(6R)-5,6,7,8-Tetrahydrobiopterin dihydrochloride (BH4 2HCl), dopamine hydrochloride (dopamine HCl), MPTP, monoclonal anti-tyrosine hydroxylase antibody, and monoclonal anti-β-actin antibody were purchased from Sigma-Aldrich (St. Louis, MO, USA). Epilson-acetamidocaproic acid (AACA) was donated by Kuhnil Pharmaceuticals (Seoul, Korea). T-PER® tissue protein extraction reagent and protease inhibitor cocktail were purchased from Thermo Scientific (St. Louis, MO, USA). IRDye® 800CW conjugated goat (polyclonal) anti-mouse IgG was purchased from LI-COR® Biosciences (Lincoln, NB, USA).

All procedures in this study were performed according to protocol approved by the Institutional Animal Care and Use Committee at Dankook University. Six-week-old C57BL/6, Balb-C, and ICR mice were purchased from Daehan-Bio Link (Seoul, Korea). They were housed in cages in groups of 10 or 11 animals under controlled environmental conditions (23±2℃; relative humidity 50±10%; 12:12-h light-dark cycle until the initiation of the experiment; food and water ad libitum) before use in experiments. Each mouse was injected subcutaneously (s.c) with MPTP (20~30mg/kg). After testing by Rota-rod and open field test systems as detailed below, mice were killed by cervical dislocation, and each brain was quickly retrieved. The striatum, substantia nigra, and hippocampus were quickly dissected on ice under a dissecting microscope. The dissected brain regions were quick-frozen in a deep-freezer (-80℃) until further analysis.

Preparation of stock solution, calibration standards, quality control, and sample preparation for LC-MS/MS system were done as previously described [20]. The liquid chromatography system was an Accela system (Thermo Fisher Scientific, Waltham, MA, USA) equipped with a nanospace SI-2 3133 solvent delivery module as an auto-sampler (Shiseido, Tokyo, Japan), and connected to a Discovery Max (Thermo Fisher Scientific) quadrupole tandem mass spectrometer coupled with electrospray ionization (ESI-MS/MS). Chromatographic separation was achieved by using hydrophilic interaction chromatography (HILIC) using a Sepax Polar-Imidazole (2.1×100 mm internal diameter, 3 µm particle size) high-pressure liquid chromatography (HPLC) column (Sepax Technologies, Newark, DE, USA) with a 4×2 mm C₁₈ guard column (Phenomenex, Torrance, CA, USA). The ESI mass spectrometer was operated in the positive ion mode. Multiple reaction monitoring (MRM) of the precursor-product ion transitions was m/z 242.1 → m/z 166.0 for BH4, m/z 154.1 → m/z 90.0 for dopamine, and m/z 174.1 → m/z 114.0 for AACA. The optimized ESI conditions could sensitively detect BH4, dopamine, and IS with positive ion detection mode. The most abundant protonated ion peaks in the Q1 mass spectra of BH4 and dopamine were at 242.1 m/z and 154.1 m/z, respectively. There was no evidence of fragmentation and adduct formation. The product ions ([M+H]⁺) in the Q3 mass spectra and proposed fragmentation patterns were BH4 at m/z 166.0 (2-amino-7,8-dihydropteridin-4(1H)-one) by loss of propane-1,2-diol, and dopamine at m/z 90.9 (butane-1,2-diol) by loss of (E)-3-methylpent-3-en-1-amine. The interpretation and analysis of data followed previously published methods [20].

Mice were trained for two consecutive days to stay on a rota-rod (Ugo Basile, Comerio VA, Italy) at least for 600 seconds (16 rpm, fixed speed) before the start of testing. On the test day, baseline data was collected and then mice were evaluated for their latency to fall after s.c administration of 20~30 mg/kg of MPTP. Rota-rod testing was performed twice per mouse with an intervening interval of at least 10 minutes. The times in seconds from the two tests were averaged.

Each mouse was put in a white or black plastic rectangular box (40×27×27 cm) after s.c administration of 20~30 mg/kg MPTP. The mice injected with the same volume of saline were used as the control group. The mice were observed with a CCD camera connected to a recording system. The recorded behavior was analyzed by EthoVision Version 2.3 (Noldus information Technology, Wageningen, The Netherlands). An automated video tracking system was used for calculation of moving distances and rearing frequencies. The mice were observed for 6 hours in normal lighting. The white or black plastic rectangular box was cleaned with 70% alcohol and water between trials.

Quick-frozen tissue samples were homogenized and analyzed via immunoblotting as previously described [21]. Briefly, the homogenized samples were boiled in Laemmli sample buffer and the proteins separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The resolved proteins were transferred to a polyvinylidene difluoride membrane (Millipore, Billerica, MA, USA). Protein transfer was confirmed by staining membranes with Ponceau Red. Each membrane was incubated with an appropriate primary antibody for overnight at 4℃. After extensive washing with Tris-buffered saline containing Tween 20, each membrane was incubated with the appropriate secondary antibody. Densitometry of protein bands was performed with the software for Odyssey Infrared Imaging System (LI-COR Biosciences).

All values given in the text are mean±SEM. Differences between means were evaluated using a Student's t-test. For comparison between strains, Two-way ANOVA with the factors "strain" (ICR, Balb C, and C57BL/6) and "day after treatment" (1, 2, and 3 days) were computed using Prism (version 5.0) (GraphPad Software, San Diego, CA, USA). Post-hoc analysis of significant differences was performed using Bonferroni post-tests for multiple comparision. Statistical significant differences were taken at the p<0.05 level. The n values given represent numbers of tissues or animals used in each experiment.

C57BL/6 mice were trained to stay on the Rota-rod for 600 seconds when the system was operating at a fixed rate of 16 rpm. The trained animals were injected with MPTP (30 mg/kg s.c.) and then evaluated their latency to fall off the Rota-rod. Fall latency 6 hours after injection was significantly decreased (Fig. 1A). The decreased latency persisted until 24 hours post-injections, although the decrease was significantly greater at 6 hours than at 18 hours (Fig. 1A). Similar findings were obtained with ICR mice (Fig. 1B). However, the latency to fall of ICR mice was greater than for C57BL/6 mice 24 hours after injection.

Multiple injections of MPTP (30 mg/kg, s.c.) were given at 24 hour intervals and mice were tested their behavioral changes (Fig. 2). This treatment produced extensive mortality and the experiment was terminated day 2, so the MPTP dosing regimens have to be changed. To limit mortality, the three different mouse strains were treated with a lower dose of MPTP (20 mg/kg, s.c), and with multiple injections of this lower dose, and were evaluated for their latency to fall on the Rota-rod. However, the injection of lower dose of MPTP (20 mg/kg, s.c.) didn't decrease the latency on rota-rod at 24 hours after MPTP injection (Fig. 1C). Moreover, multiple injection of MPTP (20 mg/kg, s.c.) at 24 hours intervals for 3 days didn't significantly decrease latency to fall on a rota-rod (data not shown). There were no significant changes in the body weights of the examined mice during the 3-day period of MPTP injection and 24 hours after MPTP injection in all three mouse strains (Fig. 1D).

We further evaluated the mice after MPTP injections with alternative methods to see whether the lower dose of MPTP was insufficient to be toxic to dopaminergic neurons or whether the Rota-rod test was not sensitive enough to detect behavioral changes. To assess the behavioral effects, the movements of C57BL/6, Balb C, and ICR mice after injecting MPTP (20 mg/kg, s.c.) were evaluated by the open field test for 6 hours. The total moving distances recorded and analyzed until 1 hour after MPTP injection were significantly decreased at day 2 and day 3 in C57BL/6 mice (Fig. 3A). However, the moving distances were still recovered after 24 hours (day 4) similarly as day 0 if the mice were not treated with MPTP (Fig. 3A).

In parallel, Balb C mice were similarly tested. The total moving distances were significantly decreased from day 1 and further decreased following the repeated injection of MPTP (20 mg/kg, s.c.). Moreover, the total moving distances at day 4 (without MPTP injection) were markedly increased by two-fold compared with day 0 (Fig. 3B). The observations were consistent with the hyperactivity of the mice at day 4. Of interest, ICR mice did not respond well at the lower dose of MPTP (20 mg/kg, s.c.) in the same experiments and showed no specific changes at day 4 (Fig. 3C).

Concerning rearing frequencies, all three strains of mice responded well to the MPTP injection (Fig. 4). The rearing frequencies until 1 hour after MPTP injection were significantly decreased from day 1 and its effects lasted until day 3. But, this decrease observed with multiple MPTP injections recovered after 24 hours (day 4) with a frequency similar to that noted on day 0 if the mice were not injected with MPTP (Fig. 4).

We further analyzed the data until 3 hours and 6 hours in the same experiments. The results showed a very similar pattern at both times in the total moving distances (Table 1) and rearing frequencies (Table 2). The total moving distances were significantly decreased following the repeated injection of MPTP (20 mg/kg, s.c.) in both C57BL/6 and Balb C mice. At the same time, the total moving distances at day 4 (without MPTP injection) were recovered to similar distances [22]. Similarly, ICR mice did not display significant differences at 3 hours or 6 hours evaluation when the lower dose of MPTP was used. Interestingly, Balb C mice at day 4 moved an extended distance (up to 3-fold) compared to day 0. These data indicated that mice, especially Balb C mice, are hyperactive during the recovery time at day 4 (Table 1).

All three strains of mice displayed significantly decreased rearing frequencies from day 1 to day 3, but still showed robust recovery of activities at day 4 (Table 2). Especially, the rearing frequencies at day 4 were markedly increased up to 5-fold in ICR mice. But, there were no increased rearing activities at day 4 either in C57BL/6 or Balb C mice. These data suggest that MPTP can differentially affect the rearing behavior of different mouse strains.

Although, repeated administrations of MPTP significantly affected the moving distances and rearing frequencies in all three mouse strains, we further analyzed the data to see mice strains are differently susceptible (Fig. 5). In both parameters, ICR strain is statistically less sensitive to MPTP than C57BL/6 or Balb C strains.

MPTP destroys dopaminergic neurons in the substantia nigra, which causes Parkinsonism [11,16]. Recently, we reported a direct detection method for tetrahydrobiopterin (BH4) and dopamine in rat brain using LC-MS/MS [20]. Dopamine and BH4 were presently measured in brain regions of the three strains of mice after administration of MPTP (Fig. 6 and 7). Three injections of MPTP (20 mg/kg s.c.) for a 24 hours interval resulted in an approximately 60% decrement of dopamine in the striatum in all three strains (Fig. 6A). In parallel, endogenous levels of dopamine in the three strains of mice were measured in the substantia nigra (Fig. 6B) and in the hippocampus (Fig. 6C). The three injections of MPTP did not statistically affect the substantia nigra in any of the strains (Fig. 6B). But, the dopamine concentration in the hippocampus of C57BL/6 mice was slightly decreased by the multiple MPTP injections (Fig. 6C).

BH4 is an essential cofactor for dopamine synthesis through its action on dopamine hydroxylase. BH4 has been intensively studied concerning the development and progression of PD [23-26]. The endogenous levels of BH4 in brain section were measured after three doses of MPTP (20 mg/kg s.c.) delivered at 24 hours intervals. No statistically significant differences were evident in the striatum, substantia nigra, and hippocampus among the three strains of mice (Fig. 7). These data suggested that the administration of MPTP can decrease the endogenous level of dopamine in mouse brain by destroying dopaminergic neurons, mainly at striatum, with no effect on BH4 levels in mouse brain.

Based on the assay of dopamine and tetrahydrobiopterin at striatum, substantia nigra, and hippocampus by LC-MS/MS, the dopamine concentration was greatest in the striatum. To ascertain the activity of tyrosine hydroxylase following MPTP administration, we quantified the total expression levels of tyrosine hydroxylase at striatum in the three strains of mice (Fig. 8). The expression levels of tyrosine hydroxylase in the striatum were significantly decreased by the systemic administration of MPTP (20 mg/kg, s.c.) in all three strains. However, the tyrosine hydroxylase decrease was least for ICR mice.