Mitochondrial Ferritin Protects Hydrogen Peroxide-Induced Neuronal Cell Damage

Oxidative stress and iron accumulation are tightly associated with neurodegenerative diseases. Mitochondrial ferritin (FtMt) is identified as an iron-storage protein located in the mitochondria, and its role in regulation of iron hemeostasis in neurodegenerative diseases has been reported. However, the role of FtMt in hydrogen peroxide (H2O2)-induced oxidative stress and iron accumulation in neuronal cells has not been studied. Here, we overexpressed FtMt in neuroblastoma SH-SY5Y cells and induced oxidative stress by treating with extracellular H2O2. We found that overexpression of FtMt significantly prevented cell death induced by H2O2, particularly the apoptosis-dependent cell death. The protective effects involved inhibiting the generation of cellular reactive oxygen species, sustaining mitochondrial membrane potential, maintaining the level of anti-apoptotic protein Bcl-2, and inhibiting the activation of pro-apoptotic protein caspase 3. We further explored the mechanism of these protective effects and found that FtMt expression markedly altered iron homeostasis of the H2O2 treated cells as compared to that of controls. The FtMt overexpression significantly reduced cellular labile iron pool (LIP) and protected H2O2-induced elevation on LIP. While in H2O2 treated SH-SY5Y cells, the increased iron uptake and reduced iron release, in correlation with levels of DMT1(-IRE) and ferroportin 1, resulted in heavy iron accumulation, the FtMt overexpressing cells didn’t show any significant changes in levels of iron transport proteins and in the level of LIP. These results implicate a neuroprotective role of FtMt on H2O2-induced oxidative stress, which may provide insights into the treatment of iron accumulation associated neurodegenerative diseases.

accommodates up to 4500 iron atoms [9,10]. Most intracellular iron is stored in the cytosol by binding to ferritin. In mammalian cells, two ferritin subtypes have been found, H-ferritin and L-ferritin. The former subtype has ferroxidase activity essential for the storage of free iron in ferritin, while the latter has a nucleation site that is involved in iron-core formation [11,12]. It has been reported that ferritins exert cellular protective roles against iron-mediated free radical damage induced by a variety of sources [10,13]. In neuronal cells, elevated ferritin expression has been shown to protect the MPTPinduced experimental PD models well [14]. Therefore, it appears that neuronal cell survival is also dependent on the cellular level of ferritin.
Mitochondrial ferritin (FtMt) is a ferritin type protein targeted to mitochondria, and has been characterized structurally and functionally analogous to the wellcharacterized cytosolic H-ferritin [15]. FtMt has been shown to modulate cellular iron metabolism dramatically [16][17][18][19]. Previous studies suggested that overexpression of FtMt caused redistribution of iron from cytosol to mitochondria [16,19], thus high levels of FtMt resulted in an iron deficient phenotype in cytosol [20]. The expression of FtMt is restricted to mitochondria of cells of testes, the central nervous system, and some other high oxygen-consumption tissues [21], indicating that the major role of FtMt may be protecting mitochondria from iron-dependent oxidative damage in cells characterized by high metabolic activity and oxygen consumption [22]. Our previous studies have found that FtMt overexpression protected 6-hydroxydopamine-induced dopaminergic cell damage, potentially playing an important neuroprotective role in Parkinson's Disease [23]. Studies by Campanella et al. revealed a protective role of FtMt in Friedreich ataxia, a disease characterized by mitochondrial iron overload and oxidative damage [24,25]. FtMt expression also inhibited tumor growth due to cytosolic iron deprivation [26]. The protective role of FtMt against oxidative stress in other disease models has also been suggested [22,[27][28][29][30]. These studies demonstrated that FtMt is not only involved in storing cellular iron, but may also play a role in protecting mitochondria from irondependent oxidative damage [22][23][24][25][26][27][28][29][30].
In this study, we aimed to investigate the role that FtMt plays against the oxidative stress to mitochondria induced by H2O2. A recent study by Dev et al. revealed the effect of H2O2 treatment on LIP level and cellular iron-uptake,storage and -release proteins in the neuroblastoma cell line SH-SY5Y [31]. They found that iron heavily accumulated in SH-SY5Y cells after H2O2 treatment, and iron-release protein FPN1 significantly decreased, whereas iron-uptake protein didn't change much [31]. Interestingly, they also found the expression of ironstorage protein H-ferritin was decreased, which was not in accordance with the regulation by the iron-regulatory protein (IRP) [32]. However, the functions of the ironstorage protein in mitochondria, FtMt, in H2O2 induced oxidative stress in neuronal cells have not been studied. We hypothesized that FtMt may play a neuroprotective role in H2O2 induced cell stress. Thus, we overexpressed FtMt gene in the neuroblastoma SH-SY5Y cells to see if an increase of FtMt expression can sequester more free iron and counter the H2O2-induced iron accumulation and cell damage. We further investigated its effects on iron metabolism and the mechanisms of neuroprotection in H2O2-induced apoptosis. This study would be useful for understanding the roles of FtMt in neurodegenerative diseases. It may provide insight into discovering new therapeutic methods for treatment of iron overload-related neurodegenerative disorders.
The WT SH-SY5Y cells, FtMt-SY5Y cells and vector-SY5Y cells were maintained in Dulbecco modified Eagle medium supplemented with 10% fatal bovine serum, 100 U/ml penicillin and 100 U/ml streptomycin. After the cells reaching ~80% confluency, H2O2 were added to a final concentration of 100 μM (or as described in each specific experiment), and cells were then incubated at 37℃ for 24 h prior to analysis.

Western blotting
After H2O2 treatment (100 μM, 24 h), cells were homogenized and lysed with RIPA buffer and protein content was determined by the Bradford assay. Aliquots of cell lysate containing approximately 30 μg of protein were immediately mixed with loading buffer and boiled for 10 minutes. Equal amounts of protein for different cells were loaded, resolved by sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a PVDF membrane. The blots were blocked by incubating with 5% nonfat milk in PBS containing 0.1% Tween 20 (PBS-T) for 1 h, and hybridized with primary antibodies. After washing 3 times for 15 minutes each with PBS-T, the blots were incubated for 1 h with peroxidase-coupled secondary antibodies, and detected with the ECL plus Western Blotting Detection Reagents (Pierce Biotechnology, Rockford IL). Six independent experiments were performed for each treatment.

Detection of cell apoptosis
After H2O2 treatment (100 μM, 24 h), cell apoptosis was detected by using annexin V/PI assay by flow cytometry as described previously [23].

Measurement of the labile iron pool (LIP) level
The LIP level was determined by the quenching of calcein fluorescence as described previously [36,37]. After treatment with or without 100 μM H2O2 for 24 h, the cells were harvested, washed, and resuspended in PBS (pH 7.4) buffer containing calcein-AM (0.25 μM final concentration), and incubated for 30 min at 37℃. The excess calcein-AM on the cell surface was washed out 3 times with PBS. The fluorescence intensity of calcein-AM was quantified by a fluorescence spectrophotometer (Hitachi F-4500), at an excitation wavelength of 488 nm and an emission wavelength of 525 nm. When the baseline was stable, SIH (100 μM final concentration) was added, and the increase in fluorescence intensity reflected the levels of calcein-bound iron. Three independent experiments were performed for each treatment.

Detection of intracellular oxidative stress-ROS assay
Intracellular ROS were examined using 2',7'dichlorofluorescein diacetate (DCF-DA) as described before [38]. After treatment with or without 100 μM H2O2 for 24 h, cells were harvested, washed, and resuspended in PBS containing DCF-DA (10 μM final concentration), and incubated for 30 min at 37℃. Cells were washed twice with PBS, and the fluorescence signals were measured by a fluorescence spectrophotometer with 488 nm excitation and 525 nm emission wavelengths. Three independent experiments were performed for each treatment. The data were expressed as a percentage of the fluorescence relative to the fluorescence of the untreated WT control cells.

Detection of mitochondrial membrane potential (MMP)
Changes in the MMP with or without H2O2 treatment (100 μM, 24 h) of SH-SY5Y cells were determined by measuring the retention of rhodamine 123 using flow cytometry [39]. The uptake of rhodamine123 into mitochondria is an indicator of the MMP. Cells were incubated with rhodamine123 at a final concentration of 5 μM for 30 min at 37℃. After washing twice with PBS, fluorescence was recorded at 488 nm excitation and 525 nm emission wavelengths. Three independent experiments were performed for each treatment. 55 Fe ( 55 FeCl3, Perkin-Elmer Life Sciences Company, Wellesley City, MA) solution was prepared by mixing 55 FeCl3 with FeSO4 in a molar ratio of 1:10 followed by a 50-fold molar excess of 2-mercaptoethanol and 0.27 M sucrose (pH 6.5) as described previously [40,41]. The H2O2 treated (100 μM, 24 h) or untreated cells (about 1×10 6 cells) were washed three times with PBS buffer, and added with 55 FeCl3 solution (1 mM) for 30 min at 37 ℃ and then washed three times with PBS. The cells were homogenized in buffer containing 1% sodium dodecyl sulphate (SDS), and aliquots of the total cell extract were assayed for released radioactivity with Liquid Scintillation Analyzer (Beckman) and protein concentrations by Lowry method. Three independent experiments were performed for each treatment.

Measurement of iron uptake and release
For measurement of iron release, the cells were incubated with 55 FeCl3 solution for 30 min and then washed three times with PBS. The cells were then incubated with 1 ml PBS at 37 ℃ for 30 min. The supernatant was collected. The cells were homogenized after washing three times with PBS. Both supernatant and cell extract were assayed for radioactivity. Percentage (%) of 55 Fe release = (cpm in supernatant) / (cpm in supernatant + cpm in cells) × 100%.

Statistical analysis
All statistical analyses were completed using SPSS 21.0 software. Results are presented as means ± SD. The statistical analyses of group differences were assessed by a two-way analysis of variance (ANOVA). P-values of <0.05 were considered to be statistically significant; P <0.01 was considered to be remarkably significant.

FtMt overexpression rescued neuronal cell death induced by H2O2 treatment
Previous studies have indicated that FtMt plays an important role in protecting cells from iron-dependent oxidative damage [23,25,27,30], particularly in neurodegenerative diseases [42,43]. In order to investigate the role of FtMt in hydrogen peroxide (H2O2)induced neuronal cell damage, a stable FtMtoverexpressing neuroblasma cell line, FtMt-SY5Y, generated previously was used [23]. Wild-type SH-SY5Y cells and pcDNA3.1(-) empty plasmid transfected cells (vector-SY5Y) were used as controls. The expression of FtMt in FtMt-SY5Y cells was confirmed (Fig. 1A). The level of overexpressed mouse FtMt was much higher than that of the endogenous human FtMt. The cell viability of the three cell lines was determined after H2O2 treatment for 24 h. Results showed that H2O2 reduced cell viability in all three cell lines in a concentration-dependent manner (Fig. 1B). However, after H2O2 treatment, at concentrations of 60, 80, 100, 120 and 140 μM, cell viability in FtMt-SY5Y cells was significantly higher than that of the vector-SY5Y cells (P < 0.01, Fig. 1B). With 100 μM H2O2 treatment, the viability of SH-SY5Y and vector-SY5Y cells were decreased to ~40%, whereas the viability of FtMt-SY5Y cells only had a ~11% decrease. 100 μM of H2O2 treatment was then used for the following assays. These results indicated that H2O2 induced cell damage and that overexpression of FtMt in SH-SY5Y cells improved cell viability.  To explore if the rescue effect of FtMt was resulted from increased resistance of FtMt-SY5Y cells against apoptosis induced by H2O2, the apoptotic rates in vector-SY5Y and FtMt-SY5Y cells were quantified by flow cytometry after annexin V and PI staining. After treated with 100 μM H2O2 for 24 h, the ratio of apoptotic vector-SY5Y cells increased from 4.7% to 39.5% (P < 0.01, Fig.  1Cii, indicated by **); the apoptotic ratio of FtMt-SY5Y cells also increased (from 4.9% to 16.5%, P < 0.01). However, when comparing the increases between FtMt overexpressing cells and vector control cells, the FtMt overexpressing cells had significantly lower number of apoptotic cells (P < 0.01, Fig. 1C, indicated by ##). The results inferred that FtMt has a significant anti-apoptotic role in H2O2-induced cell damage.

FtMt maintained Bcl-2/Bax ratio and inhibited the activation of caspase 3
Bcl-2 and Bax are anti-apoptotic and pro-apoptotic proteins, and the Bcl-2/Bax ratio has been widely used to monitor apoptosis [44]. To further confirm if the rescue effect of FtMt on cell death was resulted from decreasing cell apoptosis, the ratios of Bcl-2/Bax for vector-SY5Y and FtMt-SY5Y cells with/without H2O2 treatment were quantified by western blotting. In vector-SY5Y cells, dramatic decreases in Bcl-2 levels and increases in Bax levels were observed after H2O2 treatment as compared to that of the untreated control group, causing obviously reduced Bcl-2/Bax ratios (P < 0.01, Fig. 2A). However, the FtMt over-expressed FtMt-SY5Y cells maintained the levels of both Bcl-2 and Bax, showing a relatively small change in the Bcl-2/Bax ratio (P < 0.05, Fig. 2A). H2O2 treatment also caused caspase 3 activation in SH-SY5Y cells and vector-SY5Y cells, as shown by the increased cleaved-caspase 3 levels (Fig. 2B), whereas the activation wasn't observed in the FtMt-SY5Y cells. These findings indicate that FtMt significantly maintains the normal level of Bcl-2/Bax and prevents the activation of caspase 3 to protect the cells against H2O2-induced apoptosis, thereby protecting cells from death.

FtMt attenuated H2O2-induced increases in ROS level and H2O2-induced decreases in mitochondrial membrane potential (MMP)
Evidences have strongly suggested that apoptosis is closely linked to production of damaging ROS during electron transport [7,45], therefore we determined the effect of FtMt expression on the ROS level, by measuring the ROS-dependent fluorescence of DCF-DA using flow cytometry [38]. The relative ROS levels of different cells with/without H2O2 treatment and their differences are shown in Fig. 3A. The H2O2 treatments led to significant increases in intracellular ROS production in both SH-SY5Y and vector-SY5Y cells as compared to the untreated controls (P < 0.01). In contrast, the ROS level of FtMt-SY5Y cells only increased slightly by H2O2 treatment as compared to the untreated control. This indicates that although the overexpression of FtMt doesn't alter the intracellular ROS levels in SH-SY5Y cells under normal conditions, it can significantly attenuate the H2O2induced increase in ROS level, which therefore protects the SH-SY5Y cells from oxidative damage. The state of MMP is an indicator of the metabolic activity of mitochondria and closely relates to mitochondrial dysfunction and ROS overproductioninduced apoptosis [39]. To examine the underlying protective mechanism of FtMt on mitochondria, the MMP of the three different cell lines was measured. The results showed that H2O2 treatment decreased MMP by approximately 36% and 38% in SH-SY5Y and vector-SY5Y cells (P < 0.05, Fig. 3B), respectively, as compared with the untreated control groups; however, in FtMt-SY5Y cells, the MMP decreased only about 12% (Fig.  3B). These results indicate that FtMt maintains the MMP and protects against the mitochondrial damage induced by H2O2.

FtMt significantly decreased intracellular labile iron pool (LIP) levels
Excess ferrous ion (Fe 2+ ) can result in the generation of ROS, which damages cellular macromolecules including proteins, lipids and DNA, and consequently triggers apoptosis [6][7][8]46]. It has been shown that FtMt could mobilize iron into mitochondria and dramatically redistributes intracellular iron [16,20]. To further clarify FtMt's protective mechanism on cell death, the LIP levels in different cell lines were quantified by using ironspecific fluorescence probe calcein-AM. Accessible free iron quenched by calcein was quantified and shown in Fig. 4. The FtMt overexpressing cells indeed had lower amount of LIP as compared with the WT SH-SY5Y cells (P < 0.05, indicated by &). H2O2 treatment dramatically raised the LIP levels in WT and vector transfected SH-SY5Y cells (P < 0.01, indicated by **), but it only led to a minor increase in the LIP levels in FtMt overexpressing cells. These results infer that the largely increased LIP in SH-SY5Y cells, following H2O2 treatment, results in the increased ROS and apoptosis as observed above. The protective role of FtMt overexpression on H2O2-induced elevation in LIP is remarkably significant.

FtMt inhibited H2O2-induced elevations in iron uptake and reductions in iron release
Iron-induced oxidative stress can be very destructive because of a positive-feedback loop developed from the release of more free iron from the iron-containing proteins, such as ferritin, heme proteins and Fe-S clusters [7]. As a result, the toxic effect of iron overload in neuronal cells is exacerbated. To clarify the mechanisms of FtMt in regulating intracellular iron homeostasis and the consequent neuroprotective role in neuronal cells, the iron uptake and release were determined by 55 Fe isotope tracer experiments in vector-SY5Y cells and FtMt-SY5Y cells after treated with H2O2. The results showed that the cellular 55 Fe uptake increased significantly in all cell lines after H2O2 treatment (P < 0.01, Fig. 5A), but the increased amount in FtMt-SY5Y cells is much less than that of the vector-SY5Y controls (P < 0.05, indicated by ##). The 55 Fe release in the control vector-SY5Y cells decreased significantly (P<0.01, Fig. 5B), whereas iron release in FtMt-SY5Y cells only slightly decreased. These results suggest that H2O2 causes cells to uptake more iron, but release less; therefore, iron accumulates in cytosol. However, when FtMt overexpresses, it partially counteracts these effects and maintained a relatively low cellular iron level, protecting cells from damage.

FtMt maintained iron transport proteins at relative steady levels
To explore the protective function of FtMt on oxidative damage at molecular level, we examined the alterations of iron transport proteins induced by H2O2 treatment in different cell lines. In SH-SY5Y and vector-SY5Y cells, following H2O2 treatment, the iron efflux protein ferroportin 1 (FPN1) significantly decreased (P < 0.05, Fig. 6A). The iron uptake proteins, transferrin receptor 1 (TfR1) and divalent metal transporter 1 with iron responsive element (DMT1(+IRE)), didn't show significant changes (Fig. 6B and 6C), but DMT1 without iron responsive element (DMT1(-IRE)) increased dramatically (P < 0.01, Fig. 6D). The changes in FPN1 and DMT1(-IRE) may account for the observed intracellular iron accumulation in SH-SY5Y cells caused by H2O2 treatment [31].  55 Fe uptake levels were shown as iron concentrations. Data represent means ± SD; n=3 (*, P <0.05, and **, P < 0.01 vs. the untreated cells of same genotype; #, p < 0.05 vs. the H2O2-treated vector controls). (B) 55 Fe release levels were the percentages of iron in supernatant of cell culture against total iron (sum of supernatant and cell lysate). Data represent means±SD; n=3 (**, P < 0.01 vs. the untreated cells of same genotype; ##, p < 0.01 vs. the H2O2-treated vector controls).
In addition, we found that, prior to H2O2 treatment, the FtMt-SY5Y cells alone had lower FPN1 expression (P < 0.05, indicated by &, Fig. 6A) and higher TfR1 expression (P < 0.05) than the untreated vector-SY5Y controls. These findings were consistent with our previous observations [23] and may attribute to cellular regulations in response to low LIP level caused by FtMt overexpression.

FtMt inhibited H2O2-induced reductions in cytosolic ferritin levels
The effects of H2O2 treatment on cytosolic ferritin levels in different cells were also determined. As shown in Fig.  6E, L-ferritin levels in SH-SY5Y and vector-SY5Y cells significantly decreased after H2O2 treatment (P < 0.01). However, the FtMt overexpressing cells had low levels of cytosolic L-ferritin, despite with or without H2O2 treatment. The H-ferritin levels in all three cells decreased slightly, but not statistically significant (Fig. 6F). These results indicated that H2O2 induced downregulations of cytosolic ferritins, particularly L-ferritin, and that FtMt overexpression counteracted these effects. The FtMt-SY5Y cells had low levels of cytosolic ferritins, which were consistent with their low intracellular LIP levels, attesting the iron redistributing function of FtMt.

DISCUSSION
At normal condition, cells were in a balance between ROS and antioxidant. Once the balance was disrupted, cells would be exposed to oxidative stress damage. ROS destroys the enzymes in cytosol, induces lipid peroxidation, decreases MMP, and further leads to cell apoptosis. Iron is one of the important activators that lead to oxidative stress damage [6,46]. Thus, the iron level in the body is under strict control, and dysregulation of iron homeostasis could cause severe diseases. FtMt, as an ironstorage protein in mitochondria, has been reported to be involved in iron redistribution from cytosol to Aging and Disease • Volume 8, Number 4, August 2017 466 mitochondria [15,16,20]. Recently, FtMt was mainly recognized for its protective role in diseases associated with iron-dependent oxidative damage [23][24][25][27][28][29], although that FtMt sensitized cells to oxidative stress was also reported [47]. Our previous studies have indicated the neuroprotective role of FtMt in Parkinson's disease and Alzheimer's disease [23,27]. In this study, we explored the effects of FtMt on H2O2 induced neuronal cell damage and further investigated FtMt's functions at the molecular level.  An FtMt overexpressing neuroblastoma SH-SY5Y cell line is used to better investigate the functions of FtMt. Our results indicated that overexpression of FtMt significantly protected neuroblastoma cells from H2O2 induced apoptosis, which was due to the restriction effects of FtMt on the increase of intracellular ROS and the decrease of MMP, thus protecting cells from oxidative stressmediated apoptosis. On the molecular level, we found that H2O2 decreased the expression of Bcl-2 and increased the level of Bax dramatically in SH-SY5Y and vector-SY5Y cells, while the changes of Bcl-2/Bax ratio in FtMt-SY5Y cells were much smaller. Our results also showed that caspase 3, another important member of the apoptotic family, was activated by H2O2 treatment in SH-SY5Y and vector-SY5Y cells, leading to caspase 3-dependent cell apoptosis. However, in the FtMt overexpressing cells, no obvious increase in cleaved-caspase 3 level was observed.
It has been reported that H2O2 can modulate cellular iron metabolism [48,49]. It affects cellular iron acquisition by both IRP1-dependent and -independent mechanisms, and modulates intracellular iron distribution at a time-dependent manner by unknown mechanisms [48]. Consistent with this, our results showed that after H2O2 treatment, cellular LIP levels greatly increased in SH-SY5Y and vector-SY5Y cells, reaching more than 2times of the untreated cells. This change may attribute to two aspects. One is the observed higher iron uptake and lower release, correlating with lower expression of iron efflux protein FPN1 and higher iron absorption protein DMT1(-IRE); the other reason might be the internal iron release from various intracellular iron-containing sources, including ferritin, induced by oxidative stress after H2O2 treatment [47,48,50].
The reduction of FPN1 is consistent with the newly published study by Dev, et al. [31], in which the role of extracellular H2O2 in regulation of iron homeostasisrelated genes was exhaustively investigated. They concluded that the substantially reduced FPN1 may be responsible for iron accumulation in the H2O2 treated cells, whereas the alterations of other iron-transport proteins were not obvious. However, they only determined total DMT1 levels, which didn't alter much. Here, we found that the DMT1(+IRE) showed a statistically insignificant decrease following H2O2 treatment, which was likely due to the increased ironbinding activity of IRP1 as reported previously [47,50]. But interestingly, we found that the IRP-independent DMT1(-IRE) expressed to a significantly higher level. DMT1(-IRE) is an important protein involved in iron transport, however, its molecular mechanism was still unknown [51,52]. We hypothesized that the increased expression of DMT1(-IRE) on cell membrane may account for the increase of iron absorption observed in our 55 Fe isotope tracer experiments; in addition, the DMT1 on membrane of cell organelles, such as membranes of lysosome and mitochondria [53,54], may also elevate, likely playing important roles in iron redistribution.
In FtMt-SY5Y cells, we found the LIP levels significantly reduced. However, it seems that the iron acquisition increased and iron release decreased as indicated by the higher TfR1 and lower FPN1 expression. These suggested that the increased iron influx was preferentially transferred into mitochondria and incorporated into FtMt rather than into cytosol [16,20], which explains the decreased cytosolic LIP even with higher iron acquisition and lower iron release. Despite the differences of FtMt-SY5Y cells from WT SH-SY5Y on iron metabolism, the over-expressed FtMt protected H2O2-induced elevation in LIP in FtMt cells. Similar to the WT or vector controls, H2O2 treatment indeed induced higher iron uptake and lower iron release in FtMt-SY5Y cells, however, their LIP level didn't increase significantly because of the overexpressed FtMt. Consistent with these, both the iron-transport proteins and the cytosolic iron-storage proteins were not altered notably by the extracellular H2O2 treatment. These all indicated that overexpression of FtMt can cause dramatic redistribution of cellular iron, even at a severe condition, such as extracellular H2O2 treatment.
In summary, our results showed that FtMt played an important role in rescuing cellular damage induced by H2O2, which was achieved by regulating iron metabolism (Fig. 7). In SH-SY5Y cells, H2O2 treatment causes an increase in DMT1(-IRE) expression and decrease in FPN1 expression, resulting in the increased intracellular LIP level. The increased LIP level induces the generation of ROS, which subsequently lead cells to apoptosis, which possibly involves the decrease of Bcl-2/Bax ratio and the activation of apoptosis signal pathway protein caspase 3. In the FtMt overexpressing cells, a great amount of free iron was captured and stored in FtMt, resulting in a substantial decrease in LIP level. Therefore, under H2O2 treatment, the FtMt overexpressing cells decreased the production of ROS, conserved MMP, maintained the antiapoptotic protein Bcl-2 level, and inhibited the activation of caspase 3. Consequently, FtMt protected cells from oxidative damage.
Thus, our current study revealed a protective role of FtMt in neuronal cells against H2O2 induced oxidative damage, which was achieved by modulating the homeostasis of iron metabolism. This observation is consistent with the study by Al-Qenaei et al., in which a defensive effect of FtMt against H2O2 treatment in Jurkat T cells was implicated [30]. Since dysregulation of iron homeostasis, together with oxidative stress, has been largely demonstrated in several neurodegenerative disorders [3][4][5][6]43], modulation of FtMt expression may prevent or cure these diseases. This process can be greatly facilitated by investigating the mechanisms of regulation of FtMt expression [55,56]. This study may provide insight into the development of novel effective strategies for treatment and prevention of neurodegenerative diseases caused by iron-dependent oxidative damage.