C prevents the glutamate and erastin
C16 prevents the glutamate- and erastin-induced ROS accumulation but does not affect the decrease in GSH, indicating that prevention of ROS accumulation by C16 is not due to the restoration of GSH levels. Instead, C16 itself possessed superoxide anion scavenging activity in vitro at similar concentrations that prevent cell death. In addition to PKR inhibition, the antioxidant property of C16, an oxindole/imidazole derivative, may contribute to neuroprotective activity against oxytosis and ferroptosis. We recently reported that novel oxindole derivatives such as GIF-2165X-G1 are neuroprotective against oxytosis and ferroptosis and have superoxide scavenging activity in vitro (Hirata et al., 2018). These results suggest that oxindole structures could be useful as a part of leading compounds that function as neuroprotective agents and a rationale to develop therapeutic strategies that target PKR signaling in neurological diseases.
Real-time monitoring of OCR revealed early impairment in mitochondrial function caused by erastin was not accompanied by a decrease in ATP. This observation was consistent with the earlier study, which also noted no depletion and rather an increase in ATP concentration normalized to cell viability in erastin-treated Cathepsin Inhibitor 1 (Dixon et al., 2012). Two recent studies (Jelinek et al., 2018; Neitemeier et al., 2017) reported that ATP concentration was decreased in HT22 cells in response to glutamate or erastin. These differences can be explained by whether ATP levels were normalized to protein concentration and cell viability or not. C16 alone increased ATP levels but slightly decreased OCR and ECAR compared with those in controls. It is not clear whether the slight inhibition of OCR and ECAR by C16 could contribute to the suppression of ROS production. Further study will clarify the role of PKR in metabolic changes in endogenous oxidative stress.
Conflict of interest statement
Acknowledgments We thank Aiko Seno and Hikaru Mizutani for contributing to the initial experiments. We are very grateful to Dr. David Schubert for his generous gift of HT22 cells. This work was supported, in part, by the OGAWA Science and Technology Foundation and by Gifu University.
Introduction Intracerebral hemorrhage (ICH; bleeding in the brain) is a stroke subtype associated with hypertension, cerebral amyloid angiopathy, arteriovenous malformations, and anticoagulant use. Despite the high morbidity and mortality of ICH (van Asch et al., 2010), there are no established treatments (Keep et al., 2012). Therapeutic strategies to limit secondary damage after ICH are of intense interest (Gurol and Greenberg, 2008). Secondary damage, including neuronal death, occurs hours to days following the initial bleed (Brott et al., 1997) and is attributable to a host of factors from lysed blood, such as hemoglobin and the oxidized form of iron-rich heme (Huang et al., 2002). Recent studies demonstrate that secondary cell death in ICH is not the result of random destruction of macromolecules by iron-catalyzed oxidants, but rather by ferroptosis, a non-apoptotic, programmed cell death pathway (Karuppagounder et al., 2016, Li et al., 2017, Zille et al., 2017). Ferroptosis is triggered by the enzymatic production of oxidant lipid species and can be blocked by selective lipid peroxidation inhibitors such as ferrostatin (Dixon et al., 2012, Khanna et al., 2003, Zille et al., 2017). In addition to ICH, ferroptosis operates in erastin and sulfasalazine-induced death of cancer cells (Dixon et al., 2012, Gout et al., 2001), heat stress in plants (Distéfano et al., 2017), ischemia-reperfusion injury (Friedmann Angeli et al., 2014), traumatic brain injury (Wenzel et al., 2017), and Parkinson’s disease (Do Van et al., 2016). In cortical neurons, ferroptosis induces transcriptional responses involving the activation of the leucine zipper transcription factor ATF4, and the upregulation of genes linked to cell death including, CHOP (a prodeath transcriptional activator), TRIB3 (a pseudokinase inhibitor of Akt), or CHAC1 (an enzyme that degrades glutathione; Karuppagounder et al., 2016, Lange et al., 2008). Germline deletion of ATF4 in neurons renders them resistant to homocysteic acid (HCA)-induced ferroptosis in vitro, and sensitivity to cell death can be reinstated by wild-type ATF4, but not ATF4 with the DNA binding domain mutated (Lange et al., 2008). Subsequent studies showed that iron chelators prevent ferroptosis in neurons induced by glutamate or hemin (used to model ICH in vitro) not by inhibiting Fenton chemistry but rather by targeting a family of iron-dependent enzymes, the hypoxia-inducible factor (HIF) prolyl hydroxylases, which are necessary for ATF4-dependent prodeath transcription (Karuppagounder et al., 2016). ATF4-dependent gene expression is observed in ferroptotic cancer cells induced by erastin, but the role of ATF4 in regulating ferroptotic death in cancer cells appears context dependent (Chen et al., 2017).