N-acetyl-L-cysteine ethyl ester (NACET) as a potential therapeutic strategy for the prevention and treatment of Age-related Macular Degeneration

Age-related macular degeneration (AMD) is a multifactorial progressive chronic ocular disease. Genetics, environmental insults, and age-related issues are risk factors for the development of the disease. Dysfunction of retinal pigment epithelium (RPE) is involved in AMD and oxidative stress in RPE is one of the major causes of the etiopathogenesis of AMD. Therefore, the introduction of antioxidants may represent one of the most effective ways to delay the onset of AMD. Glutathione (GSH) is a key player in the detoxification of xenobiotics, their metabolites and of reactive oxygen species (ROS) and consequently many drugs are currently in use as GSH enhancers. N-acetyl-L-cysteine ethyl ester (NACET) is a lipophilic and cell permeable GSH prodrug that has been proposed to delay AMD progression. Here, we reported an RNA-seq transcriptome analysis of human Retinal Pigment Epithelial cells (ARPE-19) and described for the first time that NACET induces transcriptional activation of nuclear factor erythroid 2-related factor 2 (NRF2) target genes. NRF2 is a transcription factor that allows the maintenance of redox homeostasis by binding to the antioxidant responsive elements (ARE) in the upstream promoter region of many antioxidative genes and thereby inducing their transcription. By means of HPLC analysis, RT-qPCR, Western blot analysis, CRISPR-Cas9 gene editing and luciferase assay, we validated the transcriptome analysis and demonstrated that NACET increases the intracellular level of free cysteine and promotes transcriptional activation of NRF2 target genes through inhibition of NRF2 degradation. Moreover, using transcriptional profiling of retina of young and old mice orally treated with NACET, we showed that NACET rescued 57 genes impaired by aging, many of which have been correlated with retinal dysfunctions. Our study suggests that NACET, as cysteine and GSH precursor and as NRF2 activator, may be a useful tool for the treatment of oxidative stress-related retinal diseases. Although we tested NACET focusing on AMD, we also verified that NACET-induced NRF2 activation is not cell-context dependent, suggesting that NACET may be a promising agent for prevention/treatment of any pathology where oxidative stress is involved.