Drosophila strains, yeast strains, and maintenance
Drosophila strains used in this study are listed as follows: Da-Gal4 (Bloomington #8641), Elav-Gal4 (Bloomington #458), and TH-Gal4 (Bloomington #8848), all from the Bloomington Drosophila Stock Center. p Catsup RNAi (VDRC #100095) and dZnT7 RNAi (VDRC #107388) were obtained from the Vienna Drosophila RNAi Center. Transgenic fly strains used in this report have been previously described, including dZIP13 OE [37], dZIP1 OE [42], dZnT1 OE [39]. pUAST-Malvolio construct (Mvl OE) was injected into w1118 background flies following standard protocols. One line with relatively more severe neuronal phenotype when directed by ELav-Gal4 was chosen for the studies reported here.
All flies were maintained on standard cornmeal media at 25 °C with 60% humidity under a 12-h light–dark cycle unless otherwise stated. Concentrations of supplemented metals or metal chelators used were as follows: 5 mM ferric ammonium citrate (FAC; Sigma-Aldrich), 150 μM bathophenanthroline disulfonate (BPS; Sigma-Aldrich), 50 μM N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN; Sigma-Aldrich), and 2 mM ZnCl2 (Beijing Yili Fine Chemicals Ltd. Co., Beijing, China). For inhibitor studies, concentration of 3-Iodo-L-tyrosine (3-IT; Sigma-Aldrich) used was 75 μM. For the rotenone feeding experiment, 2-day-old adult flies were transferred to vials with instant Drosophila medium and exposed to 150 μM freshly prepared rotenone (Sigma-Aldrich) for 7 days. The medium was replaced every day to ensure that the rotenone was effective.
The ZHY3 strain lacking the Zrt1 and Zrt2 proteins for zinc uptake was used in this study [54].
Cell culture and plasmid transfection
For subcellular localization experiments, CHO-K1 cells were maintained in DMEM (Invitrogen) containing 10% fetal bovine serum (FBS, Gibco BRL, Gaithersburg, MD, USA) at 37 °C under 5% CO2 atmosphere. When the cells reached 80% confluence, they were transfected with pIRESneo-Catsup-FLAG using Lipofectamine™ 3000 (Invitrogen) in accordance with the manufacturer’s instructions. After 24 h, cells were fixed, stained with the FLAG antibody (1:500) and the Golgi marker (anti-GM130, 1:500) or ER marker (anti-PDI, 1:500), and imaged with the ZEISS LSM780 microscope. Anti-FLAG mouse monoclonal antibody (ab18230), anti-FLAG rabbit polyclonal antibody (ab1162), anti-GM130 rabbit polyclonal antibody (ab30637), and anti-PDI mouse monoclonal antibody (ab2792) were obtained from Abcam (Cambridge, MA, USA). Secondary antibodies include cy3-conjugated goat anti-mouse and cy3-conjugated goat anti-rabbit IgG (Zhongshan Goldenbridge Biotechnology, Beijing, China). The experiments were repeated three times.
SH-SY5Y cells were used to test the effect of metals on mammalian TH phosphorylation at Ser40. SH-SY5Y cells were maintained in DMEM (Invitrogen) with 10% FBS and 1% PS under an atmosphere of 95% air and 5% CO2 at 37 °C. To test metals’ effects, 1 μM BPS, 50 μM ZnCl2, 1 μM TPEN, and 10 μM FeCl2 (2 mg/mL vitamin C were added in the medium to inhibit iron oxidation) were respectively added in the medium. The experiments were repeated three times.
Eclosion, mobility, and longevity assays
For eclosion assays, Gal4 lines were crossed with various transgenic lines for ~ 3 days, as indicated in each experiment, and allowed to lay eggs on juice-agar plates for ~ 24 h. Newly hatched first-instar larvae were transferred to normal food or food supplemented with metals or metal chelators, as indicated. The density of each vial was controlled to 60–100 larvae, and the total number of emerging adults of each genotype was counted [30]. Assays were done with six replicates for each group, and the experiments were repeated at least three times. The results from all experiment runs were pooled together for analyses.
To assay the mobility of PD flies, 20 females were placed in a vertical glass vial. After a 1-h recovery from CO2 exposure, flies were gently tapped to the bottom of the column. The number of flies reaching the indicated height (~ 8 cm) was counted after 7 s of climbing under red light. Six parallel group tests were conducted for each genotype, and the experiments were repeated at least three times [34]. The presented data are from the results of all experiments.
For longevity assays, 3-day-old adult males or females were kept in vials (20 individuals of each vial) containing cornmeal medium and maintained at 25 °C. Flies were transferred to fresh media every 2–3 days, and the number of live flies was recorded every alternate day. Six parallel group tests were conducted for each genotype, and the experiments were repeated at least three times [34]. The data were presented as Kaplan–Meier survival distributions, and significance was determined by log-rank tests.
Eye morphology analysis
To phenotype eye morphology, flies were collected after eclosion at 18 °C and frozen to death at − 80 °C for one night. The eyes were photographed using a Zeiss imager A1 stereomicroscope. More than six flies were scored per genotype, and each experiment was repeated three times. Scanning electron microscopy analysis of fly eyes was performed as described previously with FEI Quanta 200 [70].
Alkaline phosphatase(ALP) activity assay
ALP activity assays were performed as described previously with some modifications [71]. For yeast samples, yeast was cultured in synthetic dextrose media (SD) at 30 °C to OD = 0.4, collected by centrifugation at 3000g for 5 min, and then transferred to the induction medium (synthetic glucose and raffinose (SGR) media). The yeast was cultured in SGR media at 30 °C to OD = 1, collected by centrifugation, and washed three times with ice-cold PBS. The collected yeast was homogenized in ALP lysis buffer (1.0 mM Tris–HCl pH 7.4, 0.5 mM MgCl2, and 0.1% Triton X-100), and the protein concentration was measured by the BCA kit (Thermal). Approximately 2 μg protein was added to 90 μL solution A (1.0 M diethanolamine, 0.5 mM MgCl2 pH 9.8) and 10 μL solution B (150 mM p-nitrophenyl phosphate). After incubation for 30 min at 30 °C, ALP activity was measured based on the p-nitrophenol release by its absorbance at 405 nm.
For fly samples, ∼ 80 adult heads were lysed in the ALP lysis buffer, then ∼2 μg protein was added to 90 μL solution A and 10 μL solution B. The absorbance at 405 nm was measured after incubation for 30 min at 25 °C. The experiments were repeated three times.
Luciferase activity assay
Yeast was cultured in SD media at 30 °C to OD = 0.4, collected by centrifugation at 3000g for 5 min, and then transferred to SGR media. The yeast was cultured in SGR media at 30 °C to OD = 1, collected by centrifugation, and washed three times with ice-cold PBS. The collected yeast was homogenized in cold PBS, and the protein concentration was measured by the BCA kit (Thermal). Approximately 10 μg protein was used to test the luciferase activity by Firefly Luciferase Reporter Gene Assay Kit (Beyotime, #RG027); fluorescence intensity was measured for 10 s by the fluorescence microplate reader. In order to avoid the error caused by the difference in the amount of samples, the reporter gene of Renilla luciferase was used as an internal reference.
Western blot analysis
SH-SY5Y cells were used to test the effect of metals on mammalian TH phosphorylation at Ser40. Antibodies of anti-tyrosine hydroxylase (AB152) and anti-tyrosine hydroxylase, phosphoSer 40 (AB5935) were purchased from Sigma-Aldrich (Shanghai, China). The secondary antibody HRP-conjugated goat anti-rabbit IgG was purchased from Zhongshan Goldenbridge Biotechnology (Beijing, China). For Western blot analysis, cells were homogenized in the buffer containing 1% Triton X-100 plus 10% proteinase inhibitor cocktail (Sigma), centrifuged, separated on 10% SDS-PAGE, and transferred to nitrocellulose membranes (Millipore, Watford, UK). Signals were developed with the ECL detection kit (Vigorous Biotechnology, Beijing, China). The experiments were repeated three times.
Metal sensitivity/resistance assay in yeast
Catsup was cloned into pYES2 (Invitrogen) and transformed into ZHY3. The vector pYES2 was also transformed into yeast and used as the control. For growth testing on agar plates (spotting assay), yeast grown in the culture medium (synthetic dextrose media, SD) was 10-fold serially diluted with sterile ddH2O and then spotted on SD (the transgene could not be induced on this medium) or synthetic galactose and raffinose media (SGR, the transgene could be induced on this medium) plates with or without metal or chelators. Incubation was all at 30 °C; 3 mM ZnCl2 or 20 μΜ TPEN was added in the medium, n = 6.
Metal ions binding assays of TH
A double-tagged Drosophila TH protein (dTH) expression plasmid containing N-terminal His6 and SUMO tag (pET28a-His6-SUMO-dTH) was constructed. The ensuing plasmid was transformed into E. coli BL21(DE3). Protein expression was induced by 250 μM β-D-1-thiogalactopyranoside (IPTG) with 100 μM FeSO4 supplied in LB medium for enzyme stability. Induction proceeded at 16 °C for 14 h. The His6-SUMO-dTH protein was purified with Ni-NTA resin, and the tags were removed by ULP1 digestion. Protein was then loaded onto a Superdex 200 gel filtration column for size separation. The protein composition of each fraction was determined by SDS-PAGE analysis. Fractions containing dTH were collected, and EDTA was added to a final concentration of 1 mM to remove divalent metal ions. After incubation at 4 °C for 30 min, the purified protein was concentrated by Amicon® Ultra Centrifugal Filters (type 30,000 NMWL). Then, the protein was diluted 10-fold with EDTA-free 50 mM MES buffer (pH 6.5) and applied to Ultra filters again to be concentrated to its original volume. The procedure was repeated for five times to remove EDTA in the protein buffer.
Competitive binding assays were performed as described previously with some modification [52]. Briefly, purified dTH (2.5 μM subunit) was incubated with 15 μM FeCl2, 0-25 μM ZnCl2, and 0.5 mg/mL catalase in 50 mM MES Potassium (pH 6.5) for 20 min at 4 °C. After incubation, the mixture was applied to Amicon Ultra filter devices (Merck Millipore). Free and bound metal ions were separated by centrifugation (5 min at 5000 rpm) at 4 °C. The filtrate was collected and contents of iron ion or zinc ion were determined by BPS-based colorimetric assays or ICP-MS. All the experiments were repeated at least three times.
Determination of L-dopa
For L-dopa assay in S. cerevisiae, Catsup and dTH were cloned into pYES2 (Invitrogen) and pYES3 (Invitrogen), respectively. The plasmids were transformed into ZHY3 or wild-type yeast INVSc1. The vectors pYES3 and pYES2 were also transformed into yeast and used as the controls. For L-dopa produced in yeast, a single colony was picked and grown in SD medium at 30 °C to OD600 = 1. Then, the yeast cells were collected by centrifugation at 3000g for 5 min, washed with sterile water, and transferred to SGR medium supplemented with 0.4 mg/mL tyrosine, 2.0 mg/mL ascorbic acid, and 1 mM DMPH4. In the experiments for testing metal effects, 400 mM BPS along with FeSO4 or ZnCl2 was added in the medium. The yeast was shaken at 30 °C for 18 h, and the supernatant was collected for L-dopa level determination.
For L-dopa assays in E.coli, pET28a-His6-SUMO-dTH mentioned above was transformed into BL21 (DE3). The vector pET28a was transformed into BL21(DE3) as control. Protein expression was induced by 250 μM IPTG at 37 °C. The LB medium was supplied with 400 μg/mL tyrosine, 2.0 mg/mL ascorbic acid, and 1 mM DMPH4 for L-dopa production. FeSO4 or ZnCl2 was added as indicated in each experiment. L-dopa level was determined after 8 h growth at 37 °C. L-dopa level was determined as previously described [72] with some modifications. Briefly, the colorimetric method for estimation of L-DOPA was performed in 96-well microtiter plates. Fifty microliters each of 0.5 M HCl, nitrite-molybdate reagent (composed of 10% w/v sodium nitrite and 10% w/v sodium molybdate), and 1.0 M NaOH was sequentially added to 50 μL of the sample. The absorbance was measured at 530 nm using a Thermo Multiskan GO reader. A calibration curve with different amounts of L-dopa was constructed. L-dopa level in each sample was calculated based on the standard curve or normalized to the control. All experiments were repeated at least three times.
Dopamine level determination
For sample preparation, 3rd larvae were collected and sonicated in 0.6 mL of ice-cold 0.4 M perchloric acid. After centrifugation at 13000g at 4 °C for 10 min, the supernatant was transferred into a new tube and mixed with a half supernatant volume of 20 mM potassium citrate, 300 mM K2HPO4, and 2 mM EDTA. The mixture was kept on ice in the dark for 20 min. Then, the chilled mixture was filtered through a low-binding Durapore (0.22 μm) PVDF membrane. The amounts of dopamine were measured by an HPLC-ECD system. Chromatographic separation was performed on a Diamonsil C18 column (5 μm, 250 × 4.6 mm). The mobile phase contained 60 mM KH2PO4, 350 μM C8H19NaO4S, 100 μM EDTA, and 8% formaldehyde. Dopamine levels were monitored using a HPLC system (Water e2695) equipped with an electrochemical detector (Water 2645). The experiments were repeated three times.
TH activity assay
TH activity was determined as previously described with some modifications [73, 74]. For the TH activity assay in Drosophila heads, samples were collected and homogenized in 20 mM Tris-HCl buffer, pH 7.5 by ultrasonic disintegration over ice. Protein concentrations were determined with BCA kit and adjusted for each group. Fifty microliters homogenate was further diluted with 300 μL 30 mM Tris-acetic acid containing 0.1% Triton X-100 and 25 μCi of radioactive tyrosine HCl (0.4 mCi/nMol of L-[ring-3,5-3H]tyrosine), 50 nMol of the cofactor 6, 7-dimethyl 5, 6, 7, 8-tetrahydropterin (DMPH4, Sigma, St. Louis, MO, USA), 5000 units of catalase, and 5 nM DTT in 100 mM potassium phosphate, pH 6.0 for incubation at 37 °C for 20 min. Then, aliquots were taken and placed in 5% (v/v) perchloric acid to stop the reaction. Unreacted tyrosine and the product DOPA were absorbed with 1.0 mL of an aqueous slurry of activated charcoal (in 0.1 M HCl) and the released [3H]H20 was analyzed by liquid scintillation counting. TH utilizes O2 to produce DOPA, and [3H]H20 is derived from [3H]tyrosine. The TH activity was expressed as radiation value of [3H]H20 produced per group.
For the TH activity assay in yeast, yeast was cultured in SD media at 30 °C for 24 h to OD = 1. Yeast was collected by centrifugation at 3000g for 5 min and then transferred to SGR media supplemented with metals or metal chelators, as indicated in each experiment; 500 nMol DMPH4 (Sigma, St. Louis, MO, USA) and 10 μL L-[ring-3,5-3H]tyrosine were added in the media and then the yeast was further cultured at 30 °C for 24 h. Cells were removed after centrifugation at 12,000g for 15 min. The supernatant was transferred to a new container. Unreacted tyrosine and the product DOPA were absorbed with 1.0 mL of an aqueous slurry of activated charcoal (in 0.1 M HCl) and the released [3H]H20 was analyzed by liquid scintillation counting. The TH activity was expressed as radiation value of [3H]H20 produced per group. All experiments were repeated at least three times.
Statistical analysis
All data were recorded and analyzed with Microsoft Excel and GraphPad Prism (version 6.00; La Jolla, CA, USA). Data were analyzed by Student’s t-test between the groups, and ANOVA was used for multiple comparison. Statistical results were presented as means ± SEM. Asterisks indicate critical levels of significance (*p < 0.05, **p < 0.01, ***p < 0.001).