Male C57BL/6J mice (8–10 weeks old; 23–28 g weight) were purchased from the SLRC Laboratory (Shanghai, China). The experimental procedures were approved by the Institutional Animal Care and Use Committee (IACUC) of Soochow University. All mice were housed in a SPF animal facility with a temperature at 21–25°C and a controlled light–dark cycle.

Parkinson’s disease animal models were established by intraperitoneally (i.p.) injecting the mice with MPTP (14 mg/kg, Sigma-Aldrich, St. Louis, MO, United States) four times at 2-h intervals. Saline was given as controls. To evaluate the effect of GYY (Cayman Chemical, United States), the mice were administered with GYY at individual doses (12.5, 25, and 50 mg/kg, i.p.) once daily for 3 days prior to MPTP treatment, and continued throughout the experimental period. The animals were sacrificed 2 weeks after MPTP administration for biochemical studies.

Behavioral assessments were performed at 2 weeks after MPTP injection. The data were analyzed by independent observers blinded to the treatments.

Rotarod test was performed to estimate motor balance and coordination. Before test, each mouse was trained three times on the rod with an accelerating speed at 5–20 rpm/min. During the test session, the rotational speed remained constant at 18 rpm/min. A trial was stopped if the mouse fell down or the latency to fall reached 5 min. Each mouse was subjected to three trials with an interval of 30 min during the test session, and the average value of the latency to stay on the rod was calculated and included for further analysis.

Following the behavioral tests, the animals were deeply anesthetized and perfused with 0.1 M phosphate buffer followed by 4% paraformaldehyde (PFA). Mouse brains were immediately removed from the skull and post-fixed in PFA solution overnight. After that, brains were dehydrated in a serial of alcohol solution and dimethylbenzene was applied to make the brains transparent. Brains were then embedded in paraffin wax. Next, 4 μm thickness sections were cut through SN compacta (SNpc), and every fourth slice was reserved. For staining, sections were deparaffinized and rehydrated, followed by epitope retrieval by boiling the sections in 0.01 M citrate buffer (pH 6.0) for 10–15 min and cooling at room temperature for 20–30 min. After that, sections were thoroughly washed in 0.01 M PBS three times. Endogenous peroxidase activity was blocked with 3% H₂O₂ in 0.01 M PBS for 30 min. Next, the sections were incubated in 5% BSA with 0.1% Triton X-100 in PBS for 30 min, followed by incubation with mouse monoclonal anti-TH (1:1000, Sigma, St. Louis, MO, United States) at 4°C overnight. Subsequently, the sections were washed in PBS, incubated with goat anti-mouse secondary antibody at room temperature for 1 h and stained using a DAB kit (Gene Tech, GK500705, China). The sections were observed and photographed using the Zeiss microscope (Carl Zeiss, 37081, Germany).

Striatal MPP⁺ levels were determined as previously described (Jackson-Lewis and Przedborski, 2007) with minor modifications. In brief, striatum and whole blood were collected 90 min after four injections of MPTP (14 mg/kg, i.p.) at 2-h intervals. The tissue samples were homogenized by sonication in 0.2 M chilled perchloric acid and then centrifuged at 15,000 g for 30 min at 4°C. The whole blood samples were kept at room temperature for 30 min and subjected to centrifuged at 1000 g, 30 min. Serum was harvested from the resulting supernatant, and mixed with 0.2 M perchloric acid before subjected to the second centrifuged at 15,000 g for 30 min at 4°C. The supernatants were transferred into a clean eppendorf tube for MPTP and MPP⁺ analysis.

MPTP/MPP⁺ was analyzed by HPLC equipped with UV detectors (MPTP at 295 nm; MPP⁺ at 245 nm). An aliquot (20 μl) of each supernatant was eluted through a C18 reverse phase column (5 μm, 250 mm × 4.6 mm, SHIMADZU VP-ODS) attached to the HPLC system (Waters, Milford, MA, United States) at a flow rate of 1.0 ml/min. The mobile phase consists of 15% acetonitrile, 50 mM potassium phosphate adjusted to pH 3.2 with ultrapure 18 M phosphoric acid. The detection limit is 3 ng/ml and this method provides good reproducibility.

The striatal NO content was evaluated by measuring the accumulation of nitrite and nitrate using the kit from Jiancheng Bioengineering Institute (Nanjing, Jiangsu, China). Briefly, striatum tissues were homogenized (1:10 m/v) in lysis buffer and centrifuged at 15,000 g for 30 min at 4°C. Next, an aliquote (50 μl) of the resulting supernatant was mixed with the dilution buffer at an equal volume, followed by addition with 5 μl nicotinamide adenine dinucleotide phosphate (NADPH), 10 μl flavin adenine dinucleotide (FAD) and 5 μl nitrate reductase and incubation at 37°C for 15 min. After that, 10 μl lactate dehydrogenase (LDH) was added and incubated for another 5 min. Subsequently, 50 μl Griess reagent I (0.1% naphthylethylene diaminedihydrochloride in 1% sulfanilamide) and 50 μl Griess reagent II (2.5% H₃PO₄) were added and incubated for 10 min. The optical density of the mixture was measured at 540 nm using a microplate reader (Tecan Infinite M200 PRO, Switzerland). The results were calculated by a standard curve with sodium nitrite, and expressed as μmol per gram protein. The protein concentrations of striatal homogenates were determined using the BCA protein assay kit (Thermoscientific, United States).

Whole striatum tissues were homogenized with lysis buffer and centrifuged at 13,200 rpm for 20 min at 4°C. Protein concentrations of the homogenates were determined using the BCA protein assay kit. Equal amounts of total proteins were loaded in 8–12% sodium dodecyl sulfate polyacrylamide gels and then transferred onto PVDF membranes. Membranes were incubated with specific antibodies: anti-TH (1:2000, Sigma, T1299, United States), anti-nNOS (1:1000, BD, 610308, New York, United States), anti-nitrated α-syn (1:750, Thermo Scientific, MA5-16142, United States), anti-α-syn (1:500, CST, 2642, United States), anti-p-Akt (1:500, CST, 4060s, United States), anti-Akt (1:1000, CST, 4691s, United States), and anti-β-actin (1:5000, Sigma, A3584, United States) at 4°C overnight. Blots were then briefly washed with PBS followed by incubation with the corresponding secondary antibodies (Jackson Lab, United States) for 1 h at room temperature. Protein densities were detected using an enhanced chemiluminescence kit and analyzed by ImageJ software.

PC12 cells were purchased from the Institute of Cell Biology, Chinese Academy of Sciences (China), and cultured in RPMI1640 medium with 10% fetal bovine serum and 1% penicillin/streptomycin. The cultures were maintained in a incubator with 5% CO₂/95% air at 37°C. For experimentation, cells were seeded into a 12-well plate and cultured overnight before treatment.

All data were presented as mean ± SEM of at least three independent experiments. All statistical analyses were performed with GraphPad Prism 5.0 software. The differences among groups were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s post hoc analysis. Statistical significance was set at P < 0.05.

To study the ability of GYY to affect MPTP-induced impairment in motor function, we assessed the animals’ motor coordination using rotarod, balance beam walking, and grid walking test at 2 weeks after MPTP injection. The results are shown in Figure 1. Compared to saline-treated group (control), the latency to fall off the rod significantly decreased in MPTP-injected mice (Figure 1A). Co-treatment with GYY at 25 and 50 mg/kg prolonged the time of staying on the rod compared to MPTP-only group. Balance beam walking test also showed that 50 mg/kg GYY co-treatment improved the MPTP-induced motor deficits, as indicated by a shorter time period to cross the beam compared to MPTP only group (Figure 1B). Similarly, in grid walking test we found that MPTP injection increased the number of foot slips in mice, implying that the sensorimotor function was also impaired by MPTP. This was markedly attenuated by 50 mg/kg GYY co-treatment (Figure 1C). And the treatment with GYY alone at 50 mg/kg, the maximal tested dose in this study, did not have any impact on the motor behavior.

As MPTP-caused neurotoxicity to dopaminergic neurons is dependent on the metabolism of MPTP to its active metabolite MPP⁺ via monoamine oxidase (MAO) in the nigrostriatal system, the effect of GYY on striatal MPP⁺ levels was determined in a parallel study. In this part, 10 male C57BL/6J mice (10 weeks old) were applied and randomly divided into two groups: MPTP and GYY + MPTP group (n = 5 per group). For MPTP group, the mice received four injections with MPTP (14 mg/kg, i.p., at 2 h intervals). For GYY + MPTP group, the mice were treated with GYY (50 mg/kg, i.p.) once daily for 3 days followed by MPTP administration at 1 h after the last injection with GYY. No significant changes were observed in the striatal MPP⁺ level in MPTP-treated mice with and without GYY (50 mg/kg, i.p.) pretreatment (MPTP 3.90 ± 1.98 nmol/mg tissue; GYY + MPTP group, 3.96 ± 1.46 nmol/mg tissue; P > 0.05; Figure 2), which was measured 90 min after the last MPTP injection. Measurement of serum MPP⁺ levels revealed no significant difference between the two tested groups either (MPTP 3.25 ± 0.39 μg/ml; GYY + MPTP group, 3.14 ± 0.33 μg/ml, P > 0.05). This indicates that the observed effects of GYY on MPTP-induced neurotoxicity are not caused by the reduction of MPTP conversion into MPP⁺.

The dopaminergic neuron degeneration was examined by quantifying the number of TH immunoreactive neurons in the SNpc using immunohistochemistry study. The result showed that MPTP caused an approximate 40% decrease in the number of dopaminergic neurons in the SN compared to saline, and this was attenuated in the mice co-treated with 50 mg/kg GYY (Figures 3A,B). Treatment with GYY alone did not have any impact on TH-positive staining in the SN. Western blot analysis showed that 50 mg/kg GYY co-treatment prevented the reduction of striatal TH protein level caused by MPTP (Figure 3C). These results indicate that co-treatment with GYY at a suitable dose was able to prevent dopaminergic neuron losses in the SN of MPTP-treated mice.

Overproduction of NO and its associated nitrative stress participate in PD pathology (Jimenez-Jimenez et al., 2016). In line with previous studies (Dehmer et al., 2000), we observed an accumulation of nitrite, a metabolite of NO, in the striatum of the mice receiving MPTP administration (Figure 4A). This increase of NO generation was abated in the mice co-treated with GYY at the dose of 25 and 50 mg/kg, respectively. Likewise, co-treatment with GYY at this regimen inhibited the MPTP-induced upregulation of nNOS expression in the striatum (Figure 4B). Treatment with GYY alone did not modulate NO or nNOS expression level. The result was verified by an in vitro study, which demonstrated that pre-treatment with 100 μM GYY prevented MPP⁺ (100 μM, 24 h) elicited increase of nNOS expression in PC12 cells (Figure 4C).

Excessive NO generation often results in peroxynitrite formation and tyrosine nitration in proteins. Human α-syn contains four tyrosine residues (Tyr39, Tyr125, Tyr133, and Tyr136) that can be nitrated (Schildknecht et al., 2013), which may play a role in protein aggregation in PD. Therefore, we further examined the effect of GYY on α-syn nitration by western blot using the specific antibody against nitrated α-syn (Tyr125, Tyr133). The result showed that although the total α-syn level was not altered in the striatum following MPTP injection, the nitrated α-syn level was markedly elevated in MPTP-treated mice, and this elevation was abolished by GYY co-treatment (Figure 4D).

Last, we studied the signaling pathway that underlies the inhibition by GYY on nNOS expression. Treatment with 100 μM MPP⁺ was found to reduce the phosphorylation of Akt Ser 473 in a time-dependent manner, which was detected early at 0.25 h and remained to be dramatically declined at 1 h after treatment in PC12 cells (Figure 5A). Pretreatment with 100 μM GYY almost reversed the decline of Akt phosphorylation caused by MPP⁺ exposure (Figure 5B). However, co-treatment with Akt inhibitor perifosine (5 μM) was able to attenuate the GYY-mediated inhibition on nNOS expression in MPP⁺-treated cells (Figure 5C). These results indicate that Akt signaling may be activated and thus contribute to the inhibition on nNOS expression by GYY.