Elevated intraocular pressure (IOP) is a risk factor in glaucoma progression. Wnt signaling is an essential pathway that regulates cell proliferation, survival and differentiation in the retina. Our previous work showed that the Wnt pathway is upregulated during retinal degeneration and that it protects photoreceptors from oxidative stress. In this study we investigated whether Wnt signaling protects the retinal ganglion cell line RGC-5, and explored possible factors involved in this neuroprotection.
Methods: :
Immunodetection was used to confirm expression of RGC marker genes in the RGC-5 cell line. RGC-5 cells were subjected to elevated pressure (100 mmHg, 24 hrs) in the presence of Wnt3a, DKK1 or the appropriate control. Caspase activity and Tunel assays were performed to quantitate apoptosis, and qPCR was used to investigate potential mechanisms of Wnt3a activity.
Results: :
Expression of the RGC marker genes Thy1 and Brn3b was confirmed in the RGC-5 cells in our culture conditions. Wnt3a reduced pressure-induced caspase activity to 58% of control treatment (n=8). The percentage of Tunel positive cells was significantly reduced by Wnt3a. Control treated cultures had 58% Tunel positive cells whereas Wnt3a treated cultures had 43% (n=5, p=0.0014). Addition of Wnt inhibitor, DKK1, in the presence of Wnt3a significantly increased the percentage of Tunel positive cells to 52% (n=3, p<0.05). Furthermore, the Wnt3a-treated cells up-regulated neurotrophin 3 (2.48-fold, s.e.m 0.14), bFGF (2.3-fold, s.e.m. 0.51), BDNF (1.5-fold, s.e.m. 016) and NGF (1.9 fold, s.e.m. 0.24 (n=4, p<0.05).
Conclusions: :
These data demonstrate that Wnt3a protected RGCs from apoptosis induced by elevated pressure. Increased expression of neuroprotective growth factors in the presence of Wnt3a suggests a potential mechanism of neuroprotection. These results suggest that activation of the Wnt signaling pathway by Wnt3a could be investigated further as a tool to develop therapeutic strategies for the prevention of RGC death.
Keywords: neuroprotection • ganglion cells • cell survival