N-acetyl-L-cysteine ethyl ester (NACET) induces the transcription factor NRF2 in the retina and prevents its aging and diabetic retinopathy. [III]

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: Chronic oxidative stress is widely recognized as a key factor in retinal cell damage and death in retinal degenerative diseases. With the aim of proposing NACET as a therapeutic and preventive agent against AMD, we investigated the effects of NACET in vivo on the aging retina of mice. To this end, we treated young (8-20 weeks) and old (80-110 weeks) C57BL/6J mice with or N-acetilcysteine ethyl ester (NACET) added to drinking water (daily oral intake of 50 mg/Kg). After 5 days mice have been sacrificed by CO2 euthanasia, then we proceed with the dissection of the eyes to collect the retinas as described in Simmons and Fuerst, 2018 (Simmons AB, Fuerst PG. Analysis of Retinal Vascular Plexuses and Interplexus Connections. Methods Mol Biol. 2018;1753:317-330). One retina of each mouse was used for RNA-seq analysis.