Mechanism of L-Buthionine-(S,R)-sulfoximine-induced ferroptotic cell death in mouse hippocampal neuronal cells.

Recent studies have revealed that accumulation of cellular nitric oxide (NO) resulting from protein disulfide isomerase (PDI)-catalyzed dimerization (i.e., catalytic activation) of nitric oxide synthase (NOS) is a pivotal upstream event in the induction of ferroptosis. L-Buthionine-(S,R)-sulfoximine (BSO), an inhibitor of the γ-glutamylcysteine synthase, can induce cellular glutathione depletion and ferroptosis. The present study aims to investigate whether PDI-centered signaling axis also mediates BSO-induced ferroptosis. Using the HT22 mouse hippocampal neurons as an in-vitro model, we found that BSO-induced glutathione depletion is associated with the oxidative activation of PDI, and the activated PDI mediates ferroptosis by catalyzing the dimerization of the inducible NOS (iNOS). iNOS dimerization then leads to the accumulation of cellular NO, which subsequently results in the accumulation of cellular ROS and lipid peroxides as well as mitochondrial ROS and lipid peroxides, and ultimately, oxidative ferroptosis. Pharmacological inhibition of PDI’s enzymatic activity or selective PDI knockdown can effectively abrogate BSO-induced ferroptosis. Similarly, selective inhibition of other signaling components along the PDI–iNOS–NO–ROS axis also confers strong protection against BSO-induced ferroptosis. In addition, BSO-induced ferroptosis can be selectively enhanced by chemicals that facilitate PDI activation or PDI-mediated iNOS dimerization or NO accumulation. The results of this study demonstrate that the PDI‒iNOS‒NO‒ROS signaling axis plays a pivotal role in BSO-induced ferroptosis in a cell culture model, and also offer strategies for the selective manipulation (enhancement or suppression) of ferroptosis by targeting PDI or its downstream signaling elements.