Ferroptosis is a form of regulated necrosis characterized by a chain-reaction of detrimental membrane lipid peroxidation following collapse of glutathione peroxidase 4 (Gpx4) activity. This lipid peroxidation is catalyzed by labile ferric iron. Therefore, iron import mediated via transferrin receptors and both, enzymatic and non-enzymatic iron-dependent radical formation are crucial prerequisites for the execution of ferroptosis. Intriguingly, the dynamin inhibitor dynasore, which has been shown to block transferrin receptor endocytosis, can protect from ischemia/reperfusion injury as well as neuronal cell death following spinal cord injury. Yet, it is unknown how dynasore exerts these cell death-protective effects. Using small interfering RNA suppression, lipid reactive oxygen species (ROS), iron tracers and bona fide inducers of ferroptosis, we find that dynasore treatment in lung adenocarcinoma and neuronal cell lines strongly protects these from ferroptosis. Surprisingly, while the dynasore targets dynamin 1 and 2 promote extracellular iron uptake, their silencing was not sufficient to block ferroptosis suggesting that this route of extracellular iron uptake is dispensable for acute induction of ferroptosis and dynasore must have an additional off-target activity mediating full ferroptosis protection. Instead, in intact cells, dynasore inhibited mitochondrial respiration and thereby mitochondrial ROS production which can feed into detrimental lipid peroxidation and ferroptotic cell death in the presence of labile iron. In addition, in cell free systems, dynasore showed radical scavenger properties and acted as a broadly active antioxidant which is superior to N-acetylcysteine (NAC) in blocking ferroptosis. Thus, dynasore can function as a highly active inhibitor of ROS-driven types of cell death via combined modulation of the iron pool and inhibition of general ROS by simultaneously blocking two routes required for ROS and lipid-ROS driven cell death, respectively. These data have important implications for the interpretation of studies observing tissue-protective effects of this dynamin inhibitor as well as raise awareness that off-target ROS scavenging activities of small molecules used to interrogate the ferroptosis pathway should be taken into consideration.
Many cell death pathways, including apoptosis, regulated necrosis, and ferroptosis, are relevant for neuronal cell death and share common mechanisms such as the formation of reactive oxygen species (ROS) and mitochondrial damage. Here, we present the role of the actin-regulating protein cofilin1 in regulating mitochondrial pathways in oxidative neuronal death. Cofilin1 deletion in neuronal HT22 cells exerted increased mitochondrial resilience, assessed by quantification of mitochondrial ROS production, mitochondrial membrane potential, and ATP levels. Further, cofilin1-deficient cells met their energy demand through enhanced glycolysis, whereas control cells were metabolically impaired when challenged by ferroptosis. Further, cofilin1 was confirmed as a key player in glutamate-mediated excitotoxicity and associated mitochondrial damage in primary cortical neurons. Using isolated mitochondria and recombinant cofilin1, we provide a further link to toxicity-related mitochondrial impairment mediated by oxidized cofilin1. Our data revealed that the detrimental impact of cofilin1 on mitochondria depends on the oxidation of cysteine residues at positions 139 and 147. Overall, our findings show that cofilin1 acts as a redox sensor in oxidative cell death pathways of ferroptosis, and also promotes glutamate excitotoxicity. Protective effects by cofilin1 inhibition are particularly attributed to preserved mitochondrial integrity and function. Thus, interfering with the oxidation and pathological activation of cofilin1 may offer an effective therapeutic strategy in neurodegenerative diseases.
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