Oxytosis/Ferroptosis-(Re-) Emerging Roles for Oxidative Stress-Dependent Non-apoptotic Cell Death in Diseases of the Central Nervous System

Abstract

Although nerve cell death is the hallmark of many neurological diseases, the processes underlying this death are still poorly defined. However, there is a general consensus that neuronal cell death predominantly proceeds by regulated processes. Almost 30 years ago, a cell death pathway eventually named oxytosis was described in neuronal cells that involved glutathione depletion, reactive oxygen species production, lipoxygenase activation, and calcium influx. More recently, a cell death pathway that involved many of the same steps was described in tumor cells and termed ferroptosis due to a dependence on iron. Since then there has been a great deal of discussion in the literature about whether these are two distinct pathways or cell type- and insult-dependent variations on the same pathway. In this review, we compare and contrast in detail the commonalities and distinctions between the two pathways concluding that the molecular pathways involved in the regulation of ferroptosis and oxytosis are highly similar if not identical. Thus, we suggest that oxytosis and ferroptosis should be regarded as two names for the same cell death pathway. In addition, we describe the potential physiological relevance of oxytosis/ferroptosis in multiple neurological diseases.

Keywords: brain diseases; ferroptosis; iron; oxidative stress; oxytosis; programmed cell death.

Figure 1

Ultrastructural changes in cells undergoing ferroptosis and oxytosis comprise prominent but diverse mitochondrial abnormalities while nuclear integrity is preserved. Electron micrographs of (A) mouse embryonic fibroblasts (MEF) showing a time-dependent outer mitochondrial membrane rupture (yellow arrows) upon ferroptosis induction using RSL3 (50 nM; scale bars 2 μm top row, 200 nm bottom row) while nuclear integrity is preserved, (B) of BJeLR cells treated with DMSO (10 h) or erastin (37 μM, 10 h) showing shrinkage and increased electron density of the mitochondria. (C) Similar morphological changes as in (B) in response to RSL3 in MEF. (D) HT22 cells after control treatment (panels 1/2) and after 5 mM glutamate for 10 h (panels 3–5). Panels 3/4: low- and high-power micrographs, respectively, of the same region of the same cell. Arrows indicate mitochondria. Bars in panel 1 = 5 μm; panels 2, 3, and 5 = 2 μm; and panel 4 = 1 μm. Glutamate-induced oxytosis is also characterized by preserved nuclear structure in addition to prominent swelling of the endoplasmic reticulum, Golgi apparatus and mitochondria as well as cytoplasmic vacuolization. Mitochondria showed loss of the cristae. (E) Oxytosis induced by glutamate in HT4 cells. (E, left) HT4 cells with no glutamate. Mitochondria have a regular shape and optical density. (E, middle and right) glutamate-treated cell (10 mM for 8 h). The mitochondria appeared to be swollen and degraded with low optical density. Mitochondrial outer membrane disruption is observed. Scale bars 1.85 μm (left and middle), 0.21 μm (right). [Modified and reproduced with permission from (A) (Friedmann Angeli et al., 2014), (B) (Dixon et al., 2012), (C) (Doll et al., 2017), (D) (Tan et al., 1998b), and (E) (Tirosh et al., 2000)].

Figure 2

Light microscopic changes of cellular shape during oxytosis and the nucleus during ferroptosis. (A) HT22 cells exposed for indicated durations to 5 mM glutamate and observed by phase contrast microscopy. Cells were untreated (left upper) or treated with 5 mM glutamate for 8 h (right upper), 10 h (left lower), or 14 h (right lower) before examination under a phase-contrast microscope. Inset in the right upper shows blebs on surface of cells. (B) Lack of prominent nuclear changes in erastin-treated BJ-TERT/LT/ST/RASV12 cells while camptothecin-treated cells display fragmented nuclei (arrow). [Modified and reproduced with permission from (A) (Tan et al., 1998b) and (B) (Dolma et al., 2003)].

Figure 3

Transcriptome changes in acquired oxytosis and ferroptosis resistance. The transcriptomes of HT22 cells selected for resistance to oxytosis induced by glutamate (HT22R) (A) and DU-145 human prostate cancer cells selected for resistance against ferroptosis induced by erastin (B) (Dixon et al., 2014) were compared to their parental oxytosis/ferroptosis sensitive cell lines. Using the Panther classification system, deregulated genes (= fold-change ≥± 2) are grouped into biological processes. Both cell lines have highly similar deregulation profiles, even though the total number of deregulated genes differs greatly. The erastin-resistant cell line shows (minor) deregulation in only two additional processes compared to the HT22R. These processes (cell killing and cell growth) are likely to be attributed to the cancerous origin of this cell line. Gene Ontology (GO) identifications are shown between brackets.

Figure 4

The common cell death pathway in oxytosis and ferroptosis. Uptake of cystine by system ${\text{x}}_{\text{c}}^{-}$ associated with counter-transport of glutamate (Glu) is inhibited by Glu and Erastin. This leads to depletion of glutathione (GSH) and subsequently inhibition of the GSH-dependent enzyme GSH peroxidase 4 (GPX4). GPX4 can also be directly inhibited by RSL3. GPX4 inhibition leads to activation of LOX. As a result, lipid hydroperoxides (lipid icons with OOH) accumulate probably in or very close to the endoplasmic reticulum as the initiating step of the production of reactive oxygen species (ROS). There is exponentially increasing mitochondrial ROS production associated with a hyperpolarization of the mitochondrial membrane potential (ΔΨm). Whether this is a direct effect of LOX is unknown. However, via activation of soluble guanylate cyclase (sGC) induced by LOX metabolites cGMP accumulates and calcium influx through store-operated calcium channels (SOCE) is activated. This increases cytoplasmic calcium (Ca²⁺)_cyto. There is a mutual requirement for ROS and calcium to reach their maximal levels. Lysosomes also contribute to the overall ROS production.