N-acetyl-L-cysteine ethyl ester (NACET) induces the transcription factor NRF2 in the retina and prevents its aging and diabetic retinopathy. [II]
Age-related macular degeneration (AMD) and diabetic retinopathy (DR) are leading causes of visual impairment in older people, with oxidative stress playing a central role in the development of these diseases. In fact, the cells of the retina are particularly susceptible to oxidative damage due to high metabolic activity and exposure to light. Glutathione (GSH), a key intracellular antioxidant, is essential for retinal protection but it becomes limited during aging and in diabetes patients. Cysteine (Cys), the rate-limiting precursor for GSH synthesis, can be supplemented by derivatives such as N-acetylcysteine ethyl ester (NACET), which has better bioavailability and cellular uptake compared to the widely used N-acetylcysteine (NAC). NACET effectively increases intracellular Cys and GSH levels, improves the viability of retinal pigment epithelium (RPE) cells under oxidative stress and increases in vivo GSH levels after oral administration. In this study, we show that NACET strongly stimulates NRF2 expression and activity in RPE cells, with a distinctive transcriptomic response. Using RNA interference, mass spectrometry and KEAP1 mutagenesis, we identify direct cysteinylation of sensor residues Cys226 and Cys613 on KEAP1 as the molecular mechanism underlying NRF2 activation after NACET treatment. Furthermore, in vivo administration of NACET induces NRF2 activity and increases GSH content in the retina, mitigating oxidative damage in aging and diabetic mouse models. These results position NACET as a promising therapeutic candidate for oxidative stress-related retinal diseases such as AMD and DR by targeting the KEAP1–NRF2–GSH axis. Overall design: Gene expression analysis of ARPE-19 expressing a defective NRF2, treated with H2O2 or N-acetilcysteine ethyl ester (NACET) in 1% FCS. ARPE-19 knockout for NRF2 were obtaned by transducing cells with lentiviral particles for the expression of the endonuclease Cas9 and a guide RNA (gRNA), the latter specific for the exon 5 of NFE2L2 (NM_006164.5). As negative control, other cells were transduced with similar particles, but missing the gRNA sequence in the viral genome. The two NRF2 edited alleles had a stop codon introduced in position 503 and 505, respectively. 20x10^3  cells/cm^2 were cultured in DMEM/F12 10% FCS for 40 hours and then treated with 1 mM NACET or 0.2 mM H2O2 for 8 hours..