Pamela Maher^§, Gamze Ates^§ and Jan Lewerenz^*

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 nerve cell death predominantly proceeds by regulated processes. In 1989, Murphy and colleagues reported that glutamate could induce a calcium-dependent form of delayed cell death that was associated with depletion of intracellular glutathione (GSH) and characterized by increased oxidative stress.¹ The mechanistic link between glutamate exposure and GSH depletion proved to be the glutamate-mediated inhibition of cystine uptake via system x_c^-, a cystine glutamate antiporter (Figure 1).² Importantly, the first reported inducer of ferroptosis, erastin,³ was later identified to be a system x_c^- inhibitor (Figure 1).⁴ Most of the subsequent studies addressing this delayed form of glutamate toxicity―initially called oxidative glutamate toxicity―were carried out in HT-22 cells, a hippocampal nerve cell line that was specifically selected for its sensitivity to glutamate.⁵ Using this cell-based model, the biochemical events that sequentially lead to cell death were characterized in detail. It was demonstrated that this type of cell death is associated with a large increase in reactive oxygen species (ROS) generation as well as lipoxygenase (LO) activation subsequent to GSH depletion, which are followed by a final, lethal influx of calcium.⁶ Oxytosis―a term that highlights both the ROS accumulation that is characteristic of this type of cell death as well as the fact that it is a form of regulated cell death distinct from apoptosis―was proposed as the name for this new form of non-apoptotic, regulated cell death.⁶

Further studies have revealed that the molecular pathways involved in the regulation of ferroptosis and oxytosis share many similarities.⁷ For example, the downstream players such as glutathione peroxidase 4 (GPX4) and LO and accumulation of mitochondria-derived ROS and nuclear translocation of apoptosis-inducing factor (AIF) are identical. In addition, transcriptomic changes in oxytosis- and ferroptosis-resistant cells correspond to identical pathways. However, some characteristics have been studied in more detail under the name of either oxytosis or ferroptosis (e.g., the role of cGMP and calcium during oxytosis and the generation of lipid peroxides during ferroptosis), and this literature can make them appear distinct.

Moreover, similar to ferroptosis, oxytosis in HT-22 cells can be inhibited by iron chelators^8,9 and exacerbated by different sources of iron.^9,10 Thus, both oxytosis and ferroptosis show the same dependency on iron, further suggesting that both pathways are highly similar. However, the whole concept of ferroptosis was recently challenged by a study demonstrating that at least in HT-22 cells, copper, the other important transition metal involved in redox metabolism in biological systems, exacerbates both cell death induced by glutamate, the prototypical insult that induces oxytosis in these cells, as well as cell death induced by the prototypical ferroptosis inducer erastin to a similar extent as iron.¹⁰ Thus, at least under certain conditions, transition metals other than iron have the potential to exacerbate ferroptosis.

In summary, the discrepancies that have been described in the scientific literature do not indicate that ferroptosis and oxytosis are different pathways of regulated cell death but rather result from methodological differences or cell type-specific variations on a single theme. Thus, oxytosis and ferroptosis should be regarded as two names for the same cell death pathway.

Figure 1. The common cell death pathway in oxytosis and ferroptosis. Cystine uptake by system x_c^- is inhibited by Glu and Erastin leading to the depletion of GSH resulting in reduced GSH-dependent GPX4 activity. GPX4 can also be directly inhibited by RSL3. In the absence of GPX4 activity, ROS- or LO-derived lipid hydroperoxides (lipid icons with OOH; shown as cytosolic for illustration purposes) accumulate at various membrane sites, including mitochondria where there is a concomitant hyperpolarization of the mitochondrial membrane potential (Δ_ψm). Lysosomes also contribute to the overall ROS production. Image was adapted from Lewerenz et al., 2018 with permission by CC-BY, version 4.0.

References

1. Murphy, T.H., Malouf, A.T., Sastre, A., et al. Brain Res. 444(2), 325-332 (1988).

2. Bannai, S. and Kitamura, E. J. Biol. Chem. 255(6), 2372-2376 (1980).

3. Dixon, S.J., Lemberg, K.M., Lamprecht, M.R., et al. Cell 149(5), 1060-1072 (2012).

4. Dixon, S.J., Patel, D.N., Welsch, M.E., et al. eLIFE 3, e02523 (2014).

5. Davis, J.B. and Maher, P. Brain Res. 652(1), 169-173 (1994).

6. Tan, S., Schubert, D., and Maher, P. Curr. Top. Med. Chem. 1(6), 497-506 (2001).

7. Lewerenz, J., Ates, G., Methner, A., et al. Front. Neurosci. 12, 214 (2018).

8. Liu, Y. and Schubert, D.R. J. Biomed. Res. 16(1), 98 (2009).

9. Kang, Y., Tiziani, S., Park, G., et al. Nature Commun. 5, 3672 (2014).

10. Maher, P. Free Rad. Biol. Med. 115, 92-104 (2018).

Pamela Maher (Ph.D.) is a senior staff scientist at the Salk Institute with a longstanding interest in understanding how nerve cells die and what can be done to prevent it. Gamze Ates (Pharm. D.) obtained her Ph.D. in toxicology and currently works as a post-doctoral researcher in cellular neurobiology at the Salk Institute. Jan Lewerenz studied medicine at the University of Hamburg and is currently the senior attending neurologist in the Department of Neurology of the University of Ulm and deputy head of the Huntington’s Disease Center, Ulm.