mitochondrial ferritin deletion exacerbates š½-amyloid...
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Research ArticleMitochondrial Ferritin Deletion Exacerbatesš½-Amyloid-Induced Neurotoxicity in Mice
Peina Wang, Qiong Wu, Wenyue Wu, Haiyan Li, Yuetong Guo, Peng Yu,Guofen Gao, Zhenhua Shi, Baolu Zhao, and Yan-Zhong Chang
Laboratory of Molecular Iron Metabolism, The Key Laboratory of Animal Physiology, Biochemistry and Molecular Biology ofHebei Province, College of Life Science, Hebei Normal University, Shijiazhuang, Hebei 050024, China
Correspondence should be addressed to Yan-Zhong Chang; [email protected]
Received 11 October 2016; Accepted 27 December 2016; Published 16 January 2017
Academic Editor: Rodrigo Franco
Copyright Ā© 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.
Mitochondrial ferritin (FtMt) is a mitochondrial iron storage protein which protects mitochondria from iron-induced oxidativedamage. Our previous studies indicate that FtMt attenuates š½-amyloid- and 6-hydroxydopamine-induced neurotoxicity in SH-SY5Y cells. To explore the protective effects of FtMt on š½-amyloid-induced memory impairment and neuronal apoptosis and themechanisms involved, 10-month-old wild-type and Ftmt knockout mice were infused intracerebroventricularly (ICV) with Aš½
25ā35to establish an Alzheimerās disease model. Knockout of Ftmt significantly exacerbated Aš½
25ā35-induced learning and memoryimpairment.The Bcl-2/Bax ratio inmouse hippocampi was decreased and the levels of cleaved caspase-3 and PARPwere increased.The number of neuronal cells undergoing apoptosis in the hippocampus was also increased in Ftmt knockout mice. In addition, thelevels of L-ferritin and FPN1 in the hippocampus were raised, and the expression of TfR1 was decreased. IncreasedMDA levels werealso detected in Ftmt knockout mice treated with Aš½
25ā35. In conclusion, this study demonstrated that the neurological impairmentinduced by Aš½
25ā35 was exacerbated in Ftmt knockout mice and that this may relate to increased levels of oxidative stress.
1. Introduction
Alzheimerās disease (AD) is amultifaceted neurodegenerativedisease of the elderly which is characterized by neuronal loss,neuroinflammation, and progressive memory and cognitiveimpairment [1]. Many pathogenic factors are involved in theneuropathology, including accumulation of š½-amyloid (Aš½),oxidative stress, inflammation, and metal deposition [2]. Aš½is considered to be a major factor in the pathophysiologicalmechanisms underlying AD and has been shown to directlyinduce neuronal cell death [3]. Thus, Aš½ is a useful tool forestablishing AD models and investigating the mechanismsinvolved in AD pathogenesis [4]. Aš½
25ā35 is an 11-amino acidfragment located in the hydrophobic functional domain ofthe C-terminal region of Aš½
1ā42 [5]. Single administrationof Aš½
25ā35 into the lateral ventricles of mice or rats impairsmemory and induces neurodegeneration in the hippocampus[6ā10]. We have previously shown that Aš½
25ā35, like Aš½1ā42,exerted neurotoxic effects on SH-SY5Y cells [11]. These dataare among numerous studies confirming that Aš½
25ā35 is
a useful tool for investigating AD-related mechanisms inanimal models [10].
Oxidative stress has been strongly implicated in thepathophysiology of AD [12]. Increased free radicals can dam-age proteins, lipids, and nucleic acids. The combination ofmitochondrial dysfunction and Aš½ accumulation generatesreactive oxygen species (ROS)which, in the presence ofmetalions such as Fe2+ and Cu2+, may contribute to oxidativedamage in AD brains [13]. In addition, dysregulated brainiron homeostasis also accelerates AD progression. Excessiveiron in the brain can directly lead to the generation of freeradicals that eventually cause neurodegenerative disease.
Mitochondrial ferritin (FtMt) is a recently identifiedferritin that accumulates specifically in themitochondria andpossesses a high homology to H-ferritin [14]. The functionsof FtMt include iron storage, regulating iron distributionbetween the cytosol and mitochondrial compartments, andpreventing the production of ROS generated through theFenton reaction [15, 16]. FtMt has been reported to be presentin relatively low abundance in the liver and splenocytes,
HindawiOxidative Medicine and Cellular LongevityVolume 2017, Article ID 1020357, 10 pageshttps://doi.org/10.1155/2017/1020357
2 Oxidative Medicine and Cellular Longevity
the major iron storage sites, while FtMt is found at higherlevels in the testis, kidney, heart and brain, and tissues withhigh metabolic activity. Together with the knowledge that H-ferritin expression confers an antioxidant effect, the tissuedistribution of FtMt is in line with a protective functionof FtMt in mitochondria against iron-dependent oxida-tive damage [17]. Previous results indicated that FtMt wasinvolved in the pathogenesis of neurodegenerative diseases,including AD, Parkinsonās disease, and Friedreichās ataxia[18ā20]. Increased expression of FtMt has been observedin the brains of AD patients and is associated with theantioxidant role of this protein [2]. Furthermore, our previousstudies have shown that FtMt exerted a neuroprotective effectagainst 6-hydroxydopamine- (6-OHDA-) induced dopamin-ergic cell damage [20] and FtMt overexpression attenuatedAš½-induced neurotoxicity [11].
Although abnormal iron metabolism and oxidative stresshave been reported in AD, little information is availableabout the role of FtMt in the pathogenesis of AD. In thepresent study, we investigated memory impairment and neu-ronal cell death in Aš½
25ā35-injected Ftmt knockout mice. Inaddition, we explored themolecular mechanisms responsiblefor neuronal damage in this model. Our data indicate thatFtMt deficiency exacerbated Aš½
25ā35-induced neuronal celldamage by altering intracellular iron levels in a way thatintensifies the oxidative stress caused by Aš½
25ā35.
2. Materials and Methods
2.1. Animals. C57BL/6 Ftmt-null mice were obtained fromThe Jackson Laboratory [21]. Mice were housed under condi-tions controlled for temperature (22āC) and humidity (40%),using a 12 hr/12 hr light/dark cycle [22]. Mice were fed astandard rodent diet and water ad libitum. Age-matchedC57BL/6J wild-typemalemice and Ftmt knockoutmalemice(10 months) were used in this study. All procedures werecarried out in accordance with the National Institutes ofHealth Guide for the Care and Use of Laboratory Animalsand were approved by the Animal Care and Use Committeeof the Hebei Science and Technical Bureau in China.
2.2. Antibodies and Reagents. The following antibodies andreagents were used: š½-actin (Alpha Diagnostic International,USA), TfR1 (Sigma-Aldrich, USA), FPN1, DMT1 (+IRE) andDMT1 (āIRE) (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),Aš½25ā35 peptide (Sigma-Aldrich, USA), and TUNEL in
situ Cell Death Detection Kit (Roche Diagnostics GmbH,Mannheim, Germany).
2.3. Drug Preparation and Injection. Aš½25ā35 was dissolved in
sterile saline and aggregated by incubation at 37āC for 4 daysbefore use [23]. The aggregated form of Aš½
25ā35 (7.5 nmol in5 šL saline per injection) was injected into the right lateralventricle as previously described [24]. Mice were randomlydivided into four groups: wild-type with saline injection (WT+ saline), wild-type with Aš½
25ā35 injection (WT + Aš½25ā35),
Ftmt knockout with saline injection (KO + saline), andFtmt knockout with Aš½
25ā35 injection (KO + Aš½25ā35). After
injection, the mice were housed for 15 days under normalconditions and then trained and tested in aMorriswatermaze(MWM) as described below.
2.4. Morris Water Maze Test (MWM Test). Spatial learningand memory deficits were assessed using the Morris watermaze as described previously [25, 26], with minor modifica-tion. The experimental apparatus consisted of a circular tank(diameter = 120 cm, height = 50 cm) that was divided intofour quadrants, filled with water, and maintained at 22 Ā± 2āC.At first, a visible platform test was performed, which con-firmed 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 themidpoint of the fourth quadrant, 1 cmbelow the water surface. The test was conducted four timesa day for four days, with four randomized starting points.The position of the escape platform was kept constant. Eachtrial lasted for 90 s or ended as soon as the mice reached thesubmerged platform.
2.5. Probe Test. To assess memory consolidation, a probe testwas performed 24 h after the Morris water maze test [25].For the probe test, the platform was removed and the micewere allowed to swim freely. The swimming pattern of everymouse was recorded for 90 s with a camera. Consolidatedspatial memory was estimated by the time spent in the targetquadrant area.
2.6. Assessment of Apoptosis. After the behavioral testing, theanimals were perfused with 0.9% saline under anesthesiawith 0.4% Nembutal. The brains were immediately collectedand then postfixed with 4% paraformaldehyde in 0.1Mphosphate buffer. Serial coronal sections were cut at 15 šmon a freezingmicrotome (LeicaCM1950, LeicaMicrosystems,Shanghai, China) and mounted onto slides covered withAPES (Beijing Zhongshan Biotechnology, Beijing, China).The presence of apoptosis in the dentate gyrus of mousehippocampi was assessed by the terminal deoxynucleotidyltransferase-mediated FITC-dUTP nick-end labeling method(TUNEL) following the manufacturerās protocol. Nuclei werecounterstained with DAPI. The number of TUNEL-DAPI-positive cells was counted as described previously [27]. Thecounting area was located in the same position in all groups.For each group, quantification was performed in sectionsfrom three different mice.
2.7. Western Blot Analysis. Protein expression was assessedby western blotting as previously described [28], with minormodifications. Briefly, hippocampi were homogenized andsonicated in RIPA buffer containing 1% NP40 and proteaseinhibitor cocktail tablets (Roche Diagnostics GmbH, RocheApplied Science, 68298 Mannheim, Germany). After cen-trifugation at 12,000Ćg for 20min at 4āC, the supernatantwas collected, and the whole cell lysate protein concentrationwas measured using the BCA Protein Quantification Kit
Oxidative Medicine and Cellular Longevity 3
(Yeasen Biotechnology, Shanghai, China). Protein from eachsample (40mg) was resolved by SDS-PAGE on 12% or 10%gels and then transferred to PVDF membranes. The blotswere blocked in 5% nonfat milk containing 20mM Tris-HCl(pH 7.6, 137mM NaCl, and 0.1% Tween-20; TBS-T) for 1.5 hat room temperature, followed by incubation with primaryantibody overnight at 4āC. After washing three times withTBS-T, the blots were incubated with horseradish peroxide(HRP) conjugated secondary antibody for 1.5 h at room tem-perature. Immunoreactive proteins were detected using theenhanced chemiluminescence (ECL) method and quantifiedby transmittance densitometry using volume integrationwithMultiGauge ver. 3.1 software (FUJIFILMCorporation, Tokyo,Japan).
2.8. Measurement of MDA and SOD. Malondialdehyde(MDA), a marker of lipid peroxidation, was assessed usingthe thiobarbituric acid (TBA) method [29] using a kit fromthe Nanjing Jiancheng Bioengineering Institute (Nanjing,China) according to the manufacturerās instructions. Thismethod is based on the spectrophotometric measurementof the product of the reaction of TBA with MDA. MDAconcentrations were then calculated by the absorbance ofTBA reactive substances (TBARS) at 532 nm.
Superoxide dismutases (SODs), which catalyze the dis-mutation of superoxide into oxygen and hydrogen peroxide,were determined according to xanthine oxidase methodusing a commercial kit (Nanjing Jiancheng BioengineeringInstitute, Nanjing, China) according to the manufacturerāsinstructions.The xanthine-xanthine oxidase systemproducessuperoxide ions, which can react with 2-(4-iodophenyl)-3-(4-nitrophenol-5-phenlyltetrazolium chloride) to form a redformazan dye, which can be detected by its absorbance at550 nm [29].
The levels of MDA and the total SOD (T-SOD) activitywere determined in each group. The hippocampi of micewere homogenized in ice-cold saline. The homogenate wascentrifuged at 3000Ćg at 4āC for 15min, and the supernatantwas used to determine T-SOD activity and MDA levelswith a spectrophotometer (Synergy H4, BioTek, USA) atwavelengths of 550 nm and 532 nm, respectively. Each groupcontained five mice for the MDA and SOD tests, with eachtest repeated three times.
2.9. Statistical Analysis. All data are expressed as the mean Ā±standard deviation. One-way analysis of variance was used toestimate overall significance andwas followed by Tukeyās posthoc test corrected for multiple comparisons. A probabilitylevel of 95% (š < 0.05) was considered significant. All thetests were performedwith SPSS 21.0 (IBMSPSS21.0, Armonk,New York, United States).
3. Results
3.1. Ftmt Ablation Exacerbates Aš½25ā35-Induced Spatial Mem-ory Deficits. TheMWMtest was conducted to assess learningandmemory in 10-month-old wild-typemice (WT) and Ftmtknockout mice (KO). All mice were trained with four trialsper day for 4 days. āEscape latencyā is the time to reach
the platform in the water maze and is used as a proxy formousememory. Compared towild-typemice, Ftmt knockoutmice took approximately the same time to reach the platformafter training (Figure 1(a)). After the water maze test, weperformed a probe test using the metric ātime spent inquadrantā to investigate the maintenance of memory. Thetime spent in the target quadrant was also similar in wild-type and Ftmt knockout mice (Figure 1(b)). After treatmentwith Aš½
25ā35, both the WT + Aš½25ā35 group and the KO
+ Aš½25ā35 group took a significantly longer time to reach
the platform than the groups without Aš½25ā35 injection
(Figure 1(c)). Furthermore, Aš½25ā35-infused Ftmt knockout
mice had a significantly greater memory impairment (longerescape latency time) than Aš½
25ā35-infused wild-type mice.In addition, the time spent in the target quadrant in theprobe trial was less in both the WT + Aš½
25ā35 and the KO+ Aš½25ā35 groups than in the control groups. Importantly,
the KO + Aš½25ā35 group was in the target quadrant for even
less time than theWT +Aš½25ā35 group (Figure 1(d)). Overall,
our results show that knockout of Ftmt in mice significantlyexacerbates memory deficits in the Aš½
25ā35-induced ADmodel.
3.2. Ftmt Ablation Enhances Aš½25ā35-Induced Neuronal CellApoptosis. To evaluate the neuronal apoptosis affected byFtmt gene ablation in the AD model, we used the TUNELmethod to detect apoptosis after Aš½
25ā35 stimulation. Ourresults indicated that neuronal apoptosis in the hippocampiwas increased after injecting Aš½
25ā35, especially in the den-tate gyrus. The number of apoptotic cells in the WT +Aš½25ā35 group was approximately four times greater than that
observed in the WT + saline group, and there was also anoticeable increase in theKOgroup.Thenumber of apoptoticcells in the KO + Aš½
25ā35 group was more than threefoldthat of theWT + Aš½
25ā35 group.These results confirmed thatFtmt knockout significantly enhanced neuronal apoptosiscompared to theWT+Aš½
25ā35 group (Figures 2(a) and 2(b)),suggesting that FtMt is protective against Aš½
25ā35-inducedapoptosis.
3.3. FtMtDeficiency in the ADMouseModel Elevates Proapop-totic Signals. We found that the knockout of Ftmt remarkablydecreased the ratio of Bcl-2/Bax (Figure 3(a)) and increasedthe activation of cleaved caspase-3 (Figure 3(b)) after Aš½
25ā35treatment inmice. In the apoptotic cascade, caspase-3 cleavespoly-ADP-ribose-polymerase (PARP), leading to the accu-mulation of an 89 kDa PARP fragment [30]. Caspase-3-mediated PARP cleavage was enhanced in the KO + Aš½
25ā35group compared to the WT + Aš½
25ā35 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 concomitantincrease in PARP cleavage and, ultimately, apoptosis afterAš½25ā35 injection.The activation of p38 (MAPkinase) by phosphorylation is
implicated in oxidative stress-induced cell death [31]. A highp-p38/p38 ratio can simultaneously promote Bax expressionand decrease Bcl-2 levels. Aš½
25ā35 significantly induced theactivation of p38 in the hippocampus. In the KO + Aš½
25ā35group, p-p38 levels were elevated (Figure 3(d)). Overall,
4 Oxidative Medicine and Cellular Longevity
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Figure 1: The effect of Ftmt ablation on Aš½25ā35-induced spatial memory deficits. (a) Age-matched (10 months old) Ftmt knockout mice
(š = 20) and wild-type mice (š = 23) were administered a 90 s trial four times a day to find the hidden platform.The analysis of the recordeddata 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 wasrecorded. (c) The effect of Aš½
25ā35 on escape latency. (Wild-type mice and Ftmt knockout mice were randomly divided into four groups andinjected with Aš½
25ā35 or saline. Fifteen days later, the MWM test was conducted.) (d) The time spent in the target quadrant during the probetest after injecting Aš½
25ā35. The data is presented as the mean Ā± SD. āš < 0.05 versus WT + saline group, š = 11. #š < 0.05, ##š < 0.01 versusKO + saline group, š = 10. $š < 0.05 versus WT + Aš½
25ā35 group, š = 10. KO + Aš½25ā35, š = 10.
our data demonstrate that the knockout of Ftmt in miceinjected with Aš½
25ā35 increases p-p38 levels which alters theamounts of proteins related to cell death, ultimately leadingto increased neuronal cell death in the hippocampus.
3.4. Ftmt Knockout Increases MDA Levels in ADMice withoutAltering SOD. To determine whether increased levels ofoxidative stress are responsible for the increased apoptosisin the hippocampus in the AD mouse model, we examinedthe levels of MDA and the activity of SOD in each group.Free radicals attack polyunsaturated fatty acids, leading tostructural damage tomembranes and the generation ofMDA,which is considered a marker of lipid peroxidation and thusa surrogate for oxidative damage [32]. The level of MDA
was increased in AD mice compared with controls, but thisincrease was significantly greater in Ftmt knockout mice(Figure 4(a)). SOD is a free radical scavenging enzyme thatconverts superoxide into H
2O2.The content of total SODwas
unchanged in the four groups (Figure 4(b)).
3.5. The Effects of Ftmt Knockout on the Levels of L-Ferritin,TfR1, DMT1, and FPN1. Iron is an essential cofactor inmany proteins, but excess free iron contributes to enhancedgeneration of ROS and oxidative stress [33]. When treatedwith Aš½
25ā35, the levels of L-ferritin were upregulated whilethose of TfR1 decreased significantly, compared to the controlgroups. The highest amount of L-ferritin expression wasobserved in the KO + Aš½
25ā35 group (Figures 5(a) and 5(b)).
Oxidative Medicine and Cellular Longevity 5
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Figure 2: The effect of Ftmt ablation on Aš½25ā35 -induced neuronal cell apoptosis. Apoptotic cell death was assessed by DAPI and TUNEL
staining, as described in theMaterials andMethods section. (a) Representative photographs (originalmagnification 100x) of the dentate gyrusof the hippocampus of mouse brains. (b) The statistical analysis of relative apoptotic cell levels. Data are presented as the mean Ā± SD, š = 3.āāāš < 0.001 versus WT + saline group, ###š < 0.001 versus KO + saline group, and $$$š < 0.001 versus WT + Aš½
25ā35 group.
In addition, the content of L-ferritin was also increasedin the KO + saline group when compared to the WT+ Saline group (Figure 5(a)). These observations indicatedthat Aš½
25ā35 stimulation may lead to alterations in ironhomeostasis and that FtMt deficiency may accelerate thisprocess. In addition, alterations in cellular iron distribu-tion (as detected by Perlsā staining) (see SupplementaryFigure 1 of the Supplementary Material available online athttps://doi.org/10.1155/2017/1020357) support this hypothesis.However, there was no significant difference in the expressionof DMT1 (+IRE) or DMT1 (āIRE) in any group (Figures5(c) and 5(d)), while the expression of FPN1, the iron releaseprotein, was increased in both groups treated with Aš½
25ā35(Figure 5(d)). These results suggest that injection of Aš½
25ā35into the brain disturbed iron homeostasis, possibly leading tooxidative damage, both of which were exacerbated by the lackof FtMt.
4. Discussion
Iron is an essential trace element for human health.Themetalparticipates in many biological processes. Iron homeostasisis stringently regulated in vivo as excess iron can catalyzethe generation of oxidative damage [20]. Importantly, ironis considered a contributing neurotoxic factor in severalneurodegenerative disorders, including AD [34]. Corticaliron elevation has been increasingly reported as a featureof AD [35] and may contribute to the oxidative damageobserved 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 expressedonly in mitochondria, is thought to function to protect mito-chondria from iron-dependent oxidative damage in cells withhigh metabolic activity and oxygen consumption. Previousstudies have already shown an increased FtMt expression
6 Oxidative Medicine and Cellular Longevity
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Figure 3: The effect of Ftmt deficiency on the Bcl-2/Bax ratio, cleaved caspase-3, and p38 MAPK activation in mice. Western blot andsubsequent densitometric analysis of (a) the ratio of Bcl-2/Bax, (b) the amount of cleaved caspase-3, (c) the amount of cleaved PARP, and (d)the ratio of p-p38/p38. Data are presented as the mean Ā± SD, š = 3. āš < 0.05, āāš < 0.01 versus WT + saline group, #š < 0.05, ##š < 0.01versus KO + saline group, and $š < 0.05 versus WT + Aš½
25ā35 group. MAPK: mitogen-activated protein kinase.
in the hippocampus of AD patients [36]. In addition, thedownregulation of FtMt causes severe neurodegeneration inthe Purkinje cells of the cerebellum [20]. In this study, Ftmtgene knockout mice were firstly used to study the effects ofFtMt on the behavioral changes and mechanisms of Aš½
25ā35-induced neurotoxicity.
Previous results indicate that FtMt-deficient mice arehealthy and do not show any evident phenotype underbaseline feeding conditions [21]. Here we have also found that10-month-old, wild-type, and Ftmt knockout mice show no
behavior or memory differences, as determined by MWMassays. Thus, FtMt deficiency has no obvious effects in themouse brain under normal physiological conditions. Tofurther elucidate the role of FtMt in AD pathogenesis, wefirst showed that intracerebroventricular infusion of Aš½
25ā35exacerbates memory impairment in Ftmt knockout micecompared to the Aš½
25ā35-infused controls. The number ofapoptotic cells in the hippocampus was also significantlyincreased inAš½
25ā35-infused Ftmt knockoutmice, whichmayaccount for their poorer performance in the MWM. Our
Oxidative Medicine and Cellular Longevity 7
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Figure 4: The effects of Ftmt ablation on the levels of MDA and Total SOD. (a) MDA and (b) total SOD were assayed as described in theMaterials and Methods section. Values are presented as the mean Ā± SD. āš < 0.05 versus WT + saline group, #š < 0.05 versus KO + salinegroup, and $š < 0.05 versus WT + Aš½
25ā35 group.
data suggest that FtMt is not essential in mice under normalconditions. However, when challenged, such as with amyloidbeta treatment, there appears to be a need for FtMt in aneuroprotective role.
Bcl-2 and Bax play important roles in oxidative stress-mediated neuronal apoptosis [37]. It has been reported thatBcl-2 protects neurons against oxidant stress and apoptosisin PD [38]. Bcl-2 also maintains mitochondrial integrity byblocking the release of apoptotic factors from mitochondriainto cytoplasm [20]. Bax can promote cell death by activatingelements of the caspase pathway [39], especially caspase-3[40]. As previously described, the activation of caspases, afamily of cysteine proteases, is a central mechanism in theapoptotic process. Our results show that knockout of Ftmtdecreases the ratio of Bcl-2/Bax and increases the activationof caspase-3 and PARP cleavage, which ultimately leads to celldeath.
Accumulated evidence demonstrated that Aš½-inducedneuronal injury triggers transcriptional and posttranscrip-tional processes that regulate neuronal fate, including theactivation of the MAPK pathway [41]. In this signalingcascade, p38 MAPK is activated by phosphorylation, anda high p-p38/p38 ratio can simultaneously promote Baxexpression and decrease Bcl-2 levels. Our results show asignificant elevation of p-p38 levels and its downstreamfactor Bax, strongly suggesting that this apoptotic signaltransduction pathway is enhanced in Ftmt knockout micetreated with Aš½
25ā35.An increasing number of studies have suggested that
oxidative stress is associated with AD neurodegenerationand caspase-mediated apoptosis [42]. We detected a markedincrease in the level of MDA, an indicator of oxidative dam-age, in the hippocampi of Ftmt knockout mice, indicatingthat knockout of Ftmt aggravates oxidative stress. Previousstudies indicate that, in certain antioxidant systems, theremight be a time lag between the synthesis of protein andthe expression of mRNA following neurotoxicity; the activity
of SOD is altered in the process of Aš½25ā35 induced injury.
Cu, Zn-SOD, and Mn-SOD activity in hippocampi of Aš½1ā42
treated mice returned to near vehicle levels after 10 days [43].Our data show that Ftmt ablation did not significantly affectthe activity of total SOD, although this may be related to thetime point at which SOD activity was measured.
Cellular iron homeostasis is maintained by a strict reg-ulation of various proteins that are involved in iron uptake,export, storage, and utilization [44]. Studies from our groupand others have demonstrated that aberrant iron homeostasiscan 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.Thesechanges are likely to be the result of inhibited iron-regulatoryprotein binding [45] brought about by an increase in theregulatory iron pool in the neuronal cells injected with Aš½.Consistent with this, the absence of FtMt may decrease thecells ability to sequester excess iron under stressed conditionsand enhances the degree of changes in the measured proteinsof iron metabolism. Our previous data also indicate thatāuncommittedā iron levels, commonly referred to as theālabile iron poolā (LIP), significantly increased in SH-SY5Ycells treated with Aš½
25ā35. FtMt overexpression was ableto reverse this change [11]. We propose that a larger LIP,resulting from a redistribution of iron from mitochondria tothe cytosol, especially in the absence of FtMt, is responsiblefor the oxidative stress that mediates the damage of cellcomponents in our AD model [46].
In summary, our research indicates that Aš½25ā35 elevates
the LIP and causes oxidative stress, which can be exacerbatedby the lack of FtMt. The excess iron donates electrons togenerate ROS and lipid peroxidation. These changes initiatethe programmed cell death through the p38/MAPK pathway,ultimately causing neuronal apoptosis which causes moresevere memory impairment. The alteration of iron maybeprovides the feedback regulation to the levels of TfR1 andFPN1 (Figure 6).
8 Oxidative Medicine and Cellular Longevity
WT + saline KO + saline WT + Aš½ KO + Aš½
#$
ā
āā
1.5
1.0
0.5
0.0
WT + saline KO + saline WT + Aš½ KO + Aš½
L-Fe
rriti
n ex
pres
sion
(rel
ativ
e to š½
-act
in)
š½-Actin
L-Ferritin
(a)
WT + saline KO + saline WT + Aš½ KO + Aš½
#
$
ā2.0
1.5
1.0
0.5
0.0
WT + saline KO + saline WT + Aš½ KO + Aš½
TfR1
expr
essio
n (r
elat
ive t
oš½
-act
in)
š½-Actin
TfR1
(b)
WT + saline KO + saline WT + Aš½ KO + Aš½
1.5
1.0
0.5
0.0
WT + saline KO + saline WT + Aš½ KO + Aš½
DM
T1(+
IRE)
expr
essio
n (r
elat
ive t
oš½
-act
in)
š½-Actin
DMT1 (+IRE)
(c)
WT + saline KO + saline WT + Aš½ KO + Aš½
WT + saline KO + saline WT + Aš½ KO + Aš½
WT + saline KO + saline WT + Aš½ KO + Aš½
#
$
ā
DM
T1(ā
IRE)
expr
essio
n (r
elat
ive t
oš½
-act
in)
FPN1
expr
essio
n (r
elat
ive t
oš½
-act
in)
1.0
0.8
0.6
0.4
0.2
0.0
0.8
0.6
0.4
0.2
0.0
š½-Actin
FPN1
DMT1 (āIRE)
(d)
Figure 5:The effects of Ftmt deficiency on the levels of L-ferritin, TfR1, DMT1, and FPN1.Western blotting was used to assay ironmetabolismrelated proteins in hippocampus of mice. (a) L-ferritin. (b) TfR1. (c) DMT1 (+IRE). (d) FPN1 and DMT1-IRE. The expression levels of theseproteins were normalized to š½-actin and expressed as the mean Ā± SD. āš < 0.05, āāš < 0.01 versus WT + saline group, #š < 0.05 versus KO+ saline group, and $š < 0.05 versus WT+ Aš½
25ā35 group.
Oxidative Medicine and Cellular Longevity 9
Aš½25ā35TfR1 ā
LIP āROS āMDA
p-p38 ā Bcl-2 ā
Bax ā
Apoptosis
PARP
Cleaved ācaspase-3
FtMt
Ferritin ā
FPN1ā
Figure 6: A schematic representation of the mechanism leading toneuronal cell apoptosis induced by Aš½
25ā35 in mice with a disruptedFtmt gene. Aš½
25ā35 changes the levels (LIP) and distribution ofintracellular iron, thus increasing oxidative stress. Without Ftmt tosequester excessmitochondrial iron, lipid peroxidation and the levelof LIP are significantly increased. These changes may signal the cellto begin the process of programmed death through the P38 MAPKpathway, resulting in neuronal cell death, which is enhanced in Ftmtknockout mice, leading to worsened memory impairments.
5. Conclusion
The current study supports the hypothesis that the function-ality of FtMt is not essential under normal conditions. But, incases of neuronal stress, such as Aš½
25ā35 accumulation, FtMtoffers a profoundneuroprotection through regulating cellulariron content and distribution in a way that keeps oxidativestress in check, preventing the activation of apoptosis.
Competing Interests
The authors declare that they have no competing interestsrelated to this work.
Authorsā Contributions
Peina Wang and Qiong Wu contributed equally to this study.
Acknowledgments
This work was supported by the National Science Foundationof China (31520103908, 31471035, and 31271473). The authorswish to thank Professor Greg Anderson (Iron MetabolismLaboratory, Queensland Institute of Medical Research, Aus-tralia) for critical reading.
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