Dyshomeostasis of Iron and Its Transporter Proteins in Cypermethrin-Induced Parkinson’s Disease

The etiology of Parkinson’s disease (PD) is highly complex and is still indefinable. However, a number of studies have indicated the involvement of pesticides and transition metals. Copper, magnesium, iron, and zinc have emerged as important metal contributors. Exposure to pesticides causes an accumulation of transition metals in the substantia nigra (SN) region of the brain. The cypermethrin model of PD is characterized by mitochondrial dysfunction, autophagy impairment, oxidative stress, etc. However, the effect of cypermethrin on metal homeostasis is not yet explored. The study was designed to delineate the role of metals and their transporter proteins in cypermethrin-induced animal and cellular models of PD. The level of copper, magnesium, iron, and zinc was checked in the nigrostriatal tissue and serum by atomic absorption spectroscopy. Since cypermethrin consistently increased iron content in the nigrostriatal tissue and serum after 12 weeks of exposure, the level of iron transporter proteins, such as divalent metal transporter-1 (DMT-1), ceruloplasmin, transferrin, ferroportin, and hepcidin, and their in silico interaction with cypermethrin were checked. 3,3′-Diaminobenzidine-enhanced Perl’s staining showed an elevated number of iron-positive cells in the SN of cypermethrin-treated rats. Molecular docking studies revealed a strong binding affinity between cypermethrin and iron transporter protein receptors of humans and rats. Furthermore, cypermethrin increased the expression of DMT-1 and hepcidin while reducing the expression of transferrin, ceruloplasmin, and ferroportin in the nigrostriatal tissue and human neuroblastoma cells. These observations suggest that cypermethrin alters the expression of iron transporter proteins leading to iron dyshomeostasis, which could contribute to dopaminergic neurotoxicity.


Introduction
The amalgamation of a number of factors, such as age, genetics, and environmental exposure to neurotoxicants, is found to contribute to Parkinson's disease (PD). Anatomically, PD results from the demise of dopaminergic neurons in the substantia nigra (SN) [1,2]. Epidemiological studies have also highlighted the involvement of oxidative stress, mitochondrial dysfunction, and impairment in protein clearance pathways in disease progression [3]. Besides, a variety of factors contributing to PD, the existing reports also demonstrate the involvement of transition metal ions, such as copper, iron, zinc, and magnesium, in disease progression [4]. Transition metals act as a cofactor for several physiologically essential metalloenzymes that catalyze important biological functions responsible for synaptic transmission, myelin synthesis, redox balance, and adenosine triphosphate (ATP) biosynthesis. Accumulation or deficiency of transition metal ions CSIR-IITR communication number: IITR/SEC/MS/2022/90. also serves as a contributory factor for neurodegeneration leading to PD [5]. Employing various imaging techniques, several research groups have assessed the level of transition metals in the brain regions and reported high iron content in the SN. However, conflicting reports are available for zinc, copper, and magnesium in the degenerating regions of the Parkinsonian brain [6][7][8].
Environmental factors are of major concern since environmental exposure to neurotoxicants induces irreversible or reversible degeneration of dopaminergic neurons. Neurotoxicants, such as 6-hydroxydopamine (6-OHDA), 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and rotenone, are shown to cause metal accumulation through an interaction with metal bound enzymes or by creating a free pool of metal ions that contribute to oxidative stress [9,10]. While the underlying mechanism behind the metal accumulation is elusive, several studies have shown that the accumulation of metals in various regions of the Parkinsonian brain could be due to dyshomeostasis of transporter proteins that are involved in the export, import, and redistribution of metal ions within the cell [11,12].
In 6-OHDA, MPTP, and rotenone animal models of PD, an increased level of iron was observed in the SN and ventral midbrain, which was concomitant with the increase in divalent metal transporter-1 (DMT-1) and hepcidin and reduced ferroportin, transferrin, and ceruloplasmin expression [13][14][15][16][17]. The elevated level of iron induces the generation of reactive oxygen species, alpha-synuclein aggregation, cellular fragmentation, and neuronal death. Furthermore, increased level of copper and magnesium is also reported in different areas of the dopaminergic pathway in 6-OHDA and MPTP-induced animal models of PD [18,19]. While exposure to magnesium alters the mitochondrial function and disrupts the release and function of neurotransmitters, elevated level of copper hampers ferroxidase activity of ceruloplasmin protein and induces the formation of catecholamine oxidation products, which results in DNA damage [20,21]. MPTP and 6-OHDA also promote an accumulation of zinc in the SN leading to microglial activation and neuroinflammation [22,23]. The dysregulation of metal homeostasis induces a series of detrimental events leading to the demise of dopaminergic neurons.
Cypermethrin, a pyrethroid pesticide, has been shown to be an important risk factor for PD-like symptoms in experimental rodents. Prolonged exposure to this pyrethroid at the moderate doses leads to neurodegeneration [3,24]. Cypermethrin is reported to alter the membrane potential of mitochondria, autophagy, microglial activation, and mitochondrial apoptosis leading to oxidative load and an increase in the permeability of the blood-brain barrier in experimental animals [24][25][26]. Cypermethrin is also responsible for the prolonged opening of ion channels, antagonizing gamma-aminobutyric acid (GABA)-mediated inhibition, and regulation of cholinergic transmission, which modulate the release of different neurotransmitters [25,27]. However, the effect of cypermethrin on metal accumulation and its interaction with iron transporter proteins is not yet reported. The present study was conducted to delineate the role of metals, mainly iron and its transporter proteins, in the cypermethrin model of rat and human neuroblastoma cells, SH-SY5Y. In brief, atomic absorption spectroscopy (AAS) was employed to investigate the level of zinc, copper, magnesium, and iron in the nigrostriatal region and serum of cypermethrin-treated rats. The studies were also carried out at the protein level to correlate the iron dyshomeostasis with its transporter proteins. In addition, in silico molecular docking studies were also carried out to identify the binding site and mode of interactions between cypermethrin and the active site of iron transporter proteins which can help to provide insights into the potential selectivity of cypermethrin for a specific receptor. Moreover, the outcome of in silico studies will provide insights into the comparative mode of binding affinities of cypermethrin with receptors of humans and rats. Thus, the combination of these in silico, in vitro, and in vivo studies may provide a more comprehensive understanding of the underlying mechanism of cypermethrin-induced neurotoxicity.

Materials
The ordinary/analytical grade chemicals and laboratory reagents required for experiments done in this study were purchased locally. Fine chemicals were purchased from reputed international firms and mentioned at the appropriate places in the manuscript.

In Vivo Treatment
The standard ethical guidelines were followed for experiments performed in the study. The experiments were executed by employing male Wistar rats. Protocols for performing animal experiments were approved by the institutional animal ethics committee. The experimental animals were housed appropriately under the optimum light/dark cycle, humidity, and temperature. Male pups were treated with 1.5 mg/kg cypermethrin (Thermo Fischer Scientific, Waltham, MA) during postnatal days 5-19, two times a week, intraperitoneally, along with respective control. The animals were left untreated for 2 months and then re-challenged with 15 mg/kg cypermethrin, intraperitoneally, two times a week, for 4, 8, and 12 weeks. Experimental controls treated with vehicles were also developed in parallel [25].

Serum and Brain Isolation
At the end of the study (4 weeks, 8 weeks, 12 weeks), animals were sacrificed under diethyl ether anesthesia (Merck, Darmstadt, Germany). The peripheral blood serum and brain were isolated as described elsewhere [25,28]. Blood was collected in glass tubes, centrifuged for 15 min at 1500 × g at 4 °C. After centrifugation, serum was collected and stored at − 20 °C until further use. For brain isolation, intra-cardiac perfusion was done with 0.9% normal saline at a fixed rate of 20 ml/min for 4 min. Subsequently, the brain was dissected out and kept at − 20 °C for further use [25].

Atomic Absorption Spectroscopy
The level of iron, copper, zinc, and magnesium in the nigrostriatal region of the rat's brain and serum was quantified spectroscopically as described elsewhere with minor changes [29]. In brief, the nigrostriatal region and serum were digested for 12 h in a 2.5 ml digestion mixture containing nitric acid (HNO 3 ) (Merck, Darmstadt, Germany) and perchloric acid (Thermo Fischer Scientific, Waltham, MA) in the ratio of 6:1 (v/v). Digested samples were kept on a sand bath maintained at 60 °C and re-digested until sample residue reached to 100 μl. The final residue was dissolved in 3 ml of 1% HNO 3 . The concentration of magnesium, zinc, copper, and iron in the nigrostriatal region and serum was measured using AAS (PinAAcle 900F, PerkinElmer, Waltham, MA).

Cell Culture and Lysate Preparation
Human neuroblastoma cells were indented from the National Centre for Cell Science, Pune, India. Cell culture experiments were performed as described in detail elsewhere [24]. In brief, the cells were maintained under humidified conditions at 37 °C containing 5% carbon dioxide. For western blot analysis, 1 × 10 6 cells were seeded in a T-25 flask for 24 h. Cells were exposed to cypermethrin (15 μM) for 24 h along with control. Protein was isolated employing standard procedure and estimated using Lowry's method [30].

Protein Extraction and Estimation
Protein was extracted as described somewhere else [26]. Protein content was calculated in the supernatant employing Lowry's method [30]. Approximately 100 mg of tissue was taken for making homogenate (10% w/v) in each group.

Western Blot Analysis
The sample containing 60-100 μg of protein was equally loaded on two parallel gels. The proteins depending upon the molecular weight were resolved on sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electroblotted onto polyvinylidene difluoride membrane (PVDF) (Bio-Rad Laboratories, Inc., Hercules, CA) as described elsewhere [26]. To prevent non-specific binding, the PVDF membrane was incubated with 5% skimmed milk (HiMedia Laboratories Pvt. Ltd., Mumbai, India) for 60 min. Incubation of membrane was done with the primary antibodies [anti-transferrin, anti-ferroportin, anti-DMT-1, anti-β-actin (Santa Cruz Biotechnology Inc., Dallas, TX), anti-ceruloplasmin, or anti-hepcidin (Abcam, Cambridge, UK)] for overnight followed by 1 h incubation with anti-goat/ anti-rabbit/anti-mouse (Santa Cruz Biotechnology Inc., Dallas, TX) secondary antibody conjugated with alkaline phosphatase (AP). Since the secondary antibody used in the experiment was AP-conjugated, one gel was processed for the target protein and the other for respective loading control. The protein bands were developed using AP-specific chromogenic substrates, 5-bromo-4-chloro-3-indolyl phosphate, and nitro blue tetrazolium chloride (Thermo Fischer Scientific, Waltham, MA). The band density ratio was calculated in relation to β-actin using Alpha Imager software.

Cryo-Sectioning and DAB-Enhanced Perl's Iron Staining
Cryo-sectioning and 3,3′-diaminobenzidine (DAB)enhanced Perl's iron staining were performed as mentioned elsewhere with minor changes [25,31]. In brief, rats were anesthetized using diethyl ether and perfused trans-cardially using 0.9% saline and 4% paraformaldehyde (PFA) (Sigma-Aldrich, St. Louis, MO). Brain tissue was sliced coronally across the SN into 20 μm thick sections in a cryotome (MEV cryostat, SLEE medical GmbH, Neider-Olm, Germany) maintained at − 20 °C. Brain sections were then fixed with 4% PFA for 5 min. Sections were incubated in freshly prepared solutions of 2% hydrochloric acid (Merck, Darmstadt, Germany) and 2% potassium ferrocyanide (Thermo Fischer Scientific, Waltham, MA) for 30 min. Sections were incubated with 50% methanol (Sisco Research Laboratories Pvt. Ltd., Mumbai, India) and 1% hydrogen peroxide (Merck, Darmstadt, Germany) in PBS to block endogenous peroxidase activity. The iron staining was enhanced using DAB (Sigma-Aldrich, St. Louis, MO). The sections were mounted, and images were captured at 20 × and 40 × magnifications under the microscope (DM6000, Leica Microsystems, Wetzlar, Germany). In the SN, the number of ironpositive cells was quantified using ImageJ software.  The data are expressed as a percentage of control. All control values were considered 100%. Thus, there is no error bar in control. Twoway ANOVA and Bonferroni post-test were employed. Significant changes are expressed as **p < 0.01 in comparison with the control   it is clear that all the proteins show moderate to high accuracy in terms of confidence; therefore, these proteins were used for the molecular docking study. The predicted aligned error plots for the receptors of rats are provided in the supplementary information (S1). The 3D structure of the ligand (cypermethrin) was drawn using ACD/ChemSketch [33]. The 3D structure of the ligand was then converted into pdb format using OpenBabelGUI software [34]. Initially, these transporter receptors were prepared for docking followed by the addition of the polar hydrogens and Kollaman charges to respective receptors. The receptor and ligand structures were saved in pdb format using OpenBabelGUI. The ligands were subsequently saved in pdbqt format in ADT to carry out the docking studies. The AutoDock default parameters were considered for the docking studies. In each case, ten docked conformations were generated. The energy calculations were achieved by genetic algorithms and blind docking centered on the target. Binding energies, the hydrogen bonding with the receptor and receptor-ligand interactions were the parameters used for analyzing the docked ligand conformations. Chimera 1.11 [35] was used to visualize the receptor with the ligand binding site and Discovery Studio 2021 [36] was used to analyze different types of interactions. ChimeraX software was used for the prediction of confidence score and predicted aligned error for alpha fold protein.

Statistical Analysis
Unpaired student's t-test or two-way analysis of variance (ANOVA) followed by the Bonferroni post-test was employed for comparison. The outcomes are expressed as mean ± standard error of the mean (SEM; biological replicates = 3). GraphPad Prism version 5.0 was employed to plot the bar diagrams. Changes were judged as statistically significant only when the "p"-value was less than 0.05.

Level of Copper, Iron, Magnesium, and Zinc in the Nigrostriatal Tissue and Serum of Cypermethrin-Treated Rats
The level of copper, iron, magnesium, and zinc was quantified in the nigrostriatal tissue and serum by AAS. Animals treated with cypermethrin for 4-12 weeks marked a slight increase in the level of copper, magnesium, and zinc in the nigrostriatal tissue. However, the augmented level of these metals was not statistically significant (Fig. 1a, c, d). A significant augmentation in the level of iron (378.82 ± 45.63%) after 12 weeks of cypermethrin exposure was seen as compared with that of control in the nigrostriatal region (Fig. 1b). Also, cypermethrin significantly increased the level of iron (158.06 ± 19.38%) in serum of 12 weeks treated animals in comparison with control (Fig. 2b). Whereas, no significant change was observed in the level of copper, magnesium, and zinc in the treated group for 4, 8 and 12 weeks (Fig. 2a, c, d).

Number of Iron-positive Cells in the SN
Using DAB-enhanced Perl's staining, iron-positive cells in the SN of cypermethrin-treated rats were analyzed. In the SN region of control rats, some round and small cells with iron-containing processes were present. Cypermethrin significantly increased the count of iron-containing cells (259.2 ± 24.99%) in comparison with control in the SN region of treated animals (Fig. 3a, b), which demonstrates an increased iron level in that region after cypermethrin exposure.

Docking and Interaction Analysis
To identify binding orientations, cypermethrin was docked onto the ligand binding site of iron transporter protein receptors using Autodock 4.0. The binding energies of cypermethrin  (Table 1). Besides, the binding energies of cypermethrin with the rat ceruloplasmin (5N0K) receptor, transferrin (AF-P12346-F1) receptor, ferroportin (AF-Q923U9-F1) receptor, hepcidin (AF-Q99MH3-F1) receptor, and DMT-1 (AF-O54902-F1) receptor were found to be − 5.21 kcal/mol, − 6.96 kcal/mol, − 6.00 kcal/mol, − 5.74 kcal/ mol, and − 5.48 kcal/mol, respectively ( Table 2). The summary of the docking results of cypermethrin with human and rat iron transporter proteins is mentioned in Table 1 and Table 2, respectively. The 2D and 3D representations of docking interactions revealed a strong affinity between cypermethrin and iron transporter proteins of human (Fig. 4) and rat (Fig. 5). The predicted 3D bound conformations of cypermethrin with human receptors, transferrin, and hepcidin showed one H-bonding interaction (Fig. 4b, d) while the other human receptors did not show any H-bonding interaction with cypermethrin (Fig. 4a, c, e). In the case of rat transporter proteins, one and three H-bonding interactions were observed between cypermethrin and amino acid residues of ceruloplasmin and hepcidin receptors, respectively. The ligand formed two H-bond with ferroportin and DMT-1 receptor. While no H-bonding interaction was observed with transferrin (Fig. 5b). 2D representation of docking interactions showed that cypermethrin was accommodated well inside the binding region of both human and rat receptor proteins. The ligand, cypermethrin, was stabilized by different types of molecular interaction such as Van der Waals interactions, alkyl and π-alkyl π-sigma, π-π stacking and hydrogen bond interactions (Fig. 4, 5 and Table 1, 2). The grid box coordinates used for molecular docking studies have been provided in Table 3.

Iron Transporter Proteins in the Nigrostriatal Tissue and SH-SY5Y Cells
Since cypermethrin increased the level of iron in the nigrostriatal region and serum after 12 weeks of treatment, this time point was selected to quantify the expression level of iron transporter proteins in the nigrostriatal tissue and SH-SY5Y cells in the presence/absence of cypermethrin. Cypermethrin augmented the expression of DMT-1 (Fig. 6a  and 7a) and reduced the expression of transferrin (Fig. 6b  and 7b) indicating a significant increase in the import of non-transferrin bound iron inside the cell. Expression of ferroportin and ceruloplasmin was attenuated in cypermethrinexposed rats with respect to control (Fig. 6c, d and 7c, d), which showed that export of iron was affected resulting in an increase in intracellular iron load. Besides, cypermethrin also increased the expression of hepcidin (Fig. 6e and 7e), a ligand of ferroportin, which is responsible for the internalization and degradation of ferroportin. This elevated level of hepcidin after cypermethrin exposure showed a compromised efflux of iron out of the cell.

Discussion
Copper, iron, magnesium, and zinc are essential trace elements required for normal growth, development, and cellular homeostasis. Trace elements serve as a cofactor of several enzymes involved in neuronal function. The role of such elements is decisive in the metabolic activity of the central nervous system and known to regulate biological processes, such as expression and aggregation of alpha-synuclein, generation of ROS, mitochondrial dysfunction, inflammation, cellular apoptosis, and autophagy, which are key regulators of PD progression. Excessive accumulation or deficiency of trace elements could lead to adverse health effects. For instance, dyshomeostasis of zinc leads to cognitive decline and is found to be associated with PD pathogenesis in a study [16]. Zinc serves as a cofactor in one of the key antioxidant enzymes, i.e., copper/zinc superoxide dismutase (CuZ-nSOD), which catalyzes the dismutation of superoxide ion. Zinc supplementation has been shown to increase the CuZ-nSOD thereby scavenging free radicals and protecting neurons [37,38]. Regulation of the function of ceruloplasmin, a metallothionein, is governed by copper and its deficiency leads to motor disabilities, such as resting tremor and slowness in movement, in rodents [39]. Deficiency or accumulation of magnesium is also harmful to cellular activity since magnesium serves as a cofactor for numerous enzymatic reactions, which play a central role in energy metabolism [40]. In recent research, iron accumulation, in areas primarily linked to motor function, has been widely reported in aged animals and humans. Epidemiological evidences have also suggested that iron is involved in myelin sheath formation, neurotransmitter synthesis, metabolic pathways, and other biological processes. The level of iron is found to be elevated in PD patients and contributes to the free radical generation and the death of dopaminergic neurons [41,42].
Environmental toxins, such as rotenone, MPTP, and 6-OHDA, are shown to enhance the accumulation of metal ions, such as zinc [23]. Contrary to this, zinc is shown to offer neuroprotection by decreasing lipid peroxidation in 6-OHDA-treated rats [43]. The protective effect of magnesium is reported in 1-methyl-4-phenylpyridinium-treated  [19]. The protective effect of magnesium is also found against MPTP-induced motor anomalies and loss of dopamine-producing neurons [40,44]. An increase in copper and iron levels is also reported in 6-OHDA, MPTP, and rotenone models [18,45,46]. Additionally, an elevated level of iron in the SN of MPTP and 6-OHDA animal models of PD is also shown, which is correlated with motor dysfunction, dopaminergic neurodegeneration, and decreased content of dopamine in the striatum [47][48][49].
The current study was designed to explore the role of metal accumulation in cypermethrin-induced Parkinsonism. Cypermethrin increased the level of iron in the nigrostriatal tissue as well as the serum of 12 weeks treated animals. Although the values were slightly altered in some experiments, the animals treated with cypermethrin for 4-12 weeks showed no statistically significant change in the level of copper, magnesium, and zinc in the nigrostriatal tissue and serum. The abundance of non-heme iron in the brain especially along the basal ganglia explains an increased trend of iron in the nigrostriatal tissue. Iron is found to accumulate to a much higher extent in the SN as compared with other brain areas that could lead to functional impairment of dopamine-producing neurons. Therefore, DAB-enhanced Perl's staining was done to detect the status of iron in the SN. In cypermethrin-treated animals, an increased count of iron-positive cells concentrated in the SN was observed, which indicated an increase in iron accumulation. Iron homeostasis is required to balance the normal physiological functions of the brain. An increased iron could contribute to the free labile pool of iron that acts as a potent pro-oxidant and generates positive feedback leading to oxidative stress and the demise of dopaminergic neurons via the Fenton reaction [50,51].
Iron equilibrium in the cellular system depends on the balance between the influx and efflux of iron. This process is under strict regulation of iron transporter proteins, which are accountable for the uptake and release of iron from the cell.
Accrual of iron is correlated with impairment in the expression of a few transporter proteins in rotenone, MPTP, paraquat, and 6-OHDA models of PD [52]. DMT-1 is required for the import of iron inside the cell in a non-transferrinbound form [47]. Once iron enters the cells, it can be housed in an iron storage protein, ferritin, in a non-reactive form. If the cellular iron concentration increases, iron is exported out of the cell by an exporter protein, ferroportin after getting oxidized to ferric iron via associated ferroxidase, i.e., ceruloplasmin [53]. The amount of iron exported out of the cell is under the surveillance of master regulator protein hepcidin, a small peptide that controls the amount of iron to be exported by ferroportin. The role of hepcidin in controlling iron homeostasis in systemic circulation and peripheral organs is well-established. Owing to its adequate expression in different brain areas, such as the cortex, striatum, cerebellum, and olfactory bulb, its potential to reduce iron accumulation has been postulated in the brain. Reduction in the expression of ferroportin mRNA and protein levels in rat cerebral cortex and hippocampus after hepcidin administration along with the augmented level of DMT-1 and reduced level of ceruloplasmin are also reported [14]. In order to further understand the underlying mechanism of iron accumulation, in silico approach was used to model the interaction between cypermethrin and iron-binding proteins in humans and rats. The interaction of cypermethrin with different human and rat iron-binding receptors was analyzed individually. A molecular docking study illustrated a strong and stable binding of docked conformation of cypermethrin with DMT-1, ferroportin, ceruloplasmin, transferrin, and hepcidin proteins.
The expression level of iron transporter proteins was also checked by employing western blotting with or without cypermethrin treatment. Cypermethrin augmented the level of DMT-1 and hepcidin whereas reduced the level of ferroportin, ceruloplasmin, and transferrin in the nigrostriatal region of rats as compared with the control. Expression of transporter protein was also measured in SH-SY5Y cells   sity ratio with respect to loading control (β-actin). An unpaired student's t-test was used. Significant changes are expressed as *p < 0.05, **p < 0.01, and ***p < 0.001 in comparison with respective control in the presence/absence of cypermethrin. Similar to nigrostriatal tissue, the expression of DMT-1 and hepcidin was increased while the expression of ferroportin, ceruloplasmin, and transferrin proteins was reduced. The outcomes were consistent with the experimental findings, which reported that the knockdown of hepcidin protects neuroblastoma cells from cellular iron load and oxidative damage caused by 6-OHDA treatment through up-regulation of ferroportin expression [54]. The results are also in accordance with an increase in the expression of DMT-1 in the SN of 6-OHDA, MPTP, and rotenone-induced PD model and PD patients [13,31,47]. In addition, treatment with transferrin and ceruloplasmin is already shown to ameliorate iron accumulation and motor impairment in MPTP-induced mouse model of PD [17]. The study concludes that cypermethrin increases the cellular iron load and dysregulates iron transporter proteins, DMT-1, hepcidin, ferroportin, transferrin, and ceruloplasmin. An alteration in transporter proteins could lead to impairment of iron homeostasis in the nigrostriatal tissue of cypermethrin-exposed animals and SH-SY5Y cells.