Transgenic Mice Overexpressing the Divalent Metal Transporter 1 Exhibit Iron Accumulation and Enhanced Parkin Expression in the Brain
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Exposure to divalent metals such as iron and manganese is thought to increase the risk for Parkinson’s disease (PD). Under normal circumstances, cellular iron and manganese uptake is regulated by the divalent metal transporter 1 (DMT1). Accordingly, alterations in DMT1 levels may underlie the abnormal accumulation of metal ions and thereby disease pathogenesis. Here, we have generated transgenic mice overexpressing DMT1 under the direction of a mouse prion promoter and demonstrated its robust expression in several regions of the brain. When fed with iron-supplemented diet, DMT1-expressing mice exhibit rather selective accumulation of iron in the substantia nigra, which is the principal region affected in human PD cases, but otherwise appear normal. Alongside this, the expression of Parkin is also enhanced, likely as a neuroprotective response, which may explain the lack of phenotype in these mice. When DMT1 is overexpressed against a Parkin null background, the double-mutant mice similarly resisted a disease phenotype even when fed with iron- or manganese-supplemented diet. However, these mice exhibit greater vulnerability toward 6-hydroxydopamine-induced neurotoxicity. Taken together, our results suggest that iron accumulation alone is not sufficient to cause neurodegeneration and that multiple hits are required to promote PD.
Keywords6-OHDA Dopamine Oxidative stress Parkin Parkinson’s disease
Parkinson disease (PD) is a prevalent neurodegenerative movement disorder that is characterized pathologically by the rather selective loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). Although most cases of PD occur in a sporadic manner, a subset of PD cases is inheritable and attributable to mutations in specific genes (Chai and Lim 2013). For example, mutations in α-synuclein and Parkin are causative of autosomal dominant and recessive forms of PD, respectively. Notwithstanding this, it is important to recognize that even in individuals harboring PD-linked mutations, it typically takes several decades for the disease to surface, suggesting that additional factors (likely present in the environment) are involved in the pathogenesis of PD. Notably, exposure to metals such as iron and manganese has consistently been implicated by epidemiological studies to increase the risk for PD, particularly in view of their preferential accumulation in the SN and their role in promoting harmful oxidative reactions (Dexter et al. 1989). Moreover, aging, which is an unequivocal risk factor for PD, promotes iron accumulation in the brain. Notably, Zecca and colleagues reported that iron concentration increases linearly with age in the SN, while the amount in the locus coeruleus remains comparatively much lower even in individuals aged 80 and above (Zecca et al. 2004). However, because iron and manganese are both essential to human physiology, their levels are normally regulated by homeostatic mechanisms that constantly adjust the intake and excretion rates to avoid intoxication (or deficiency). The major transport protein responsible for the uptake of iron and manganese is divalent metal transporter 1 (DMT1, also known as NRAMP2 and SLC11A2), which exists in two isoforms—DMT1A and DMT1B (Mims and Prchal 2005). The 1A isoform is mostly found in the duodenum, whereas DMT1B is ubiquitously expressed. Both forms of DMT1 are capable of transporting several divalent cations in a pH-dependent manner and in the respective order of preference Cd2+ > Fe2+ > Mn2+, Co2+ ≫ Cu2+ (Mackenzie et al. 2007; Illing et al. 2012). Of relevance to PD is the finding that DMT1 and iron staining are tightly correlated in the basal ganglia (Huang et al. 2004). Accordingly, alterations in DMT1 levels may underlie the abnormal accumulation of metal ions in the SN and thereby PD pathogenesis. Supporting this, DMT1 expression has been reported to increase with age (Ke et al. 2005) and in the SNpc DA neurons of PD patients compared to age-matched controls, which correlates with a rise in iron content (Salazar et al. 2008). Further, mice intoxicated with the parkinsonian neurotoxin MPTP also register an increase in DMT1 levels in the ventral mesencephalon concomitant with iron accumulation, oxidative stress and DA neuronal loss (Salazar et al. 2008). Conversely, rodents carrying a mutant DMT1 that impairs transport are significantly protected against MPTP-induced neurotoxicity (Salazar et al. 2008). Together, these studies suggest a role for DMT1 in PD pathogenesis.
To examine the potential role of DMT1 in PD, we have generated transgenic mice overexpressing monkey DMT1B under the direction of a mouse prion promoter, which express in several regions of the brain. When these mice are fed with iron-supplemented diet, they exhibit robust accumulation of iron especially in the SN. Interestingly, the treated mice also display markedly enhanced level of Parkin (but not α-synuclein) that might explain their lack of an overt phenotype by virtue of the neuroprotective role of Parkin. As an extension of this study, we have also generated and characterized DMT1-expressing mice against a Parkin null background. Interestingly, these double-mutant mice continue to resist a disease phenotype even when fed with iron- or manganese-supplemented diet, although they exhibit greater vulnerability toward 6-hydroxydopamine (6-OHDA)-induced neurotoxicity. Taken together, our results suggest that multiple hits are required to promote PD.
Materials and Methods
The following antibodies were used: anti-β-actin (Sigma), anti-c-myc (Roche), anti-DMT1 (Abnova), anti-ferroportin (Novus Biologicals, Littleton, CO), anti-GAPDH (Abcam), anti-Parkin clone PRK8 (Covance, Princeton, NJ), anti-α-synuclein (BD Biosciences), anti-TH (Pel-Freez Biologicals, Rogers, AR), anti-transferrin receptor (Abcam) and HRP-conjugated anti-mouse and anti-rabbit (Sigma).
Generation of DMT1 Transgenic Mice
Iron- and Manganese-Supplemented Feed
DMT1 transgenic animals and WT littermates were aged to 9 months on a normal diet (AIN-93M with 35 mg/kg Fe and 10 mg/kg Mn (Reeves et al. 1993)) and then given either normal feed or feed supplemented with 1000 mg/kg of iron (TD110540, Harlan Laboratories) for another 9 months. DMT1/Parkin knockout double-mutant mice were given either normal diet, feed supplemented with 1000 mg/kg of iron or 1000 mg/kg of manganese (TD110541, Harlan Laboratories).
Mouse brains were harvested and lysed in PBS buffer containing 1% SDS (w/v) with added protease inhibitor (Roche). Lysates were homogenized using a handheld Teflon glass homogenizer for 15 strokes on ice and then sonicated for 15 s. Centrifugation was then carried out at 13,500 rpm for 15 min and the supernatant collected. Protein content was determined using the Bradford assay (Pierce). Approximately 55 to 100 µg of total protein was resolved on 10–15% SDS-PAGE gel before transferring onto PVDF membrane (Millipore). Detection was carried out using ECL reagent (Pierce).
Mice were anaesthetized and transcardially perfused with saline (0.9% NaCl, w/v) followed by 4% PFA (w/v). The brains were removed and post-fixed for another 24 h before placing into cryoprotectant containing 15% sucrose (w/v) in PBS for 24 h and then 30% sucrose for another 24 h. Brains were sectioned in the coronal plane at 20 µm thickness and mounted on histology slides (Marienfeld, Germany). Primary and secondary antibodies were diluted in blocking buffer containing 0.5% BSA (w/v), 0.5% FBS (w/v) and 0.1% Tween 20 (v/v) in PBS. Primary antibody incubations were done overnight at 4 °C while secondary antibodies were incubated for 30 min at room temperature. Sections were washed three times after primary and secondary antibodies with TNT wash buffer consisting of 0.1M Tris-HCl (pH 7.5), 0.15M NaCl and 0.05% Tween 20 (v/v). For immunohistochemistry, antibody signals were amplified with VectorElite ABC Kit (Vector Laboratories) and developed using DAB (Vector Laboratories) according to the manufacturer’s protocol. The sections were then dehydrated under alcohol gradient (75, 90 and 100%) and subsequently two changes of xylene for 3 min each. The sections were then mounted using DPX mounting medium and viewed using a BX61 microscope (Olympus).
Perls Prussian Blue Stain
Fixed brain sections were immersed in Perls solution consisting of 2% HCl (v/v) and 2% potassium ferrocyanide (w/v) in a 1:1 ratio for 30 min at room temperature. Sections were washed and counterstained with Nuclear Fast Red solution (Sigma) for 5 min. The sections were dehydrated under alcohol gradient (75, 90 and 100%) followed by two changes of xylene. Finally, the sections were mounted using DPX and viewed with a BX61 microscope (Olympus).
T2-Weighted Magnetic Resonance Imaging (MRI)
Mice were anesthetized with ketamine/xylazine (10 mg/kg) and transcardially perfused with saline (0.9% NaCl, w/v) and thereafter with 4% PFA (w/v). The brains were then harvested and embedded in 1% agarose. T2-weighted MRI was acquired on a 9.4 Tesla scanner (Agilent Technologies, Santa Clara, CA) using a volume transmit/receive coil and the following parameters: multislice fast spin echo; 100-µm isotropic resolution; 256 × 192 × 192 matrix size; 25.6 × 19.2 × 19.2 mm3 field-of-view; 2000 ms TR; 8 ETL; 39.15 ms effective TE; and 1 average.
Immunohistochemistry and Stereological Assessment of TH-Positive Neurons
Mice were given a lethal overdose of anesthesia and were perfused through the heart with cold saline, followed by 4% PFA (w/v). Brains were collected, post-fixed overnight and transferred to a 15% and then 30% sucrose solution overnight. Every fourth section (40 μm) through the perfusion-fixed ventral midbrain was collected on CM3050S cryostat (Leica Biosystems) and placed in free-floating PBS. Endogenous peroxidase activity of each section was quenched with 0.3% H2O2 (Sigma–Aldrich) for 30 min, followed by washing with TNT buffer. Brain sections were then blocked with TNT blocking buffer containing 5% normal goat serum (Jackson ImmunoResearch Laboratories) for another 30 min. Subsequently, sections were incubated in rabbit polyclonal anti-TH antibody (Pel-Freez Biologicals, Rogers, AR) in 5% NGS TNT blocking buffer overnight at 4 °C and washed with TNT buffer the following day. Brain sections were then incubated in secondary biotinylated antibody (Vector Laboratories) for 1 h prior to incubation with ABC reagent kit (Vector Laboratories) for 30 min at room temperature. TH+ cells were made visible via DAB staining (Vector Laboratories) before the sections were mounted on glass slides for visualization. To analyze the TH+ neurons in the left and right pars compacta regions of each brain section, unbiased stereological methodology was employed using a computer-assisted system consisting of Axio Imager 2 microscope (Carl Zeiss), equipped with a motorized ASI MS-2000 stage (Applied Scientific Instrumentation, Eugene, OR), a IEEE 1394 camera (Imi Tech, Encinitas, CA) and interfaced with Stereologer software (Stereology Resource Centre, St. Petersburg, FL).
Measurement of Iron in Urine and Feces
Iron concentrations in the urine and feces were measured using the QuantiChrom Iron Assay Kit (BioAssay Systems, Hayward, CA) according to the manufacturer’s protocol. Air-dried feces were suspended in 1 ml of water for an hour, and the resulting suspension assayed in a 96-well plate. Urine samples were analyzed directly without further dilution. Final reading was measured with a plate reader at 590 nm (Tecan, Switzerland).
Parkin Real-time PCR and Stable Cell Lines
PCR for Parkin and GAPDH mRNA levels, as well as generation of vector and Parkin overexpression SH-SY5Y stable cell lines have previously been described (Wang et al. 2005).
Generation of 6-OHDA Lesion Mouse PD Model and Apomorphine-Induced Rotation Assessment
Mice of 4–6 months of age were anesthetized with ketamine/xylazine and mounted on a stereotaxic frame (Narishige, Japan). A small hole was drilled in the skull with coordinates AP +0.5 mm to Bregma, ML 2.0 mm left to the midline. 3 ul of 2.5 ug/ul 6-hydroxydopamine (6-OHDA, Sigma–Aldrich) was delivered to the striatum with a Hamilton syringe. Once injection was complete, the needle was left in place for 5 min to allow the pressure of the injected volume to dissipate. The needle was then slowly retracted and the wound closed by suturing. 6 weeks after 6-OHDA injection, the apomorphine-induced rotation assay was carried out. Apomorphine (Sigma–Aldrich) was dissolved in saline with 0.1% ascorbic acid and administered by IP injection into the mouse (1.5 mg/kg). 5 min after apomorphine administration, the mouse was transferred to a circular container and the number of rotations made in 3 min was counted.
Motor performance assays were carried out using a rotarod (Ugo Basile, Italy). Briefly, the mice were placed on the rotarod with a rotating speed of 4 rpm. Once the mice were ready on the beam, the speed was accelerated to 40 rpm within 4 min and the time spent on the rotarod was recorded. Each mouse was given three trials with 10–15-min rest after every trial.
All data are expressed as mean ± S.E.M. Statistical analysis was performed using Student’s two-tailed t test, one-way or two-way ANOVA and statistical significance was considered when p < 0.05.
DMT-1 Expressing Mice Exhibit Iron Accumulation in the Brain
To generate transgenic mice that overexpress DMT1, we cloned the cDNA of monkey DMT1 into the MoPrP.Xho expression vector (Fig. 1a), which drives high expression of the transgene in most CNS neurons via the murine prion promoter. Transgenic founders were backcrossed for six generations, and the DMT1 transgene was subsequently maintained in the heterozygous state. Analysis of transgene genotype via PCR (Fig. 1b) and protein immunoblotting (Fig. 1c) show the presence and robust expression of DMT1 transgene in the brains of these mice. Consistent with this, strong DMT1 immunoreactivity was observed in all the six brain regions surveyed, i.e., olfactory bulb, hippocampus, substantia nigra, striatum, cortex and cerebellum, relative to their control counterparts (Fig. 1d).
Parkin is Upregulated in the Brain of DMT1-Expressing Mice
Generation and Characterization of DMT1/Parkin Knockout Double-Mutant Mice
The observation that DMT1 upregulation correlates with iron accumulation in the SN of PD patients (Salazar et al. 2008) has prompted us to investigate the role of the DMT1 transporter in brain metal accumulation and neurodegeneration. For this purpose, we have generated and characterized transgenic mice overexpressing DMT1 in the brain via the murine prion promoter in this study. We found that DMT1-expressing mice express the transgene robustly in various regions of the brain but exhibit rather selective accumulation of iron in the SN when treated with iron-supplemented diet. However, these mice do not display obvious motoric deficits even with age, suggesting that iron accumulation in the SN alone is insufficient to promote DA neurodegeneration. In part, this may be due to the upregulation of Parkin, a potent neuroprotectant, in the brain of these mice. Curiously, DMT1/Parkin KO double-mutant mice treated with iron- or manganese-supplemented also fail to exhibit robust Parkinsonism phenotype, but are nonetheless more susceptible to 6-OHDA-induced neurotoxicity. Taken together, our results support the suggestion that multiple hits are required for DA neuronal loss in PD (Sulzer 2007).
Iron accumulation is widely thought to be detrimental for cellular survival. It is well known that the iron-catalyzed Fenton reaction can covert mitochondrial-related H2O2 to the highly reactive hydroxyl radical (·OH) that can damage intracellular molecules such as DNA, proteins and lipids. Accordingly, brain regions that have a high burden of iron accumulation are likely to be more susceptible to oxidative stress. The situation is further aggravated in DA neurons as iron can promote the oxidation of dopamine and facilitate the formation of dopamine quinone as well as the neurotoxic 6-OHDA (Hare and Double 2016). Iron can also enhance the aggregation of α-synuclein, which is particularly toxic to DA neurons (Levin et al. 2011). Given these, one could therefore readily appreciate the impact of iron accumulation on DA neuronal survival. However, it remains unclear whether iron accumulation represents a cause or consequence of neurodegeneration in PD (Daugherty and Raz 2015). Notably, several studies have revealed the accumulation of Fe3+ (rather than the more reactive Fe2+) in the SN of PD brains compared to controls (Dusek et al. 2015). Specifically, Mössbauer spectroscopy can distinguish between Fe2+, which generates free radicals via the Fenton reaction, and Fe3+ which is usually found stored in ferritin. Using this technique, Galazka-Friedman conducted a study in PD individuals and age-matched controls but found no evidence of Fe2+ in the SN, suggesting that iron is stored as the more inert Fe3+ form in ferritin (Galazka-Friedman et al. 2012). Similarly, our results showed increased Fe3+ in the SN of DMT1 mice compared to WT controls, as detected using Perls staining. Finally, a study that involved MPTP-treated monkeys showed a reduction in TH-positive cells within a week of injection, but increase in iron levels was only observed after 4.5 months (He et al. 2003). This again suggests that iron accumulation may not be a direct cause of neuronal death, and could explain why accumulation of iron is generally observed but not always present in cases of neurodegeneration (Dashtipour et al. 2015). This may also explain why DMT1-expressing mice do not develop any overt signs of disease-associated phenotype despite exhibiting robust accumulation of iron, especially in the SN when fed with iron-supplemented diet. The lack of neurotoxicity in the transgenic mice could also be due in part to the upregulation of Parkin, which was observed in DMT1 mice treated with iron-supplemented diet. Parkin is a multifunctional ubiquitin ligase that is widely regarded to be a broad spectrum neuroprotectant capable of protecting the cells against a plethora of toxic insults (Zhang et al. 2015). Interestingly, a recent study by Roth and colleagues demonstrated that Parkin regulates metal transport via promoting proteasomal degradation of DMT1B (Roth et al. 2010). Consistent with this, Parkin overexpression affords considerable protection to cells treated with manganese (Higashi et al. 2004) and iron (this study). In light of these findings, it was intuitive for us to investigate whether the ablation of Parkin expression would promote a PD phenotype in DMT1-expressing mice. As described above, the double-mutant mice also fail to exhibit robust signs of Parkinsonism, even when treated with iron-supplemented diet until they reached 18 months of age. This reinforces the suggestion that iron accumulation may not be the culprit in PD. Supporting this, we have recently shown that low concentrations of iron can mitigate manganese induced cytotoxicity rather than having deleterious effects (Tai et al. 2016). Unlike iron, chronic manganese exposure is well documented to be the cause of motoric disturbances known as manganism (Chen et al. 2015). Curiously, even when treated with manganese-enriched diet, DMT1/Parkin KO double-mutant mice appear largely normal, although they tend to display lower motor performance on a rotarod in the initial months following treatment. Thus, despite lending itself as a “gene (i.e., Parkin KO)—environmental (i.e., DMT1-mediated accumulation of metal ions)” model of PD, the double-mutant mice are relatively resistant to neurodegeneration. This is somewhat reminiscent of the scenario with the triple Parkin/PINK1/DJ-1 knockout mice, which exhibit no evidence of Parkinsonism (Kitada et al. 2009). Notwithstanding this, DMT1/Parkin KO double-mutant mice are more vulnerable to 6-OHDA-induced neurotoxicity. This appears to be brought about by the combined effects of DMT1 overexpression and Parkin gene ablation as mice expressing DMT1 alone do not exhibit significantly different motoric phenotype from WT mice when treated with 6-OHDA. Similarly, Parkin-deficient mice alone are not more sensitive to the toxin (Perez et al. 2005).
Although many genetic mutations are known to cause or modulate the risk of PD, the low penetrance of some of these mutations together with the low disease concordance in relatives led to the suggestion that there must be interactions among multiple factors for PD to manifest (Sulzer 2007). Our results indicate that a combination of factors is required to promote a disease phenotype in mice and thus support the “multiple hit” hypothesis for the pathogenesis of PD. At the same time, our results may also help to explain why mouse models of PD based on single-gene mutation generally lack robust signs of Parkinsonism.
This work was supported by the following grants from the National Medical Research Council—CBRG12nov078 (STW), EDG10nov065 (LKL) and Translational Clinical Research Program in Parkinson’s disease (LKL and STW).
Compliance with Ethical Standards
Conflict of interest
The authors declare that they have no conflict of interest.
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