Abstract:

Purpose: Glycogen synthase kinase-3β (Gsk-3β) participates in various essential cellular biological activities and signal transduction. Pharmacological inhibition of Gsk-3β attenuates DNA damage, thus improving cell survival in neurons under pathological conditions. Here, we sought to investigate its underlying mechanism. 

Methods: An in vitro serum starvation retinal neuron model and an in vivo ischemia-reperfusion (I/R) retina injury rat model were established and were treated with or without SB216763, a Gsk-3β inhibitor. Real-time PCR and western blotting were performed. γ-H2AX staining was used to label DNA double strand breaks (DSBs) in situ. A luciferase assay was used to analyze the involvement of Gsk-3β in the Ligase IV transcription mechanism. siRNA interference was used to downregulated Ligase IV expression in retinal neurons. MTT assay was used to monitored the cell viability. 

Results: Inhibiting Gsk-3β activity by SB216763 dramatically decreased the formation of γ-H2AX foci and improved cell viability in ischemic retinal neurons. Moreover, our data suggested that interference of Ligase IV diminished the protected effect of SB216763 on cell viability. SB216763 upregulated expression of P-Cr eb1, a Ligase IV transcription factor, and significantly increased the transcriptional activity of Ligase IV in ischemic retinal neurons, as evidenced by the luciferase assay. These results were confirmed in rat retinas following I/R injury. Furthermore, we found that, unlike lithium chlorine (another well-known Gsk-3β inhibitor), which directly inhibited Gsk-3β, SB216763 inhibits Gsk-3β activity by suppressing the phosphorylation level of Gsk-3β. 

Conclusion: Taken together, our results suggest that Gsk-3β inhibition enhances DNA DSBs repair by upregulating Ligase IV expression in ischemic retinal neurons.

Key words: Gsk-3β, SB216763, Ligase IV , LiCl, DNA double strand repair, retinal neurons, γ-H2AX, P-Creb1, ischemic, neuroprotective