Neuroprotective role of Nrf2 for retinal ganglion cells in ischemia-reperfusion

Abstract

Retinal ischemia plays a critical role in multiple vision-threatening diseases and leads to death of retinal neurons, particularly ganglion cells. Oxidative stress plays an important role in this ganglion cell loss. Nrf2 (NF-E2-related factor 2) is a major regulator of the antioxidant response, and its role in the retina is increasingly appreciated. We investigated the potential retinal neuroprotective function of Nrf2 after ischemia-reperfusion (I/R) injury. In an experimental model of retinal I/R, Nrf2 knockout mice exhibited much greater loss of neuronal cells in the ganglion cell layer than wild-type mice. Primary retinal ganglion cells isolated from Nrf2 knockout mice exhibited decreased cell viability compared to wild-type retinal ganglion cells, demonstrating the cell-intrinsic protective role of Nrf2. The retinal neuronal cell line 661W exhibited reduced cell viability following siRNA-mediated knockdown of Nrf2 under conditions of oxidative stress, and this was associated with exacerbation of increase in reactive oxygen species. The synthetic triterpenoid CDDO-Im (2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide), a potent Nrf2 activator, inhibited reactive oxygen species increase in cultured 661W under oxidative stress conditions and increased neuronal cell survival after I/R injury in wild-type, but not Nrf2 knockout mice. Our findings indicate that Nrf2 exhibits a retinal neuroprotective function in I/R and suggest that pharmacologic activation of Nrf2 could be a therapeutic strategy. Oxidative stress is thought to be an important mediator of retinal ganglion cell death in ischemia-reperfusion injury. We found that the transcription factor NF-E2-related factor 2 (Nrf2), a major regulator of oxidative stress, is an important endogenous neuroprotective molecule in retinal ganglion cells in ischemia-reperfusion, exerting a cell-autonomous protective effect. The triterpenoid 2-Cyano-3,12-dioxooleana-1,9-dien-28-imidazolide (CDDO-Im) reduces neurodegeneration following ischemia-reperfusion in an Nrf2-dependent fashion. This suggests that Nrf2-activating drugs including triterpenoids could be a therapeutic strategy for retinal neuroprotection.

Keywords: Nrf2; ganglion cell; ischemia; oxidative stress; retina.

Figure 1

Exacerbated neuronal cell loss in ganglion cell layer (GCL) in Nrf2^−/− mice after I/R. (a) The number of survived neuronal cells in GCL were counted 7 days after I/R. n=4. *, p<0.05, **, p<0.01. (b) Representative confocal images of NeuN-stained (green) GCL neuronal cells in flat-mount retinas were shown.

Figure 2

Nrf2 is critical for RGC survival in vitro. (a) Primary RGCs from Nrf2^+/+ and Nrf2^−/− were cultured for 4 days, and cell survival were detected using Celltiter-Glo luminescent cell viability assay. (b) Calcein AM, hoechst 33342 and ethidium homodimer triple staining of live cells showed decreased cell number of RGCs deficient of Nrf2. n=4–8. *, p< 0.05; **, p<0.01.

Figure 3

Nrf2 protects against oxidant-induced ROS increase and cell survival in 661W cells. (a) Western blot analysis of Nrf2 expression after 48 h of Nrf2 siRNA transfection. (b) Down-regulation of Nrf2 by Nrf2 siRNA affected TBH-induced oxidative stress in 661W cells. 48 h after siRNA transfection, cells were challenged by different doses of TBH for 1 h. ROS levels were determined by DCF assay. n=4, *p < 0.05 and **p < 0.01 vs control siRNA with TBH treatment. (c) Down-regulation of Nrf2 exacerbates TBH-caused decrease of cell viability. 48 h after siRNA transfection, cells were challenged by TBH for 24 h. n=3, **p < 0.01 vs control siRNA treated with TBH. White bars: control siRNA; black bars: Nrf2 siRNA. (d) Calcein AM, hoechst 33342 and ethidium homodimer triple staining showed further cell loss in Nrf2 siRNA-transfected cells. n=5, **p < 0.01.

Figure 4

The CDDO-Im’s protective effect against TBH-induced oxidative stress in 661W cells. (a) Representative western blot analysis of Nrf2 nuclear translocation showed increased nuclear localization after 24 hours treatment of CDDO-Im. (b) Quantitation of western blot analysis of Nrf2 nuclear translocation by densitometry indicated significant induction by CDDO-Im (n = 3, *p < 0.05) (c) Dose dependent increase of Nrf2-responsive antioxidant gene expression in 661W cells by CDDO-Im (n=4, *p < 0.05; **p < 0.01). (d) Dose response effect of CDDO-Im on inhibiting TBH-induced ROS increase. Cells were pretreated with CDDO-Im for 24 hours before challenged by TBH for 1 h. ROS levels were determined by DCF assay. n=3, *p < 0.05 vs PBS group. (e) Same dose range of CDDO-Im did not cause cell toxicity. n=4, *p < 0.05 and **p < 0.01 vs TBH treatment.

Figure 5

CDDO-Im rescues I/R-induced retinal neuronal cell loss in GCL in wild-type mice but not in Nrf2^−/− mice. (a–f) Up-regulation of retinal Nrf2 target gene expression by CDDO-Im in wild-type mice but not in Nrf2^−/− mice. 6 hours after one intraperitoneal injection of 3 µmol/kg CDDO-Im, RNA was extracted from retinas for q-PCR analysis of Nrf2 target gene expression. n=4–6, **, p < 0.01. (g) The number of NeuN-positive neuronal cells in GCL was counted. White bar: control group; Black bar: I/R group. n=4–6. *, p < 0.05, **, p < 0.001, NS, not significant. (h) Representative confocal images of NeuN-labelled (green) retina flat-mounts were shown.