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. 2020 Feb 28:11:183.
doi: 10.3389/fphar.2020.00183. eCollection 2020.

LY354740 Reduces Extracellular Glutamate Concentration, Inhibits Phosphorylation of Fyn/NMDARs, and Expression of PLK2/pS129 α-Synuclein in Mice Treated With Acute or Sub-Acute MPTP

Yang Tan  1 , Yan Xu  1 , Chi Cheng  1 , Cong Zheng  2 , Weiqi Zeng  1 , Ji Wang  1 , Xiaoqian Zhang  1 , Xiaoman Yang  1 , Jialing Wang  1 , Xiaomei Yang  1 , Shuke Nie  3 , Xuebing Cao  1
Affiliations

LY354740 Reduces Extracellular Glutamate Concentration, Inhibits Phosphorylation of Fyn/NMDARs, and Expression of PLK2/pS129 α-Synuclein in Mice Treated With Acute or Sub-Acute MPTP

Yang Tan et al. Front Pharmacol. .

Abstract

Glutamate overactivity in basal ganglia critically contributes to the exacerbation of dopaminergic neuron degeneration in Parkinson's disease (PD). Activation of group II metabotropic glutamate receptors (mGlu2/3 receptors), which can decrease excitatory glutamate neurotransmission, provides an opportunity to slow down the degeneration of the dopaminergic system. However, the roles of mGlu2/3 receptors in relation to PD pathology were partially recognized. By using mGlu2/3 receptors agonist (LY354740) and mGlu2/3 receptors antagonist (LY341495) in mice challenged with different cumulative doses of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), we demonstrated that systemic injection of LY354740 reduced the level of extracellular glutamate and the extent of nigro-striatal degeneration in both acute and sub-acute MPTP mice, while LY341495 amplified the lesions in sub-acute MPTP mice only. LY354740 treatment improved behavioral dysfunctions mainly in acute MPTP mice and LY341495 treatment seemed to aggravate motor deficits in sub-acute MPTP mice. In addition, ligands of mGlu2/3 receptors also influenced the total amount of glutamate and dopamine in brain tissue. Interestingly, compared with normal mice, MPTP-treated mice abnormally up-regulated the expression of polo-like kinase 2 (PLK2)/pS129 α-synuclein and phosphorylation of Fyn/N-methyl-D-aspartate receptor subunit 2A/2B (GluN2A/2B). Both acute and sub-acute MPTP mice treated with LY354740 dose-dependently reduced all the above abnormal expression. Compared with MPTP mice treated with vehicle, mice pretreated with LY341495 exhibited much higher expression of p-Fyn Tyr416/p-GluN2B Tyr1472 and PLK2/pS129 α-synuclein in sub-acute MPTP mice models. Thus, our current data indicated that mGlu2/3 receptors ligands could influence MPTP-induced toxicity, which supported a role for mGlu2/3 receptors in PD pathogenesis.

Keywords: 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Fyn kinase; NMDA receptor; Parkinson’s disease; Polo-like kinase; metabotropic glutamate receptor; pS129 α-synuclein.

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Figures

Figure 1
Figure 1
Experimental designs and the effect of chronic treatment with ligands of mGlu2/3 receptors on locomotor functions in acute or sub-acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease (PD) mice models. (A) Molecular formula of LY354740. (B) Molecular formula of LY341495. (C) Schematic representation of the sub-acute MPTP-induced PD mice model experimental scheme. (D) Experimental design of acute-MPTP induced PD mice models. Representative analysis of rotarod tests (E) and pole tests (G) in sub-acute MPTP mice models. Behavioral tests were performed 30 min after the last treatment of drug intervention in sub-acute MPTP mice models on the fourteenth day. #p < 0.05, ##p < 0.01 compared with the MPTP + saline group; &p < 0.05, &&p < 0.01 compared with MPTP + LY341495 group. N = 8. Error bars represent SEM. Analysis of rotarod tests (F) and pole tests (H) on the seventh day of the experiment in acute MPTP mice models. Behavioral tests were performed 30 min after the last treatment of drug intervention. #p < 0.05, ##p < 0.01 compared with the MPTP + saline group; &p < 0.05, &&p < 0.01 compared with the MPTP + LY341495 group. N = 8. Error bars represent SEM.
Figure 2
Figure 2
Ligands of mGlu2/3 receptors regulate the levels of dopamine (DA), homovanillic acid (HVA), and glutamate in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) treated mice. Levels of DA (A) and HVA (B) detected in the striatum and levels of glutamate detected in the (D) substantia nigra (SN), (E) striatum. Data are expressed as ng/µg or ng/mg wet weight tissue and represent the mean ± SEM (n = 3). #p < 0.05, ##p < 0.01 compared to the normal + saline group; *p < 0.05, **p < 0.01. (C) Sample chromatogram of standard solution. Peaks in order: DA (11 min), HVA (15 min). Illustration of the glutamate standard peak (5 min). (F, G) Optical density analysis and immunofluorescence of glial fibrillary acidic protein (GFAP) in SN. Scale bar = 50 µm. **p < 0.01. N = 3. Error bars represent SEM.
Figure 3
Figure 3
Ligands of mGlu2/3 receptors influence expression of tyrosine hydroxylase (TH) in striatum and substantia nigra (SN) of sub-acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. (A, B) Immunohistochemical images and corresponding TH positive cell counts in SN from each group. Scale bar = 100 μm. All data were expressed as % of the normal + saline group. *p < 0.05, **p < 0.01. N = 3 per group. Error bars represent SEM. (C, E, F, H) Immunoblots present the levels of TH in SN of each group. * p < 0.05, **p < 0.01; &&p < 0.01 vs. the other the two groups. N = 5 per group in (E); N = 4 per group in (F). (D, G, I, J) Relative quantifications of expression of TH in striatum of each group were presented. **p < 0.01. N = 5 per group in (G); N = 3 per group in (J). The band intensity of particular target proteins was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin level. Data in (E, G) were expressed as % of normal + saline group. Data in (F, J) were expressed as % of the MPTP + saline group. Error bars represent SEM.
Figure 4
Figure 4
Ligands of mGlu2/3 receptors influence expression of phosphorylated Fyn and subunits of N-methyl-D-aspartate receptors (NMDAR) in substantia nigra (SN) in sub-acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Western blot analysis showed the levels of p-GluN2A tyr1325 (A), p-GluN2B Tyr1472 (C) and p-Fyn Tyr416 (E) in SN of each group. Quantification of ratios of p-GluN2A tyr1325/GluN2A (B), p-GluN2B Tyr1472/GluN2B (D). *p < 0.05, **p < 0.01; #p < 0.05 vs. LY354740 + saline group. N = 3 per group in (B); N = 4 per group in (D). Quantification of the ratio of p-Fyn Tyr416/Fyn (F) of each group. *p < 0.05, **p < 0.01; #p < 0.05 vs. the normal + saline group. N = 3. Relative band intensities were calculated as a ratio of the phosphorylated protein to total protein. The data above were expressed as % of the normal + saline group and error bars represent SEM. Western blot analysis showed the levels of p-GluN2A tyr1325, p-Fyn Tyr416 (G) and p-GluN2B Tyr1472 (I) in SN of each group. (J) Quantification of ratios of p-GluN2A tyr1325/GluN2A and p-GluN2B Tyr1472/GluN2B of each group. *p < 0.05, **p < 0.01. N = 3. (H) Quantification of ratios of p-Fyn Tyr416/Fyn of each group. *p < 0.05; &&p < 0.01 vs. the other two groups. N = 3. Relative band intensities were calculated as a ratio of the phosphorylated protein to total protein. Data in (H, J) were expressed as % of the saline group and error bars represent SEM.
Figure 5
Figure 5
Ligands of mGlu2/3 receptors regulate expression of polo-like kinase 2 (PLK2) and phosphorylated α-synuclein at Ser129 in substantia nigra (SN) of sub-acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease (PD) mice model. Western blots represent the expression of PLK2 (A) and pS129 α-synuclein (B) in SN of each group. The band intensity of particular target proteins was normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) or β-actin level (C, D). * p < 0.05, ** p < 0.01; # p < 0.05 vs. LY354740 + saline group. N = 3 in (C); N = 4 in (D). Data were expressed as % of the normal + saline group and shown as the mean ± SEM. Immunoblots (G) and corresponding histograms (H) show expression of PLK2 and pS129 α-synuclein in SN. && p < 0.01 compared with other two groups; * p < 0.05. N = 3. Data were expressed as % of the MPTP + saline group and shown as the mean ± SEM. (E, F) Immunofluorescence and analysis of PLK2 positive cell counts in SN. Scale bar = 50 µm. * p < 0.05, ** p < 0.01 compared with Normal+Saline group; # p < 0.05 compared with MPTP+LY354740 group. N = 3. Data were shown as the mean ± SEM.
Figure 6
Figure 6
The effect of ligands of mGlu2/3 receptors on expression of tyrosine hydroxylase (TH) in substantia nigra (SN) and striatum in acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s disease (PD) mice models. (A) Immunohistochemical images of tyrosine hydroxylase (TH) in SN of each group. Scale bar = 100 μm. (B) Analysis of differences in TH positive cell counts in SNc from each group. ** p < 0.01. N = 3. Error bars represent SEM. Western blot analysis showed levels and relative quantification of TH in acute-MPTP mice in SN (C–F) of each group. * p < 0.05, ** p < 0.01, N = 4 in (D); ** p < 0.01 vs. other groups; N = 3 in (F). Western blot analysis showed the levels and relative quantification of expression of TH in striatum (G–J) of acute-MPTP mice treated with different doses of LY354740. * p < 0.05 vs. the other groups; ** p < 0.01. N = 4 in (J), N = 3 in (H). Data are expressed as % of the normal + saline group in (D, J). Data are expressed as % of the MPTP + saline group in (F, H). Error bars represent SEM.
Figure 7
Figure 7
mGlu2/3 receptor ligands regulate expression of phosphorylation of Fyn/N-methyl-D-aspartate receptors (NMDAR) in acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mice models. Immunoblots and relevant histograms show expression of phosphorylated NMDAR subunit GluN2A at Tyr1325 (A, B), GluN2B at Tyr1472 (B, C), Fyn at Tyr416 (D, F) in substantia nigra (SN) of acute MPTP-treated mice with different doses of LY354740. The band intensity of particular target proteins was normalized to GluN2A, GluN2B or Fyn levels. *p < 0.05, **p < 0.01. N = 4. Western blots represent the expression of phosphorylated NMDAR subunit GluN2A at Tyr1325 (G, H), GluN2B at Tyr1472 (G, I) and Fyn at Tyr416 (E, J) in acute MPTP-treated mice with saline, LY341495 or LY354740. The band intensity of particular target proteins was normalized to GluN2A, GluN2B or Fyn levels. *p < 0.05, **p < 0.01 vs. the other two groups. N = 3. Data in (B, D) were expressed as % of the normal + saline group, and data in (E, G) were expressed as % of the MPTP + saline group. Error bars represent SEM.
Figure 8
Figure 8
The effect of ligands of mGlu2/3 receptors on expression of polo-like kinase 2 (PLK2) and phosphorylated α-synuclein at Ser129 in substantia nigra (SN) of acute 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced Parkinson’s Disease (PD) mice model. Immunoblots (A) and relevant histograms (C, D) represent expression of PLK2 and phosphorylated α-synuclein at Ser129 in SN of each group. Band intensity of particular target proteins were normalized to β-actin or glyceraldehyde 3-phosphate dehydrogenase (GAPDH) level. **p < 0.01. N = 6 in (C); N = 3 in (D). Data were expressed as % of the normal + saline group and shown as the mean ± SEM. (B, E) Western blots represent expression of PLK2 and pS129 α-synuclein in SN of acute MPTP-treated mice with different drug intervention. *p < 0.05 compared with the other two groups. N = 3. Data were expressed as % of the MPTP + saline group and shown as the mean ± SEM.
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