Mitochondrial Ferritin Deletion Exacerbates <i>β</i>-Amyloid-Induced Neurotoxicity in Mice

Wang, Peina; Wu, Qiong; Wu, Wenyue; Li, Haiyan; Guo, Yuetong; Yu, Peng; Gao, Guofen; Shi, Zhenhua; Zhao, Baolu; Chang, Yan-Zhong

doi:https://doi.org/10.1155/2017/1020357

Oxidative Medicine and Cellular Longevity

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Abstract Introduction Materials and Methods Results Discussion Conclusion Authors’ Contributions Acknowledgments Supplementary Materials References Copyright Related Articles

Research Article | Open Access

Volume 2017 | Article ID 1020357 | https://doi.org/10.1155/2017/1020357

Mitochondrial Ferritin Deletion Exacerbates β-Amyloid-Induced Neurotoxicity in Mice

Peina Wang,¹Qiong Wu,¹Wenyue Wu,¹Haiyan Li,¹Yuetong Guo,¹Peng Yu,¹Guofen Gao,¹Zhenhua Shi,¹Baolu Zhao,¹and Yan-Zhong Chang¹

Academic Editor: Rodrigo Franco

Received11 Oct 2016

Accepted27 Dec 2016

Published16 Jan 2017

Abstract

Mitochondrial ferritin (FtMt) is a mitochondrial iron storage protein which protects mitochondria from iron-induced oxidative damage. Our previous studies indicate that FtMt attenuates β-amyloid- and 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells. To explore the protective effects of FtMt on β-amyloid-induced memory impairment and neuronal apoptosis and the mechanisms involved, 10-month-old wild-type and Ftmt knockout mice were infused intracerebroventricularly (ICV) with A to establish an Alzheimer’s disease model. Knockout of Ftmt significantly exacerbated A-induced learning and memory impairment. The Bcl-2/Bax ratio in mouse hippocampi was decreased and the levels of cleaved caspase-3 and PARP were increased. The number of neuronal cells undergoing apoptosis in the hippocampus was also increased in Ftmt knockout mice. In addition, the levels of L-ferritin and FPN1 in the hippocampus were raised, and the expression of TfR1 was decreased. Increased MDA levels were also detected in Ftmt knockout mice treated with A. In conclusion, this study demonstrated that the neurological impairment induced by A was exacerbated in Ftmt knockout mice and that this may relate to increased levels of oxidative stress.

1. Introduction

Alzheimer’s disease (AD) is a multifaceted neurodegenerative disease of the elderly which is characterized by neuronal loss, neuroinflammation, and progressive memory and cognitive impairment [1]. Many pathogenic factors are involved in the neuropathology, including accumulation of β-amyloid (Aβ), oxidative stress, inflammation, and metal deposition [2]. Aβ is considered to be a major factor in the pathophysiological mechanisms underlying AD and has been shown to directly induce neuronal cell death [3]. Thus, Aβ is a useful tool for establishing AD models and investigating the mechanisms involved in AD pathogenesis [4]. A is an 11-amino acid fragment located in the hydrophobic functional domain of the C-terminal region of A [5]. Single administration of A into the lateral ventricles of mice or rats impairs memory and induces neurodegeneration in the hippocampus [6–10]. We have previously shown that A, like A, exerted neurotoxic effects on SH-SY5Y cells [11]. These data are among numerous studies confirming that A is a useful tool for investigating AD-related mechanisms in animal models [10].

Oxidative stress has been strongly implicated in the pathophysiology of AD [12]. Increased free radicals can damage proteins, lipids, and nucleic acids. The combination of mitochondrial dysfunction and Aβ accumulation generates reactive oxygen species (ROS) which, in the presence of metal ions such as Fe²⁺ and Cu²⁺, may contribute to oxidative damage in AD brains [13]. In addition, dysregulated brain iron homeostasis also accelerates AD progression. Excessive iron in the brain can directly lead to the generation of free radicals that eventually cause neurodegenerative disease.

Mitochondrial ferritin (FtMt) is a recently identified ferritin that accumulates specifically in the mitochondria and possesses a high homology to H-ferritin [14]. The functions of FtMt include iron storage, regulating iron distribution between the cytosol and mitochondrial compartments, and preventing the production of ROS generated through the Fenton reaction [15, 16]. FtMt has been reported to be present in relatively low abundance in the liver and splenocytes, the major iron storage sites, while FtMt is found at higher levels in the testis, kidney, heart and brain, and tissues with high metabolic activity. Together with the knowledge that H-ferritin expression confers an antioxidant effect, the tissue distribution of FtMt is in line with a protective function of FtMt in mitochondria against iron-dependent oxidative damage [17]. Previous results indicated that FtMt was involved in the pathogenesis of neurodegenerative diseases, including AD, Parkinson’s disease, and Friedreich’s ataxia [18–20]. Increased expression of FtMt has been observed in the brains of AD patients and is associated with the antioxidant role of this protein [2]. Furthermore, our previous studies have shown that FtMt exerted a neuroprotective effect against 6-hydroxydopamine- (6-OHDA-) induced dopaminergic cell damage [20] and FtMt overexpression attenuated Aβ-induced neurotoxicity [11].

Although abnormal iron metabolism and oxidative stress have been reported in AD, little information is available about the role of FtMt in the pathogenesis of AD. In the present study, we investigated memory impairment and neuronal cell death in A-injected Ftmt knockout mice. In addition, we explored the molecular mechanisms responsible for neuronal damage in this model. Our data indicate that FtMt deficiency exacerbated A-induced neuronal cell damage by altering intracellular iron levels in a way that intensifies the oxidative stress caused by A.

2. Materials and Methods

2.1. Animals

C57BL/6 Ftmt-null mice were obtained from The Jackson Laboratory [21]. Mice were housed under conditions controlled for temperature (22°C) and humidity (40%), using a 12 hr/12 hr light/dark cycle [22]. Mice were fed a standard rodent diet and water ad libitum. Age-matched C57BL/6J wild-type male mice and Ftmt knockout male mice (10 months) were used in this study. All procedures were carried out in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and were approved by the Animal Care and Use Committee of the Hebei Science and Technical Bureau in China.

2.2. Antibodies and Reagents

The following antibodies and reagents were used: β-actin (Alpha Diagnostic International, USA), TfR1 (Sigma-Aldrich, USA), FPN1, DMT1 (+IRE) and DMT1 (−IRE) (Alpha Diagnostic International, USA), L-ferritin (Abcam Inc., SF, USA), cleaved PARP, caspase-3, phospho-p38 (p-p38) and p38 (Cell Signaling Technology, USA), Bcl-2 and Bax (Santa Cruz Biotechnology, USA), A peptide (Sigma-Aldrich, USA), and TUNEL in situ Cell Death Detection Kit (Roche Diagnostics GmbH, Mannheim, Germany).

2.3. Drug Preparation and Injection

A was dissolved in sterile saline and aggregated by incubation at 37°C for 4 days before use [23]. The aggregated form of A (7.5 nmol in 5 μL saline per injection) was injected into the right lateral ventricle as previously described [24]. Mice were randomly divided into four groups: wild-type with saline injection (WT + saline), wild-type with A injection (WT + A), Ftmt knockout with saline injection (KO + saline), and Ftmt knockout with A injection (KO + A). After injection, the mice were housed for 15 days under normal conditions and then trained and tested in a Morris water maze (MWM) as described below.

2.4. Morris Water Maze Test (MWM Test)

Spatial learning and memory deficits were assessed using the Morris water maze as described previously [25, 26], with minor modification. The experimental apparatus consisted of a circular tank (diameter = 120 cm, height = 50 cm) that was divided into four quadrants, filled with water, and maintained at °C. At first, a visible platform test was performed, which confirmed that there were no significant differences in sensory, motor, or motivational activities among these four groups. Then, hidden platform tests were performed in succession. For the hidden platform test, a round platform (diameter = 9 cm) was placed at the midpoint of the fourth quadrant, 1 cm below the water surface. The test was conducted four times a day for four days, with four randomized starting points. The position of the escape platform was kept constant. Each trial lasted for 90 s or ended as soon as the mice reached the submerged platform.

2.5. Probe Test

To assess memory consolidation, a probe test was performed 24 h after the Morris water maze test [25]. For the probe test, the platform was removed and the mice were allowed to swim freely. The swimming pattern of every mouse was recorded for 90 s with a camera. Consolidated spatial memory was estimated by the time spent in the target quadrant area.

2.6. Assessment of Apoptosis

After the behavioral testing, the animals were perfused with 0.9% saline under anesthesia with 0.4% Nembutal. The brains were immediately collected and then postfixed with 4% paraformaldehyde in 0.1 M phosphate buffer. Serial coronal sections were cut at 15 μm on a freezing microtome (Leica CM1950, Leica Microsystems, Shanghai, China) and mounted onto slides covered with APES (Beijing Zhongshan Biotechnology, Beijing, China). The presence of apoptosis in the dentate gyrus of mouse hippocampi was assessed by the terminal deoxynucleotidyl transferase-mediated FITC-dUTP nick-end labeling method (TUNEL) following the manufacturer’s protocol. Nuclei were counterstained with DAPI. The number of TUNEL-DAPI-positive cells was counted as described previously [27]. The counting area was located in the same position in all groups. For each group, quantification was performed in sections from three different mice.

2.7. Western Blot Analysis

Protein expression was assessed by western blotting as previously described [28], with minor modifications. Briefly, hippocampi were homogenized and sonicated in RIPA buffer containing 1% NP40 and protease inhibitor cocktail tablets (Roche Diagnostics GmbH, Roche Applied Science, 68298 Mannheim, Germany). After centrifugation at 12,000 ×g for 20 min at 4°C, the supernatant was collected, and the whole cell lysate protein concentration was measured using the BCA Protein Quantification Kit (Yeasen Biotechnology, Shanghai, China). Protein from each sample (40 mg) was resolved by SDS-PAGE on 12% or 10% gels and then transferred to PVDF membranes. The blots were blocked in 5% nonfat milk containing 20 mM Tris-HCl (pH 7.6, 137 mM NaCl, and 0.1% Tween-20; TBS-T) for 1.5 h at room temperature, followed by incubation with primary antibody overnight at 4°C. After washing three times with TBS-T, the blots were incubated with horseradish peroxide (HRP) conjugated secondary antibody for 1.5 h at room temperature. Immunoreactive proteins were detected using the enhanced chemiluminescence (ECL) method and quantified by transmittance densitometry using volume integration with Multi Gauge ver. 3.1 software (FUJIFILM Corporation, Tokyo, Japan).

2.8. Measurement of MDA and SOD

Malondialdehyde (MDA), a marker of lipid peroxidation, was assessed using the thiobarbituric acid (TBA) method [29] using a kit from the Nanjing Jiancheng Bioengineering Institute (Nanjing, China) according to the manufacturer’s instructions. This method is based on the spectrophotometric measurement of the product of the reaction of TBA with MDA. MDA concentrations were then calculated by the absorbance of TBA reactive substances (TBARS) at 532 nm.

Superoxide dismutases (SODs), which catalyze the dismutation of superoxide into oxygen and hydrogen peroxide, were determined according to xanthine oxidase method using a commercial kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s instructions. The xanthine-xanthine oxidase system produces superoxide ions, which can react with 2-(4-iodophenyl)-3-(4-nitrophenol-5-phenlyltetrazolium chloride) to form a red formazan dye, which can be detected by its absorbance at 550 nm [29].

The levels of MDA and the total SOD (T-SOD) activity were determined in each group. The hippocampi of mice were homogenized in ice-cold saline. The homogenate was centrifuged at 3000 ×g at 4°C for 15 min, and the supernatant was used to determine T-SOD activity and MDA levels with a spectrophotometer (Synergy H4, BioTek, USA) at wavelengths of 550 nm and 532 nm, respectively. Each group contained five mice for the MDA and SOD tests, with each test repeated three times.

2.9. Statistical Analysis

All data are expressed as the mean ± standard deviation. One-way analysis of variance was used to estimate overall significance and was followed by Tukey’s post hoc test corrected for multiple comparisons. A probability level of 95% () was considered significant. All the tests were performed with SPSS 21.0 (IBM SPSS21.0, Armonk, New York, United States).

3. Results

3.1. Ftmt Ablation Exacerbates Aβ_25–35-Induced Spatial Memory Deficits

The MWM test was conducted to assess learning and memory in 10-month-old wild-type mice (WT) and Ftmt knockout mice (KO). All mice were trained with four trials per day for 4 days. “Escape latency” is the time to reach the platform in the water maze and is used as a proxy for mouse memory. Compared to wild-type mice, Ftmt knockout mice took approximately the same time to reach the platform after training (Figure 1(a)). After the water maze test, we performed a probe test using the metric “time spent in quadrant” to investigate the maintenance of memory. The time spent in the target quadrant was also similar in wild-type and Ftmt knockout mice (Figure 1(b)). After treatment with A, both the WT + A group and the KO + A group took a significantly longer time to reach the platform than the groups without A injection (Figure 1(c)). Furthermore, A-infused Ftmt knockout mice had a significantly greater memory impairment (longer escape latency time) than A-infused wild-type mice. In addition, the time spent in the target quadrant in the probe trial was less in both the WT + A and the KO + A groups than in the control groups. Importantly, the KO + A group was in the target quadrant for even less time than the WT + A group (Figure 1(d)). Overall, our results show that knockout of Ftmt in mice significantly exacerbates memory deficits in the A-induced AD model.

(a)

(b)

(c)

(d)

Figure 1

The effect of Ftmt ablation on A-induced spatial memory deficits. (a) Age-matched (10 months old) Ftmt knockout mice () and wild-type mice () were administered a 90 s trial four times a day to find the hidden platform. The analysis of the recorded data shows the changes in latency to find the hidden platform over the four consecutive days of training. (b) Ftmt knockout mice and wild-type mice were assessed in the probe test one day after the hidden platform test. The time spent in the target quadrant within the 90 s was recorded. (c) The effect of A on escape latency. (Wild-type mice and Ftmt knockout mice were randomly divided into four groups and injected with A or saline. Fifteen days later, the MWM test was conducted.) (d) The time spent in the target quadrant during the probe test after injecting A. The data is presented as the mean ± SD. versus WT + saline group, . , versus KO + saline group, . versus WT + A group, . KO + A, .

3.2. Ftmt Ablation Enhances Aβ_25–35-Induced Neuronal Cell Apoptosis

To evaluate the neuronal apoptosis affected by Ftmt gene ablation in the AD model, we used the TUNEL method to detect apoptosis after A stimulation. Our results indicated that neuronal apoptosis in the hippocampi was increased after injecting A, especially in the dentate gyrus. The number of apoptotic cells in the WT + A group was approximately four times greater than that observed in the WT + saline group, and there was also a noticeable increase in the KO group. The number of apoptotic cells in the KO + A group was more than threefold that of the WT + A group. These results confirmed that Ftmt knockout significantly enhanced neuronal apoptosis compared to the WT + A group (Figures 2(a) and 2(b)), suggesting that FtMt is protective against A-induced apoptosis.

(a)

(b)

3.3. FtMt Deficiency in the AD Mouse Model Elevates Proapoptotic Signals

We found that the knockout of Ftmt remarkably decreased the ratio of Bcl-2/Bax (Figure 3(a)) and increased the activation of cleaved caspase-3 (Figure 3(b)) after A treatment in mice. In the apoptotic cascade, caspase-3 cleaves poly-ADP-ribose-polymerase (PARP), leading to the accumulation of an 89 kDa PARP fragment [30]. Caspase-3-mediated PARP cleavage was enhanced in the KO + A group compared to the WT + A group (Figure 3(c)). These results indicate that the lack of FtMt can affect the Bcl-2/Bax ratio, leading to caspase-3 activation and a concomitant increase in PARP cleavage and, ultimately, apoptosis after A injection.

(a)

(b)

(c)

(d)

The activation of p38 (MAP kinase) by phosphorylation is implicated in oxidative stress-induced cell death [31]. A high p-p38/p38 ratio can simultaneously promote Bax expression and decrease Bcl-2 levels. A significantly induced the activation of p38 in the hippocampus. In the KO + A group, p-p38 levels were elevated (Figure 3(d)). Overall, our data demonstrate that the knockout of Ftmt in mice injected with A increases p-p38 levels which alters the amounts of proteins related to cell death, ultimately leading to increased neuronal cell death in the hippocampus.

3.4. Ftmt Knockout Increases MDA Levels in AD Mice without Altering SOD

To determine whether increased levels of oxidative stress are responsible for the increased apoptosis in the hippocampus in the AD mouse model, we examined the levels of MDA and the activity of SOD in each group. Free radicals attack polyunsaturated fatty acids, leading to structural damage to membranes and the generation of MDA, which is considered a marker of lipid peroxidation and thus a surrogate for oxidative damage [32]. The level of MDA was increased in AD mice compared with controls, but this increase was significantly greater in Ftmt knockout mice (Figure 4(a)). SOD is a free radical scavenging enzyme that converts superoxide into H₂O₂. The content of total SOD was unchanged in the four groups (Figure 4(b)).

(a)

(b)

3.5. The Effects of Ftmt Knockout on the Levels of L-Ferritin, TfR1, DMT1, and FPN1

Iron is an essential cofactor in many proteins, but excess free iron contributes to enhanced generation of ROS and oxidative stress [33]. When treated with A, the levels of L-ferritin were upregulated while those of TfR1 decreased significantly, compared to the control groups. The highest amount of L-ferritin expression was observed in the KO + A group (Figures 5(a) and 5(b)). In addition, the content of L-ferritin was also increased in the KO + saline group when compared to the WT + Saline group (Figure 5(a)). These observations indicated that A stimulation may lead to alterations in iron homeostasis and that FtMt deficiency may accelerate this process. In addition, alterations in cellular iron distribution (as detected by Perls’ staining) (see Supplementary Figure 1 of the Supplementary Material available online at https://doi.org/10.1155/2017/1020357) support this hypothesis. However, there was no significant difference in the expression of DMT1 (+IRE) or DMT1 (−IRE) in any group (Figures 5(c) and 5(d)), while the expression of FPN1, the iron release protein, was increased in both groups treated with A (Figure 5(d)). These results suggest that injection of A into the brain disturbed iron homeostasis, possibly leading to oxidative damage, both of which were exacerbated by the lack of FtMt.

(a)

(b)

(c)

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4. Discussion

Iron is an essential trace element for human health. The metal participates in many biological processes. Iron homeostasis is stringently regulated in vivo as excess iron can catalyze the generation of oxidative damage [20]. Importantly, iron is considered a contributing neurotoxic factor in several neurodegenerative disorders, including AD [34]. Cortical iron elevation has been increasingly reported as a feature of AD [35] and may contribute to the oxidative damage observed in AD brains. In addition, abnormalities in iron-regulatory proteins occur in the brains of AD sufferers [19]. FtMt, a recently identified H-ferritin-like protein expressed only in mitochondria, is thought to function to protect mitochondria from iron-dependent oxidative damage in cells with high metabolic activity and oxygen consumption. Previous studies have already shown an increased FtMt expression in the hippocampus of AD patients [36]. In addition, the downregulation of FtMt causes severe neurodegeneration in the Purkinje cells of the cerebellum [20]. In this study, Ftmt gene knockout mice were firstly used to study the effects of FtMt on the behavioral changes and mechanisms of A-induced neurotoxicity.

Previous results indicate that FtMt-deficient mice are healthy and do not show any evident phenotype under baseline feeding conditions [21]. Here we have also found that 10-month-old, wild-type, and Ftmt knockout mice show no behavior or memory differences, as determined by MWM assays. Thus, FtMt deficiency has no obvious effects in the mouse brain under normal physiological conditions. To further elucidate the role of FtMt in AD pathogenesis, we first showed that intracerebroventricular infusion of A exacerbates memory impairment in Ftmt knockout mice compared to the A-infused controls. The number of apoptotic cells in the hippocampus was also significantly increased in A-infused Ftmt knockout mice, which may account for their poorer performance in the MWM. Our data suggest that FtMt is not essential in mice under normal conditions. However, when challenged, such as with amyloid beta treatment, there appears to be a need for FtMt in a neuroprotective role.

Bcl-2 and Bax play important roles in oxidative stress-mediated neuronal apoptosis [37]. It has been reported that Bcl-2 protects neurons against oxidant stress and apoptosis in PD [38]. Bcl-2 also maintains mitochondrial integrity by blocking the release of apoptotic factors from mitochondria into cytoplasm [20]. Bax can promote cell death by activating elements of the caspase pathway [39], especially caspase-3 [40]. As previously described, the activation of caspases, a family of cysteine proteases, is a central mechanism in the apoptotic process. Our results show that knockout of Ftmt decreases the ratio of Bcl-2/Bax and increases the activation of caspase-3 and PARP cleavage, which ultimately leads to cell death.

Accumulated evidence demonstrated that Aβ-induced neuronal injury triggers transcriptional and posttranscriptional processes that regulate neuronal fate, including the activation of the MAPK pathway [41]. In this signaling cascade, p38 MAPK is activated by phosphorylation, and a high p-p38/p38 ratio can simultaneously promote Bax expression and decrease Bcl-2 levels. Our results show a significant elevation of p-p38 levels and its downstream factor Bax, strongly suggesting that this apoptotic signal transduction pathway is enhanced in Ftmt knockout mice treated with A.

An increasing number of studies have suggested that oxidative stress is associated with AD neurodegeneration and caspase-mediated apoptosis [42]. We detected a marked increase in the level of MDA, an indicator of oxidative damage, in the hippocampi of Ftmt knockout mice, indicating that knockout of Ftmt aggravates oxidative stress. Previous studies indicate that, in certain antioxidant systems, there might be a time lag between the synthesis of protein and the expression of mRNA following neurotoxicity; the activity of SOD is altered in the process of A induced injury. Cu, Zn-SOD, and Mn-SOD activity in hippocampi of A treated mice returned to near vehicle levels after 10 days [43]. Our data show that Ftmt ablation did not significantly affect the activity of total SOD, although this may be related to the time point at which SOD activity was measured.

Cellular iron homeostasis is maintained by a strict regulation of various proteins that are involved in iron uptake, export, storage, and utilization [44]. Studies from our group and others have demonstrated that aberrant iron homeostasis can generate ROS which can eventuate in AD pathogenesis [2, 11]. In the present study, we observed upregulated L-ferritin and FPN1 and a simultaneous decrease in TfR1. These changes are likely to be the result of inhibited iron-regulatory protein binding [45] brought about by an increase in the regulatory iron pool in the neuronal cells injected with Aβ. Consistent with this, the absence of FtMt may decrease the cells ability to sequester excess iron under stressed conditions and enhances the degree of changes in the measured proteins of iron metabolism. Our previous data also indicate that “uncommitted” iron levels, commonly referred to as the “labile iron pool” (LIP), significantly increased in SH-SY5Y cells treated with A. FtMt overexpression was able to reverse this change [11]. We propose that a larger LIP, resulting from a redistribution of iron from mitochondria to the cytosol, especially in the absence of FtMt, is responsible for the oxidative stress that mediates the damage of cell components in our AD model [46].

In summary, our research indicates that A elevates the LIP and causes oxidative stress, which can be exacerbated by the lack of FtMt. The excess iron donates electrons to generate ROS and lipid peroxidation. These changes initiate the programmed cell death through the p38/MAPK pathway, ultimately causing neuronal apoptosis which causes more severe memory impairment. The alteration of iron maybe provides the feedback regulation to the levels of TfR1 and FPN1 (Figure 6).

Figure 6

A schematic representation of the mechanism leading to neuronal cell apoptosis induced by A in mice with a disrupted Ftmt gene. A changes the levels (LIP) and distribution of intracellular iron, thus increasing oxidative stress. Without Ftmt to sequester excess mitochondrial iron, lipid peroxidation and the level of LIP are significantly increased. These changes may signal the cell to begin the process of programmed death through the P38 MAPK pathway, resulting in neuronal cell death, which is enhanced in Ftmt knockout mice, leading to worsened memory impairments.

5. Conclusion

The current study supports the hypothesis that the functionality of FtMt is not essential under normal conditions. But, in cases of neuronal stress, such as A accumulation, FtMt offers a profound neuroprotection through regulating cellular iron content and distribution in a way that keeps oxidative stress in check, preventing the activation of apoptosis.

Competing Interests

The authors declare that they have no competing interests related to this work.

Authors’ Contributions

Peina Wang and Qiong Wu contributed equally to this study.

Acknowledgments

This work was supported by the National Science Foundation of China (31520103908, 31471035, and 31271473). The authors wish to thank Professor Greg Anderson (Iron Metabolism Laboratory, Queensland Institute of Medical Research, Australia) for critical reading.

Supplementary Materials

Serial sections (15 μm) were cut using a cryostat microtome (Leica CM1950) and mounted on slides. Prior to staining, sections were treated with methanol containing 3% hydrogen peroxide for 10 minutes. After washing with 0.01 M PBS, sections were stained with Prussian blue, mixing 1% potassium ferrocyanide with 1% HCl for 8 hours at 37^°C. Then sections were washed in 0.01 M PBS and treated with DAB solution for 5 minutes. PBS, phosphate buffered saline.

Supplementary Material

References

V. A. Campbell and A. Gowran, “Alzheimer's disease; taking the edge off with cannabinoids?” British Journal of Pharmacology, vol. 152, no. 5, pp. 655–662, 2007.
View at: Publisher Site | Google Scholar
H. Yang, H. Guan, M. Yang et al., “Upregulation of mitochondrial ferritin by proinflammatory cytokines: implications for a role in Alzheimer's disease,” Journal of Alzheimer's Disease, vol. 45, no. 3, pp. 797–811, 2015.
View at: Publisher Site | Google Scholar
D. J. Selkoe, “Alzheimer's disease: genes, proteins, and therapy,” Physiological Reviews, vol. 81, no. 2, pp. 741–766, 2001.
View at: Google Scholar
A. Stéphan and A. G. Phillips, “A case for a non-transgenic animal model of Alzheimer's disease,” Genes, Brain and Behavior, vol. 4, no. 3, pp. 157–172, 2005.
View at: Publisher Site | Google Scholar
B. A. Yankner, L. K. Duffy, and D. A. Kirschner, “Neurotrophic and neurotoxic effects of amyloid β protein: reversal by tachykinin neuropeptides,” Science, vol. 250, no. 4978, pp. 279–282, 1990.
View at: Publisher Site | Google Scholar
T. Maurice, B. P. Lockhart, and A. Privat, “Amnesia induced in mice by centrally administered β-amyloid peptides involves cholinergic dysfunction,” Brain Research, vol. 706, no. 2, pp. 181–193, 1996.
View at: Publisher Site | Google Scholar
M. Y. Stepanichev, Y. V. Moiseeva, N. A. Lazareva, M. V. Onufriev, and N. V. Gulyaeva, “Single intracerebroventricular administration of amyloid-beta (25–35) peptide induces impairment in short-term rather than long-term memory in rats,” Brain Research Bulletin, vol. 61, no. 2, pp. 197–205, 2003.
View at: Publisher Site | Google Scholar
M. Y. Stepanichev, I. M. Zdobnova, I. I. Zarubenko et al., “Amyloid-β(25–35)-induced memory impairments correlate with cell loss in rat hippocampus,” Physiology and Behavior, vol. 80, no. 5, pp. 647–655, 2004.
View at: Publisher Site | Google Scholar
Y. Yamaguchi and S. Kawashima, “Effects of amyloid-β-(25–35) on passive avoidance, radial-arm maze learning and choline acetyltransferase activity in the rat,” European Journal of Pharmacology, vol. 412, no. 3, pp. 265–272, 2001.
View at: Publisher Site | Google Scholar
M. Y. Stepanichev, M. V. Onufriev, A. A. Yakovlev et al., “Amyloid-β (25-35) increases activity of neuronal NO-synthase in rat brain,” Neurochemistry International, vol. 52, no. 6, pp. 1114–1124, 2008.
View at: Publisher Site | Google Scholar
W. S. Wu, Y. S. Zhao, Z. H. Shi et al., “Mitochondrial ferritin attenuates β-amyloid-induced neurotoxicity: reduction in oxidative damage through the Erk/P38 mitogen-activated protein kinase pathways,” Antioxidants & Redox Signaling, vol. 18, no. 2, pp. 158–169, 2013.
View at: Google Scholar
A. S. Darvesh, R. T. Carroll, A. Bishayee, W. J. Geldenhuys, and C. J. Van Der Schyf, “Oxidative stress and Alzheimer's disease: dietary polyphenols as potential therapeutic agents,” Expert Review of Neurotherapeutics, vol. 10, no. 5, pp. 729–745, 2010.
View at: Publisher Site | Google Scholar
D.-Y. Choi, Y.-J. Lee, J. T. Hong, and H.-J. Lee, “Antioxidant properties of natural polyphenols and their therapeutic potentials for Alzheimer's disease,” Brain Research Bulletin, vol. 87, no. 2-3, pp. 144–153, 2012.
View at: Publisher Site | Google Scholar
S. Levi, B. Corsi, M. Bosisio et al., “A human mitochondrial ferritin encoded by an intronless gene,” Journal of Biological Chemistry, vol. 276, no. 27, pp. 24437–24440, 2001.
View at: Publisher Site | Google Scholar
B. Corsi, A. Cozzi, P. Arosio et al., “Human mitochondrial ferritin expressed in HeLa cells incorporates iron and affects cellular iron metabolism,” Journal of Biological Chemistry, vol. 277, no. 25, pp. 22430–22437, 2002.
View at: Publisher Site | Google Scholar
J. Drysdale, P. Arosio, R. Invernizzi et al., “Mitochondrial ferritin: a new player in iron metabolism,” Blood cells, molecules & diseases, vol. 29, no. 3, pp. 376–383, 2002.
View at: Publisher Site | Google Scholar
P. Santambrogio, G. Biasiotto, F. Sanvito, S. Olivieri, P. Arosio, and S. Levi, “Mitochondrial ferritin expression in adult mouse tissues,” Journal of Histochemistry and Cytochemistry, vol. 55, no. 11, pp. 1129–1137, 2007.
View at: Publisher Site | Google Scholar
G. Gao and Y.-Z. Chang, “Mitochondrial ferritin in the regulation of brain iron homeostasis and neurodegenerative diseases,” Frontiers in Pharmacology, vol. 5, article 19, 2014.
View at: Publisher Site | Google Scholar
H. Yang, M. Yang, H. Guan et al., “Mitochondrial ferritin in neurodegenerative diseases,” Neuroscience Research, vol. 77, no. 1-2, pp. 1–7, 2013.
View at: Publisher Site | Google Scholar
Z.-H. Shi, G. Nie, X.-L. Duan et al., “Neuroprotective mechanism of mitochondrial ferritin on 6-hydroxydopamine- induced dopaminergic cell damage: implication for neuroprotection in parkinson's disease,” Antioxidants and Redox Signaling, vol. 13, no. 6, pp. 783–796, 2010.
View at: Publisher Site | Google Scholar
T. B. Bartnikas, D. R. Campagna, B. Antiochos, H. Mulhern, C. Pondarré, and M. D. Fleming, “Characterization of mitochondrial ferritin-deficient mice,” American Journal of Hematology, vol. 85, no. 12, pp. 958–960, 2010.
View at: Publisher Site | Google Scholar
L.-H. You, F. Li, L. Wang et al., “Brain iron accumulation exacerbates the pathogenesis of MPTP-induced Parkinson's disease,” Neuroscience, vol. 284, pp. 234–246, 2015.
View at: Publisher Site | Google Scholar
T. Mamiya, T. Asanuma, M. Kise et al., “Effects of pre-germinated brown rice on beta-amyloid protein-induced learning and memory deficits in mice,” Biological and Pharmaceutical Bulletin, vol. 27, no. 7, pp. 1041–1045, 2004.
View at: Google Scholar
S. H. Park, J. H. Kim, S. S. Bae et al., “Protective effect of the phosphodiesterase III inhibitor cilostazol on amyloid β-induced cognitive deficits associated with decreased amyloid β accumulation,” Biochemical and Biophysical Research Communications, vol. 408, no. 4, pp. 602–608, 2011.
View at: Publisher Site | Google Scholar
C. Chen, X. Li, P. Gao et al., “Baicalin attenuates Alzheimer-like pathological changes and memory deficits induced by amyloid β_1–42 protein,” Metabolic Brain Disease, vol. 30, no. 2, pp. 537–544, 2015.
View at: Publisher Site | Google Scholar
S. Takeda, N. Sato, K. Niisato et al., “Validation of Aβ1-40 administration into mouse cerebroventricles as an animal model for Alzheimer disease,” Brain Research, vol. 1280, pp. 137–147, 2009.
View at: Publisher Site | Google Scholar
W. Huang, W.-B. Xie, D. Qiao et al., “Caspase-11 plays an essential role in methamphetamine-induced dopaminergic neuron apoptosis,” Toxicological Sciences, vol. 145, no. 1, pp. 68–79, 2015.
View at: Publisher Site | Google Scholar
L.-H. You, Z. Li, X.-L. Duan, B.-L. Zhao, Y.-Z. Chang, and Z.-H. Shi, “Mitochondrial ferritin suppresses MPTP-induced cell damage by regulating iron metabolism and attenuating oxidative stress,” Brain Research, vol. 1642, pp. 33–42, 2016.
View at: Publisher Site | Google Scholar
G.-X. Mao, L.-D. Zheng, Y.-B. Cao et al., “Antiaging effect of pine pollen in human diploid fibroblasts and in a mouse model induced by D-galactose,” Oxidative Medicine and Cellular Longevity, vol. 2012, Article ID 750963, 10 pages, 2012.
View at: Publisher Site | Google Scholar
D. W. Nicholson, A. Ali, N. A. Thornberry et al., “Identification and inhibition of the ICE/CED-3 protease necessary for mammalian apoptosis,” Nature, vol. 376, no. 6535, pp. 37–43, 1995.
View at: Publisher Site | Google Scholar
X. Wang, J. L. Martindale, Y. Liu, and N. J. Holbrook, “The cellular response to oxidative stress: influences of mitogen-activated protein kinase signalling pathways on cell survival,” Biochemical Journal, vol. 333, no. 2, pp. 291–300, 1998.
View at: Publisher Site | Google Scholar
M. A. Lovell and W. R. Markesbery, “Oxidative damage in mild cognitive impairment and early Alzheimer's disease,” Journal of Neuroscience Research, vol. 85, no. 14, pp. 3036–3040, 2007.
View at: Publisher Site | Google Scholar
R. J. Ward, F. A. Zucca, J. H. Duyn, R. R. Crichton, and L. Zecca, “The role of iron in brain ageing and neurodegenerative disorders,” The Lancet Neurology, vol. 13, no. 10, pp. 1045–1060, 2014.
View at: Publisher Site | Google Scholar
C. Mancuso, G. Scapagnini, D. Currò et al., “Mitochondrial dysfunction, free radical generation and cellular stress response in neurodegenerative disorders,” Frontiers in Bioscience, vol. 12, no. 3, pp. 1107–1123, 2007.
View at: Publisher Site | Google Scholar
S. van Rooden, N. T. Doan, M. J. Versluis et al., “7T T2*-weighted magnetic resonance imaging reveals cortical phase differences between early- and late-onset Alzheimer's disease,” Neurobiology of Aging, vol. 36, no. 1, pp. 20–26, 2015.
View at: Publisher Site | Google Scholar
L. Wang, H. Yang, S. Zhao et al., “Expression and localization of mitochondrial ferritin mRNA in Alzheimer's disease cerebral cortex,” PLoS ONE, vol. 6, no. 7, Article ID e22325, 2011.
View at: Publisher Site | Google Scholar
Y. Oshima, T. Akiyama, A. Hikita et al., “Pivotal role of Bcl-2 family proteins in the regulation of chondrocyte apoptosis,” Journal of Biological Chemistry, vol. 283, no. 39, pp. 26499–26508, 2008.
View at: Publisher Site | Google Scholar
A. Tortosa, E. López, and I. Ferrer, “Bcl-2 and Bax proteins in lewy bodies from patients with Parkinson's disease and Diffuse Lewy body disease,” Neuroscience Letters, vol. 238, no. 1-2, pp. 78–80, 1997.
View at: Publisher Site | Google Scholar
P. Golstein, “Controlling cell death,” Science, vol. 275, no. 5303, pp. 1081–1082, 1997.
View at: Publisher Site | Google Scholar
T. S. Zheng, S. Hunot, K. Kuida et al., “Deficiency in caspase-9 or caspase-3 induces compensatory caspase activation,” Nature Medicine, vol. 6, no. 11, pp. 1241–1247, 2000.
View at: Publisher Site | Google Scholar
Z. H. Yang, K. Sun, W. H. Suo et al., “N-stearoyltyrosine protects primary neurons from Aβ-induced apoptosis through modulating mitogen-activated protein kinase activity,” Neuroscience, vol. 169, no. 4, pp. 1840–1847, 2010.
View at: Publisher Site | Google Scholar
I. I. Kruman, A. Nath, and M. P. Mattson, “HIV-1 protein tat induces apoptosis of hippocampal neurons by a mechanism involving caspase activation, calcium overload, and oxidative stress,” Experimental Neurology, vol. 154, no. 2, pp. 276–288, 1998.
View at: Publisher Site | Google Scholar
J. H. Jhoo, H.-C. Kim, T. Nabeshima et al., “β-Amyloid (1-42)-induced learning and memory deficits in mice: involvement of oxidative burdens in the hippocampus and cerebral cortex,” Behavioural Brain Research, vol. 155, no. 2, pp. 185–196, 2004.
View at: Publisher Site | Google Scholar
R. Santamaria, F. Fiorito, C. Irace et al., “2,3,7,8-Tetrachlorodibenzo-p-dioxin impairs iron homeostasis by modulating iron-related proteins expression and increasing the labile iron pool in mammalian cells,” Biochimica et Biophysica Acta—Molecular Cell Research, vol. 1813, no. 5, pp. 704–712, 2011.
View at: Publisher Site | Google Scholar
D. J. R. Lane, A. M. Merlot, M. L.-H. Huang et al., “Cellular iron uptake, trafficking and metabolism: key molecules and mechanisms and their roles in disease,” Biochimica et Biophysica Acta—Molecular Cell Research, vol. 1853, no. 5, pp. 1130–1144, 2015.
View at: Publisher Site | Google Scholar
M. Valko, K. Jomova, C. J. Rhodes, K. Kuča, and K. Musílek, “Redox- and non-redox-metal-induced formation of free radicals and their role in human disease,” Archives of Toxicology, vol. 90, no. 1, pp. 1–37, 2016.
View at: Publisher Site | Google Scholar

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Copyright © 2017 Peina Wang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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Oxidative Medicine and Cellular Longevity

Mitochondrial Ferritin Deletion Exacerbates β-Amyloid-Induced Neurotoxicity in Mice

Abstract

1. Introduction

2. Materials and Methods

2.1. Animals

2.2. Antibodies and Reagents

2.3. Drug Preparation and Injection

2.4. Morris Water Maze Test (MWM Test)

2.5. Probe Test

2.6. Assessment of Apoptosis

2.7. Western Blot Analysis

2.8. Measurement of MDA and SOD

2.9. Statistical Analysis

3. Results

3.1. Ftmt Ablation Exacerbates Aβ25–35-Induced Spatial Memory Deficits

3.2. Ftmt Ablation Enhances Aβ25–35-Induced Neuronal Cell Apoptosis

3.3. FtMt Deficiency in the AD Mouse Model Elevates Proapoptotic Signals

3.4. Ftmt Knockout Increases MDA Levels in AD Mice without Altering SOD

3.5. The Effects of Ftmt Knockout on the Levels of L-Ferritin, TfR1, DMT1, and FPN1

4. Discussion

5. Conclusion

Competing Interests

Authors’ Contributions

Acknowledgments

Supplementary Materials

References

Copyright

3.1. Ftmt Ablation Exacerbates Aβ_25–35-Induced Spatial Memory Deficits

3.2. Ftmt Ablation Enhances Aβ_25–35-Induced Neuronal Cell Apoptosis