The role of hydroxyindoles in protecting neuronal cultures from ferroptosis

Hydroxyindoles are organic compounds characterized by a hydroxyl group attached to an indole ring. One notable example is 5-hydroxyindole, which can be found in humans, plants, and microorganisms. The structure of 5-hydroxyindole is integral to molecules such as melanin, serotonin and 5-hydroxyindoleacetic acid (a serotonin metabolite). Ferroptosis is a regulated form of cell death driven by uncontrolled phospholipid peroxidation, which has been linked to the pathogenesis of neurodegenerative diseases, including Alzheimer's and Parkinson's. The impact of hydroxyindoles on ferroptosis remains largely unexplored. This study tests the hypothesis that different hydroxyindoles can modulate ferroptosis in neuronal cultures through specific structure-activity relationships. We used various pathway-specific inducers, including erastin, RSL3, and FINO2, to induce ferroptosis. Cytotoxicity was evaluated using calcein AM, MTT (thiazolyl blue tetrazolium bromide), and LDH (lactate dehydrogenase) release assays. Glutathione levels were measured with the monochlorobimane assay, and intracellular ATP (adenosine triphosphate) levels were quantified using the ATP-Glo™ Bioluminometric cell viability assay. We also performed the ABTS (2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) assay to evaluate the radical-trapping antioxidant activity of the compounds. Our findings indicate that hydroxyindoles function as a class of ferroptosis inhibitors in cell cultures. Among the hydroxyindole analogs studied, 3-hydroxyindole emerged as the most potent inhibitor of ferroptosis in both HT-22 (mouse hippocampal neurons) and N27 (rat dopaminergic neurons) cell lines. In contrast, 5-hydroxyindole and its specific analogs, such as serotonin and 5-hydroxyindoleacetic acid, were found to be less effective in inhibiting ferroptosis in HT-22 cells. Further investigations into the underlying mechanisms revealed that hydroxyindoles inhibit ferroptosis through their intrinsic radical-trapping antioxidant activity. In conclusion, several hydroxyindole analogs, including 3-hydroxyindole, 6-hydroxyindole, and 7-hydroxyindole, have been identified as inhibitors of ferroptosis, highlighting their potential as therapeutic agents for conditions involving neuronal loss caused by ferroptosis.