Transient acceleration of autophagic degradation by pharmacological Nrf2 activation is important for retinal pigment epithelium cell survival

Abstract

Non-exudative age-related macular degeneration (AMD) is mainly caused by the accumulation of lipofuscin and drusen on the retinal pigment epithelium (RPE). Both oxidative stress and autophagic dysfunction accelerate the deposition of lipofuscin at the RPE. One of the key regulators in the response against oxidative stress is the NF-E2-Related Factor 2 (Nrf2)-kelch like ECH associated protein 1 (Keap1) axis, which is also closely associated with the autophagy pathway. Nrf2 activation upregulates the expression levels of certain anti-oxidative enzymes [e.g. Heme oxygenase-1 (HO-1)], which attenuates oxidative damage. However, until now, the relationship between cytoprotective effects of Nrf2 activation and autophagic degradation remain unclear. To address these questions, we investigated the effects of a novel Nrf2 activator, RS9, on RPE damage. We found that RS9 protected ARPE-19 cells against NaIO3-induced oxidative damage, and that the protective effects of RS9 were inhibited by co-treatment with zinc protoporphyrin, an HO-1 inhibitor. Next, we examined the involvement of autophagic degradation in the protective effects of RS9. Co-treatment with RS9 and chloroquine, a lysosomal acidification inhibitor, inhibited the protective effect. Furthermore, western blotting and immunostaining showed that RS9 accelerated autophagy flux and induced transient upregulation of p62 [also known as sequestosome 1 (SQSTM1)]. Co-treatment with chloroquine and RS9 also inhibited the degradation of autophagosomes. Transient upregulation of SQSTM1 by RS9 was unaltered by HO-1 knockdown using siRNA. RS9 and chloroquine had the same actions in light damaged adult zebrafish retina as those in vitro. In conclusion, we clarified the relationship between acceleration of the autophagy pathway and the cytoprotective effects of Nrf2 activation in RPE cells and zebrafish retina. These findings indicated that Nrf2 activation could be a promising therapeutic approach for non-exudative AMD by supporting RPE maintenance.

Autophagy in Age-Related Macular Degeneration: A Regulatory Mechanism of Oxidative Stress

“…Notably, levels of the autophagy substrate p62/SQSTM1 were transiently elevated in the NaIO 3 treatment group at 6 h after treatment, and the levels of LC3-I were upregulated at 24 h after NaIO 3 treatment, implying that RS9 accelerated autophagy via transient induction of SQSTM1 expression. They also employed an intense light injury model in zebrafish to mimic in vivo AMD, and the results were in line with the in vitro experiments using ARPE-19 cells [106]. This study supports the idea that the NFE2L2 pathway plays an important role in regulating autophagy and hence preventing oxidative stress in AMD.…”

Oxidative Stress in Age-Related Macular Degeneration: Nrf2 as Therapeutic Target

“…RS9 decreases light-induced retinal cell death in vivo and in vitro ( Inoue et al, 2017 ). Very recently it has been reported that RS9 protects RPE cells from sodium iodate-induced OS and adult zebrafish retina from light-induced damage by increasing Nrf2-dependent HO-1 expression ( Saito et al, 2018 ). RTA 408, a synthetic triterpenoid able to activate Nrf2, protects cultured RPE cells from OS ( Liu et al, 2016a ).…”

Apigenin Protects Mouse Retina against Oxidative Damage by Regulating the Nrf2 Pathway and Autophagy

“…Polyunsaturated fatty acids rapidly increase the generation of ROS in RPE and upregulate the expression of Nrf2 and SQSTM1/p62 proteins [11]. Studies have shown that many inducers of the Nrf2 pathway can also induce autophagy [12][13][14]. Correspondingly, a deficiency of Nrf2 reduces SQSTM1/p62 expression [15].…”