Sustained dysregulation of iron and glutathione homeostasis induces chronoferroptosis, a persistent ferroptotic adaptation in neuronal cells.

Although iron accumulates in brain regions impacted by neurodegenerative diseases such as Alzheimer's and Parkinson's, how chronic elevated iron levels contribute to neuronal dysfunction remains unclear. Here, we show that sustained iron overload, but not acute exposure, leads to a state of ferroptotic stress where nerve cells remain viable but become hypersensitive to oxidative injury. Retinoic acid-differentiated SH-SY5Y neuronal cells were exposed to acute (6-8 h) or chronic (9 days) iron loading to model transient versus prolonged age-related iron stress. While acute iron exposure produced minimal biochemical changes and did not sensitize cells to oxidative or ferroptotic challenges, chronic iron exposure induced ferritin upregulation, mitochondrial superoxide accumulation, suppression of GPX4 expression, elevated lipid peroxidation and loss of cellular glutathione (GSH). In addition, chronic but not acute GSH depletion by buthionine sulfoximine (BSO) recapitulated the iron-induced phenotype. Cells under chronic ferroptotic stress exhibited increased sensitivity not only to the ferroptosis inducer RSL-3 but also to hydrogen peroxide. Ferrostatin-1 significantly mitigated these effects suggesting that lipid peroxidation drives this state. Together, these findings demonstrate that, in contrast with acute exposure, chronic disruption of iron homeostasis with consequent GSH depletion remodels cellular redox homeostasis over time, inducing a state we term chronoferroptosis: a persistent ferroptotic adaptation characterized by coordinated alterations in iron-handling and antioxidant defense proteins that may represent early vulnerability to neurodegenerative pathology. Thus, these studies highlight the importance of sustained stress paradigms for modeling the progressive nature of neurodegenerative diseases. Graphical abstract illustrating how prolonged iron or BSO exposure drives a persistent ferroptotic stress state in RA differentiated SH-SY5Y cells. These cells were subjected to either acute exposure for 6-8 h or chronic exposure for 9 days. Acute iron or BSO treatment caused minimal biochemical changes and did not increase vulnerability to oxidative or ferroptotic challenges. In contrast, chronic exposure stimulated ferritin production, mitochondrial superoxide buildup, GSH depletion, reductions in GPX4 expression and increased lipid peroxidation without altering cell viability. However, these chronically stressed neuronal cells became markedly more sensitive to secondary stressors such as RSL-3 or hydrogen peroxide resulting in decreases in cell viability, whereas viability was not altered in acutely treated cells.