脊髓损伤
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Figure 1｜Schematic illustrating key elements of the ferroptosis pathway.
A PubMed search revealed that 930 papers on cuprizone were published in the past 55 years since it was first shown to cause damage to the CNS. Interestingly, there were few papers until the turn of this century, with only 4 papers published in the year 2000 but the numbers rose sharply to 119 papers in 2019. There is therefore an increasing interest in the use of the cuprizone model. Although 180 papers dealt with mechanisms underlying demyelination and remyelination, the precise mechanism that trigger oligodendrocyte cell death and demyelination has remained elusive. We became interested in whether copper chelation by cuprizone will dysregulate iron homeostasis because of the long-standing work in our lab showing that ferroxidases (ceruloplasmin and hephaestin) play an important role in iron efflux or export from cells, and that lack or deficiency in these ferroxidases leads to iron accumulation in the CNS (Jeong and David, 2006; Schulz et al., 2011). These two ferroxidases are homologues that differ mainly in their membrane-anchoring regions. They are among the most copper-rich enzymes, each molecule containing 6 coppers, which are essential for electron transfer reactions and ferroxidase activity (Bento et al., 2007). In the CNS, ceruloplasmin is expressed in astrocytes, while hephaestin is expressed in oligodendrocytes. Ceruloplasmin knockout mice first showed presence of iron accumulation in astrocytes at 18 months of age (Jeong and David, 2006), while hephaestin mutant mice show iron deposition in oligodendrocytes by 2 months of age but it may likely occur even earlier (Schulz et al., 2011). These findings show that there are differences in iron cycling and utilization in these two glial cell types. Iron deposition in CNS cells can be detected in these conditions by iron histochemistry, as well as by increased expression of ferritin, an iron storage protein, which is a sensitive indicator of iron. It was therefore reasonable to ask if copper chelation by cuprizone would lead to iron accumulation and iron-mediated loss of oligodendrocytes. Our analysis showed that 60% of oligodendrocytes are lost as early as 2 days after start of the cuprizone diet (Jhelum et al., 2020). Such rapid loss of oligodendrocytes has also been supported by some earlier reports. However, at this timepoint we did not detect increased iron by iron histochemistry or increased ferritin staining. Instead, we found a loss of ferritin staining at day 1, the day before the loss of oligodendrocytes is detected. This decrease in ferritin correlated with increased expression in oligodendrocytes of NCOA4, a cargo receptor that binds to ferritin and shuttles it to autophagosomes for degradation (ferritinophagy) (Mancias et al., 2014; Quiles Del Rey and Mancias, 2019). Interestingly, double immunofluorescence labeling revealed that cells that expressed NCOA4 had low levels or no ferritin, while cells with high levels of ferritin expressed little or no NCOA4. Increased expression of NCOA4 will result in ferritinophagy and the release of bioactive iron, which because of the lack of ferritin will fuel generation of free radicals via the Fenton reaction. Other changes that can add to increase bioactive iron load in oligodendrocytes is reduced expression of the oligodendrocyte-specific ferroxidase, hephaestin; reduction of which will lead to reduced iron efflux from oligodendrocytes. In addition, we also detected increased expression of transferrin receptor 1 in oligodendrocytes that will increase iron uptake into these cells. Additionally, intracellular iron load can also be increased by the expression of heme oxygenase-1 that releases iron from heme. The potential rapid increase in iron load from these mechanisms can lead to lipid peroxidation in the CNS (Figure 1). In fact, we detected a rapid increase in lipid peroxidation in the corpus callosum, as seen by a 3-fold increase in 4-hydroxy-2-nonenal, a lipid peroxidation product, as early as 2 days after start of the cuprizone treatment (Jhelum et al., 2020). In addition, double immunofluorescence staining of tissue sections showed that 4-hydroxy-2-nonenal and malondialdehyde, an end-product of polyunsaturated fatty acid peroxidation, are localized to oligodendrocytes in the corpus callosum at this early time period. NCOA4 expression, ferritinophagy, and lipid peroxidation are considered hallmarks of ferroptosis. As mentioned above, another hallmark of ferroptosis is insufficiency of the antioxidant GSH which neutralizes lipid radicals. Two molecules in the GSH pathway were found to be reduced during the first week after start of the cuprizone diet. These include (i) reduction in system Xc, the cystine antiporter, required for influx of cystine into cells for GSH synthesis; and (ii) reduction in GPX4, the enzyme that catalyzes the reaction between GSH and lipid peroxides, and in the process converting reduced GSH to its oxidized from, GSSG. Deficiency in these molecules is also an important feature of ferroptosis (Figure 1). Direct evidence that ferroptosis is responsible for cuprizone-induced loss of oligodendrocytes was obtained from function blocking experiments in which this cell loss was rescued by treatment with a lipid radical scavenging molecule, ferrostatin-1. We also showed that demyelination in the corpus callosum induced by cuprizone can also be rescued by ferrostatin-1. There is a delay of several weeks after the early loss of oligodendrocytes (80% loss by 7 days) before significant myelin loss is seen (4–5 weeks). The slow clearance of myelin, which is also seen in Wallerian degeneration in the CNS, is due to the slow phagocytic response in the adult mammalian CNS. Nevertheless, we show using a novel spectral imaging technique (Nile Red solvatochromism) that there are rapid changes in the molecular composition of myelin with loss of non-polar lipids, as early as 2 days after start of the cuprizone diet (Jhelum et al., 2020). This imaging technique could be very useful to detect early myelin pathology in regions of that look normal with conventional histological techniques, such as in normal appearing white matter in MS, as well as conditions such as stroke, concussion, and other types of CNS trauma.