Iron Overload Causes Ferroptosis But Not Apoptosis in MO3.13 Oligodendrocytes

Oligodendrocytes are the most iron-rich cells in the brain. Studies have shown that oligodendrocytes are very sensitive to oxidative stress, and iron overload is more likely to cause damage to oligodendrocytes. The purpose of this experiment was to investigate the damaging effect and mechanism of ferric ammonium citrate (FAC) on MO3.13 oligodendrocytes. In FAC treatment group, the intracellular iron concentration and intracellular reactive oxygen species were increased. There were no obvious changes in nucleus and chromatin, but increased mitochondrial membrane density, decreased mitochondrial cristae and mitochondrial length were observed. Glutathione peroxidase 4 (GPX4) expression was decreased, but the ratio of Bcl-2/Bax protein levels and cleaved caspase-3 expression did not change. Moreover, the iron chelator deferoxamine (DFO) and the ferroptosis inhibitor ferrostatin-1(Fer-1) could inhibit the upregulation of GPX4, which indicating that DFO and Fer-1 could inhibit ferroptosis in MO3.13 oligodendrocytes induced by iron overload. Furthermore, the phosphorylation level of p53 was not changed, while the ratio of protein expressions of p-Erk1/2/Erk1/2 were markedly increased. Taken together, our data suggest that iron overload induces ferroptosis but not apoptosis in oligodendrocytes. The mechanism may be related to mitogen-activated protein kinase pathway activation rather than p53 pathway activation.


Introduction
Parkinson's disease (PD) is a common neurodegenerative disease characterized by the loss of dopaminergic neurons in the substantia nigra (SN) [1]. Neuroimaging and postmortem examination reported that iron selective aggregation in the SN, suggesting that nigral iron accumulation plays a key role in the pathogenesis of PD [2]. Iron plays important roles in the central nervous system, including myelin production, mitochondrial respiration, neurotransmission and metabolism, DNA synthesis, and more. When iron is present in excess in cells and tissues, it disrupts redox homeostasis and catalyzes excessive production of reactive oxygen species, leading to oxidative stress [3,4].
Recent studies have revealed that lineage of oligodendrocytes plays important role in PD. PD upregulation genes are significantly enriched in oligodendrocyte lineages, and intrinsic gene alterations in oligodendrocytes occur in the early stages of the disease, preceding the emergence of dopaminergic neurons loss in the SN [5]. Oligodendrocytes, differentiated from oligodendrocyte precursor cells (OPCs) are myelin-forming cells in the central nervous system [6]. Oligodendrocytes require sufficient iron to form myelin sheaths and are the most iron-rich cells in the brain [4]. Studies have shown that oligodendrocytes are very sensitive to oxidative stress, and iron load is more likely to cause damage to oligodendrocytes, and PD patients have demyelinating changes in the brain [7,8]. However, the damaging effect of iron loading on oligodendrocytes and its possible mechanism remain unclear.
The term ferroptosis, coined in 2012 [9], is a distinct form of cell death that describes the inhibition of cystine input by the small molecule erastin, leading to glutathione (GSH) Yinghui Li and Bingjing Wang contributed equally to this work. depletion and inactivation of the phospholipid peroxidase glutathione peroxidase 4 (GPX4), inducing cell death [10]. As the primary enzyme, GPX4 is critical in preventing ferroptosis by converting lipid hydroperoxides into non-toxic lipid alcohols [11]. Recent studies have revealed that ferroptosis is involved in neurodegenerative diseases [12], and that the midbrains of patients with PD are characterized by high-iron, GSH depletion and increased reactive oxygen species (ROS) production of lipid peroxidation, suggesting that the pathogenesis of PD is closely related to ferroptosis [13]. At present there is no effective cure for PD, and the current treatment is merely symptomatic based on substitution of dopaminergic deficit. Ferroptosis could be inhibited by ferrostatin-1 (Fer-1), desferrioxamine (DFO), vitamin E, coenzyme Q10 (CoQ10) and etc., which may bring us closer to this goal [12,14]. Apoptosis is the process of programmed cell death characterized by blebbing, cell shrinkage, nuclear fragmentation, chromatin condensation, and chromosomal DNA fragmentation [15]. Apoptosis is characterized by decreased ratio of Bcl-2/Bax, and finally initiated by increased level of cleaved caspase-3 [16]. Evidence for the role of apoptotic cell death in PD arises from morphological, as well as molecular, studies in cell cultures, animal models for PD, as well as human studies on postmortem brains from PD patients [17]. New ''antiapoptotic'' compounds may offer a means of protecting neurons from cell death and of slowing the rate of neurodegeneration and disease progression [18].
Previous studies have found that iron overload in dopaminergic neurons causes both apoptosis and ferroptosis [19], but the mechanism of iron overload on oligodendrocytes damage is unclear. In the present study, we observed that FAC treatment induced ferroptosis but not apoptosis in MO3.13 oligodendrocytes. Moreover, iron overload induced ferroptosis might be related to mitogen-activated protein kinase (MAPK) signaling pathway, independent of p53 signaling pathway.

Cell Culture
The rodent MO3.13 cell line was purchased from Shanghai Bialy Biotechnology Co., LTD. MO3.13 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) (BI, USA), added 10% fetal bovine serum, 100 units/mL of penicillin and 100 units/mL of streptomycin in a humidified atmosphere containing 5% CO 2 at 37 °C. were dissolved in DMSO. The concentration of DMSO used to dissolve the drug proved to have no effect on the cells.

Iron Assay Kit
MO3.13 cells were seeded in 6-well plates, pre-incubated for 24 h with FAC. Culture supernatants were collected, and the concentrations of hepcidin were determined using Iron Assay Kit (Sigma, St. Louis, MO, USA) as described in the manufacturers' instructions. Absorbance at 593 nm was determined on Spectra Max Plus 384 Microplate Reader.

Transmission Electron Microscopy Measurement of Changes in Mitochondrial Morphology
Transmission electron microscopy was performed using a Tecnai 10 microscope (FEI, Hillsboro, OR) at the Electron Microscopy Core Facility, Qingdao University. Mitochondrial length (along the long axis) was measured and summarized. A minimum of 90 mitochondria per treatment condition were examined.

ROS Detection
For quantification of intracellular ROS levels, MO3.13 cells were loaded with 5 μM2′,7′-dichlorodihydrofluorescein diacetate (Sigma, USA) in the dark for 30 min at 37 °C, 5% CO 2 in phosphate-buffered saline. Cells were collected, washed twice with HEPES Buffered Saline buffer (HBS, Sigma, USA), and suspended in 500 μL HBS buffer, then analyzed by a flow cytometer. Fluorescent intensity was measured using FACS Calibur (BD

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Biosciences, CA, USA) and analyzed by Cell Quest Pro 5.2 according to the manufacturer's protocol.

Western Blots
Samples from cells and animals were lysed in RIPA lysis buffer containing protease inhibitor and phosphatase inhibitor cocktail. The protein concentration was detected by BCA kits (Thermo Fisher Scientific, USA). Samples were separated by SDS-poly acrylamide gel electrophoresis and transferred to PVDF membrane. After blocked with 100 g/L non-fat milk for 2 h at room temperature, the membranes were incubated with primary antibody overnight at 4 °C, and then with secondary antibodies coupled to horseradish peroxidase. The primary antibodies were used for Western blot analysis: anti-phosphorylated extracellular signal-regulated kinase (p-Erk) 1/2, anti-Erk1/2, anti-phosphorylated p53, anti-p53 and anti-β-actin were purchased from Cell Signaling Technology (Boston, MA). Anti-Gpx4, anti-Bax, anti-Bcl-2 and anti-cleved caspase-3 were purchased from Abcam (UK). The secondary antibodies were used for Western blot analysis: anti-rabbit IgG-HRP and anti-mouse IgG-HRP (Santa Cruz Biotechnology, Dallas, TX, USA). Cross-reactivity was visualized using ECL Western blotting detection reagents and analyzed by scanning densitometry using a UVP Vision Works™ LS Software (UVP, Cambridge, UK) and quantified with ImageJ Software.

Statistical Analysis
Statistical analysis was conducted using Prism 6 statistical software. All results were presented as mean ± SEM. Oneway analysis of variance (ANOVA) followed by the Tukey test was used to compare group differences. P < 0.05 was considered to be statistically significant.

Iron Contents Increased in MO3.13 Cells with FAC Treatment
We first detected the cell viability of MO3.13 cells treated with different concentrations of FAC by CCK-8. The cell viability did not change significantly at 100 mg/L, but decreased significantly at 200 mg/L, 500 mg/L, 1000 mg/L and 2000 mg/L (Fig. 1A). Therefore, FAC concentrations of 100 mg/L, 200 mg/L and 500 mg/L were selected for subsequent experiments. The intracellular ferrous iron, ferric iron and total iron concentrations were increased in FAC treatment group compared with the control group ( Fig. 1B-D).

FAC Treatment Induced Ferroptosis But Not Apoptosis in MO3.13 Cells
In order to determine the mechanism of FAC-induced cell death, we selected 20 µmol/L erastin and 1 µmol/L staurosporine as ferroptosis and apoptosis positive controls, respectively. Using transmission electron microscopy, we observed that the morphology of nucleus in MO3.13 cells was unchanged in FAC-treated groups and erastin groups, whereas the morphology of mitochondria changed. The distinctive morphological feature of FAC-treated cells was changed in mitochondria that appeared smaller than normal with increased membrane density and decreased mitochondrial crest, consistent with the erastin-treated group. With increased concentration of FAC treatment, the ferroptosis phenomenon becomes more and more obvious but the characteristic morphological features associated with apoptosis not appeared. (Fig. 2A). In all FAC treated groups, smaller mitochondria were observed compared with that of the control, and the length of mitochondrial in 200 mg/L and 500 mg/L FAC groups, as well as erastin treated groups decreased in comparison with 100 mg/L FAC group (Fig. 2B). ROS levels were increased in FAC and erastin treated groups compared with that of the control (Fig. 2C). As a marker of ferroptosis, Gpx4 protein levels were also found decreased in cells treated with FAC and erastin groups (Fig. 1D). Meanwhile, we detected the expression of Bax and Bcl-2 protein. Western blots results showed that Bcl-2/Bax ratio was not change in FAC and staurosporine treated groups compared with control group (Fig. 2E). Caspase-3 activation was elevated in staurosporine treated group, but did not change in FAC treated groups (Fig. 2F). Based on the above results, we found that ferroptosis occurred rather than apoptosis after FAC treatment in MO3.13 cells.

Iron Chelator DFO and Ferroptosis Inhibitor Fer-1 Could Inhibit the Ferroptosis Induced by FAC in MO3.13 Cells
In order to confirm the occurrence of ferroptosis, we blocked ferroptosis using iron chelator DFO and ferroptosis specific inhibitor Fer-1 respectively. First, we pretreated MO3.13 cells with DFO which could combine with Fe 3+ to form a stable, non-toxic, water-soluble ferric ammonium, to clear iron-induced oxidative stress for 1 h, and added different concentrations of FAC. Pretreatment of cells with 200 µmol/L DFO completely blocked the decreased of Gpx4 protein (Fig. 3A). When MO3.13 cells were pretreated with ferroptosis specific inhibitor Fer-1 for 1 h, and then added different concentrations of FAC, the changes of Gpx4 protein expression that induced by FAC were blocked (Fig. 3B). Therefore, Fer-1 and DFO pretreatment could significantly antagonize FAC induced changes of ferroptosis. Taking these together, the above results indicated that the FAC induced ferroptosis in MO3.13 cells.

FAC Induced Ferroptosis was Dependent on MAPK Pathway Rather Than p53 Pathway
Previous studies reported that the MAPK and p53 signaling pathway were involved in ferroptosis, we detected the changes of the molecules involved in these signaling pathways in FAC induced ferroptosis. The phosphorylation level of p53 was not changed when cells were treated with FAC, compared with control group (Fig. 4A). Whereas the phosphorylation level of Erk1/2 was markedly increased when cells were treated with FAC, compared with the control group. Especially the ratio of protein expressions of p-Erk1/2/Erk1/2 increased when cells were treated with 200 mg/L and 500 mg/L FAC compared with 100 mg/L FAC treatment group compared with that of the control. This conformed that the occurrence of ferroptosis caused by iron overload might be related to the phosphorylation of Erk1/2 signaling pathway (Fig. 4B).

Discussion
The forms of cell death in PD include apoptosis, necrosis and ferroptosis etc. Ferroptosis is performed by phospholipid peroxidation, a process that relies on transition metal iron, reactive oxygen species (ROS), and phospholipids containing polyunsaturated fatty acid chains (PUFA-PLs) [20]. When cells are treated with classic ferroptosis activator erastin or RSL3 inducers, features that differ from other modes of death occur, such as smaller mitochondrial atrophy and increased membrane density; Accumulation of lipid peroxides in cell membranes; Reduced GSH depletion and iron aggregation, etc. In terms of molecular mechanisms, the upstream signaling pathways of ferroptosis ultimately affect the activity of GPX4 by directly or indirectly [17,18,21,22]. Due to its high metabolic rate and relatively low antioxidant defenses, and the presence of high concentrations of polyunsaturated fatty acids in membrane-rich structures, ROS in the brain increases, leading to oxidative damage, and high levels of peroxidation are the driving force behind ferroptosis [23]. Gpx4 inhibits lipid peroxidation by directly reducing hydroperoxides from membrane lipids and has been shown to regulate ferroptosis [20,21,24,25]. As we predicted, in this study, MO3.13 oligodendrocytes were treated with FAC to simulate iron overload during the onset of PD, and after 24 h of treatment with 100 mg/L, 200 mg/L, 500 mg/L, 500 mg/L FAC, the concentration of intracellular divalent iron, trivalent iron and total iron increased, the length of mitochondrial morphology decreased, the density of mitochondrial membrane increased, the mitochondrial crest decreased, the GPX4 expression decreased, and the level of reactive oxygen species increased significantly, Similar to the cell morphology after treatment with 1 3 ). E Bcl-2 and Bax protein levels and statistical analysis of the ratio of Bcl-2/Bax. Bcl-2/Bax ratio was not change in FAC and staurosporine treated groups compared with control group (n = 6, F = 0.72). F Immunoblotting and statistical analysis of Cleaved caspase-3 protein levels. Cleaved caspase-3 protein levels were increased in staurosporine treated group, but did not change in FAC treated groups (n = 6, F = 7.97). *P < 0.05, **P < 0.01, ***P < 0.001, compared with the control; # P < 0.05, ### P < 0.001, compared with the FAC 100 mg/L 1 3 the ferroptosis inducer erastin. The above indicates that iron overload causes ferroptosis in MO3.13 oligodendrocytes. Since oligodendrocytes are the cells with the highest iron content, ferroptosis occurs mainly in oligodendrocytes, as well as leads to iron deposition and myelin loss [26].
Apoptosis is a type of programmed cell death. Changes in the ratio of pro-apoptotic protein Bax to apoptosis protein Bcl-2 can regulate the process of apoptosis [27,28]. Initiation of apoptosis relies on activation of a series of cysteineaspartate proteases (called caspase), leading to activation of nucleic acid endonucleases, destruction of nucleoproteins  A Immunoblotting and statistical analysis of the ratio of p-p53/p53 level. FAC treatment did not change the ratio of p-p53/p53 level. B Immunoblotting and statistical analysis of the ratio of p-Erk1/2/Erk1/2 level, FAC treatment increased the ratio of p-Erk1/2/Erk1/2 level. *P < 0.05, ***P < 0.001, compared with the control group; # P < 0.05, compared with the FAC 100 mg/L, n = 4, F = 0.64, 17.14 1 3 and cytoskeletons, crosslinking of proteins, expression of phagocyte ligands, and formation of apoptotic bodies leading to DNA breakages, and activation of caspase-3 is considered the main hallmark of early and late apoptosis [29]. The morphological characteristics of apoptosis are cell contraction, chromatin concentration, DNA breakage, mitochondrial swelling, and apoptotic body formation [30]. MES23.5 cells characterized by dopaminergic neurons have been found to have apoptosis under iron overload conditions [19]. In our experiments, there were no obvious changes in Bcl-2/Bax and cleaved caspase-3 after treatment of 100 mg/L, 200 mg/L, and 500 mg/L FAC, and the nuclear membrane and chromatin morphology were observed by electron microscopy, while no apoptosis features such as nucleus fragmentation of chromatin concentrate were found. We used staurosporine-treated oligodendrocytes as a positive control group, whose expression of cleaved caspase-3 increased significantly, but Bcl-2/Bax did not change. Studies have reported that the Bcl-2/Bax ratio is reduced in staurosporine induced apoptosis in pancreatic cancer cells, but Bcl-2/Bax is unchanged while caspase-3 activity enhanced in staurosporine induced apoptosis in suckling mouse cardiomyocytes. Therefore, the ratio of Bcl-2/Bax may be related to the cell specificity in staurosporine induced apoptosis [31].
Ferroptosis can be prevented by enzymatic reactions from two major antioxidant systems, including GPX4-mediated, GSH-specific catalyzing lipid peroxides to inhibit ferroptosis, and the recently discovered ferroptosis inhibitory protein FSP1 catalyzing the regeneration of ubiquinone [11]. As a hallmark of ferroptosis, the expression of GPX4 increased, indicating the occurrence of ferroptosis. The process of ferroptotic cell death is defined by the accumulation of lethal lipid species derived from the peroxidation of lipids, which can be prevented by iron chelators (e.g., deferiprone, deferoxamine) and small lipophilic antioxidants (e.g., ferrostatin, liproxstatin) [32]. As the first ferroptosis inhibitor to be found in cancer cells, the anti-ferroptotic activity of fer-1 is actually due to the scavenging of initiating alkoxyl radicals produced, together with other rearrangement products, by ferrous iron from lipid hydroperoxides [33]. Iron chelator-triggered mitophagy could conceivably prevent neuronal degeneration in PD and other diseases by enhancing the removal of mitochondria that are responsible for producing damaging levels of ROS [3]. There are also studies oligodendrocyte iron-induced cell death and myelination is rescued by iron chelation in PMD pre-clinical models [34]. In our experiments, we also found that using Fer-1 can indeed antagonize the decline in GPX4 caused by iron overload, and DFO can chelate iron to reduce the intracellular iron content, so that GPX4 expression decreases, achieving the effect of inhibiting ferroptosis in cells.
Solute carrier family 7 member 11 (SLC7A11) and GPX4 are key factors in ferroptosis. SLC7A11 encodes an element of the cystine/glutamate antiporter complex, which promotes GSH synthesis by mediating cystine uptake and glutamate release [11,32,35,36]. Recently, further investigations have also revealed that reactive oxygen species-induced ferroptosis could potentially be suppressed by high levels of SLC7A11. P53 is a transcription factor that usually activates gene expression [37]. Some recent studies have shown that p53 activation reduces cystine uptake by inhibiting SLC7A11 transcription, which in turn limits the production of intracellular GSH (the primary cellular antioxidant). Thus, the sensitivity of ROS-induced ferroptosisis markedly increased in p53-activated cells [38][39][40]. However, our results show that after 100 mg/L, 200 mg/L, and 500 mg/L FAC treatment, ferroptosis occurs in MO3. 13 oligodendrocytes, but there is no significant change in intracellular p53 phosphorylation levels, indicating that ferroptosis of MO3.13 oligodendrocytes caused by iron overload may not depend on the p53 pathway. In cell metabolism, the RAS/RAF/MEK/ERK pathway (also known as the MAPK/ ERK pathway) is one of the most important signaling pathways. Iron stimulates ryanodine receptor-mediated calcium release through ROS produced via the Fenton reaction leading to stimulation of the ERK signaling pathway [41]. In Pancreatic ductal adenocarcinoma (PDAC), the activation of the MAPK/ERK pathway is required for erastin-induced STAT3 activation and subsequent ferroptosis, and ERK is an upstream kinase for STAT3 activation in response to various stressors [42][43][44]. By examining the ERK pathway, we found that the ERK phosphorylation level of MO3.13 oligodendrocytes treated with 100 mg/L, 200 mg/L and 500 mg/L FAC indicated that ferroptosis of MO3.13 oligodendrocytes caused by iron overload may be dependent on the ERK pathway.
In summary, Iron overload causes ferroptosis but does not induce apoptosis in oligodendrocytes. The iron chelator DFO and the ferroptosis inhibitor Fer-1 could inhibit the ferroptosis of oligodendrocytes induced by iron overload. The mechanism of ferroptosis in oligodendrocytes induced by iron overload may be related to Mitogen-activated protein kinase pathway activation rather than p53 pathway activation (Fig. 5). This study investigated the occurrence and sequence of different types of cell death modalities following the addition of excess iron in MO3.13 cells, as well as the drugs that inhibit different death modalities, providing a reliable experimental basis for understanding the mechanism of iron overload-induced damage to oligodendrocytes and offering new ideas for the clinical treatment of PD demyelination. oligodendrocytes. FAC induces ROS generation and accelerates lipid peroxidation, which mimics the erastin-induced ferroptosis. In cells, Gpx4 could convert GSH to GSSH, which reduces toxic peroxides to non-toxic hydroxyl compounds, thereby protecting the structure and function of cell membranes from peroxide interference and damage. In opposite, inactivation of Gpx4 blocks GSH synthesis, which lead to the accumulation of membrane lipid ROS and ferroptosis. Excessive free iron induces ROS generation through Fenton reaction, which in turn results in depletion of GSH and the activation of ERK pathway. When the GSH in the cells is depleted, the ability of the cells to clear ROS is decreased, resulting in membrane oxidation by excessive ROS and ultimate ferroptosis. DFO and Fer-1 could inhibit ferroptosis in MO3.13 oligodendrocytes induced by iron overload