GSK-3β Contributes to Parkinsonian Dopaminergic Neuron Death: Evidence From Conditional Knockout Mice and Tideglusib

Abstract

Glycogen synthase kinase-3 (GSK-3) dysregulation has been implicated in nigral dopaminergic neurodegeneration, one of the main pathological features of Parkinson's disease (PD). The two isoforms, GSK-3α and GSK-3β, have both been suggested to play a detrimental role in neuronal death. To date, several studies have focused on the role of GSK-3β on PD pathogenesis, while the role of GSK-3α has been largely overlooked. Here, we report in situ observations that both GSK-3α and GSK-3β are dephosphorylated at a negatively acting regulatory serine, indicating kinase activation, selectively in nigral dopaminergic neurons following exposure of mice to 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP). To identify whether GSK-3α and GSK-3β display functional redundancy in regulating parkinsonian dopaminergic cell death, we analysed dopaminergic neuron-specific Gsk3a null (Gsk3a ^ΔDat ) and Gsk3b null (Gsk3b ^ΔDat ) mice, respectively. We found that Gsk3b ^ΔDat , but not Gsk3a ^ΔDat , showed significant resistance to MPTP insult, revealing non-redundancy of GSK-3α and GSK-3β in PD pathogenesis. In addition, we tested the neuroprotective effect of tideglusib, the most clinically advanced inhibitor of GSK-3, in the MPTP model of PD. Administration of higher doses (200 mg/kg and 500 mg/kg) of tideglusib exhibited significant neuroprotection, whereas 50 mg/kg tideglusib failed to prevent dopaminergic neurodegeneration from MPTP toxicity. Administration of 200 mg/kg tideglusib improved motor symptoms of MPTP-treated mice. Together, these data demonstrate GSK-3β and not GSK-3α is critical for parkinsonian neurodegeneration. Our data support the view that GSK-3β acts as a potential therapeutic target in PD and tideglusib would be a candidate drug for PD neuroprotective therapy.

Keywords: GSK-3α; GSK-3β; MPTP; Parkinson’s disease; neuroprotection; tideglusib.

Figure 1

Both Glycogen synthase kinase-3α (GSK-3α) and GSK-3β are activated selectively in 1-methyl-4-pheny-1,2,3,6-tetrahydropyridine (MPTP)-treated mice. Mice were treated with 1, 3, and 5 doses of MPTP and sacrificed 6 h post-injection (M1×, M3×, M5×). (A,B) Immunofluorescent detection of p-GSK-3α (Ser21; green) in TH-positive dopaminergic neurons (red) by immunofluorescence (A) and quantitative data are shown (B). Scale bar: 50 μm. Data are expressed as mean ± SEM (n = 4–5 per group). ***p < 0.001 vs. saline-treated mice. (C,D) The detection of p-GSK-3β (Ser9; green) in TH-positive dopaminergic neurons (red) by immunofluorescence (C) and quantitative data are shown (D). Scale bar: 50 μm. Data are expressed as mean ± SEM (n = 4–5 per group). ***p < 0.001 vs. saline-treated mice.

Figure 2

Evaluation of the role of GSK-3α in dopaminergic neurons in contribution to MPTP-induced parkinsonian neurodegeneration. (A) Immunofluorescence analysis for GSK-3α (green) in the ventral midbrains from MPTP-injected littermate control and Gsk3a^ΔDat mice. Tyrosine hydroxylase (TH; red) staining marks dopaminergic neurons in the substantia nigra. Scale bar: 50 μm. n = 3 per group. (B,C) Immunohistochemical staining of TH on the midbrain sections from saline- and MPTP-injected control littermates and Gsk3a^ΔDat mice (A) and cell counts of TH-positive neurons of the whole SNpc are shown (C). Scale bar: 100 μm. Data show the mean ± SEM (n = 5–8 per group). ***p < 0.001 vs. saline-treated mice of the same genotype. n.s. no significance vs. control littermates as indicated. (D,E) Nissl staining on the midbrain sections from saline- and MPTP-injected control littermates and Gsk3a^ΔDat mice (D) and cell counts of Nissl-positive neurons of the whole SNpc are shown (E). Scale bar: 100 μm. Data show the mean ± SEM (n = 5–8 per group). ***p < 0.001 vs. saline-treated mice of the same genotype. n.s. = no significance vs. control littermates as indicated. (F,G) Immunohistochemical staining of TH on striatal sections from saline- and MPTP-injected control littermates and Gsk3a^ΔDat mice (F) and measured Integrated Optical Density (IOD) of striatal TH immunostaining (G). Scale bar: 500 μm. Data show the mean ± SEM (n = 5–8 per group). *p < 0.05 vs. saline-treated mice of the same genotype. n.s. = no significance vs. control littermates as indicated.

Figure 3

Dopaminergic neuron-associated GSK-3β contributes to MPTP-induced parkinsonian neurodegeneration. (A) Immunofluorescence analysis for GSK-3β (green) in the ventral midbrains from MPTP-injected littermate control and Gsk3b^ΔDat mice. TH (red) staining marks dopaminergic neurons in the substantia nigra. Scale bar: 50 μm. n = 3 per group. (B,C) Immunohistochemical staining of TH on the midbrain sections from saline- and MPTP-injected control littermates and Gsk3b^ΔDat mice (A) and cell counts of TH-positive neurons of the whole SNpc are shown (C). Scale bar: 100 μm. Data show the mean ± SEM (n = 5–6 per group). *p < 0.05, ***p < 0.001 vs. saline-treated mice of the same genotype. ^#p < 0.05 vs. control littermates as indicated. (D,E) Nissl staining on the midbrain sections from saline- and MPTP-injected control littermates and Gsk3b^ΔDat mice (D) and cell counts of Nissl-positive neurons of the whole SNpc are shown (E). Scale bar: 100 μm. Data show the mean ± SEM (n = 5–6 per group). **p < 0.01, ***p < 0.001 vs. saline-treated mice of the same genotype. ^##p < 0.01 vs. control littermates as indicated. (F,G) Immunohistochemical staining of TH on striatal sections from saline- and MPTP-injected control littermates and Gsk3b^ΔDat mice (F) and measured IOD of striatal TH immunostaining (G). Scale bar: 500 μm. Data show the mean ± SEM (n = 5–6 per group). *p < 0.05, ***p < 0.001 vs. saline-treated mice of the same genotype. ^#p < 0.05 vs. control littermates as indicated.

Figure 4

Tideglusib administration exerts neuroprotection against MPTP lesions. (A) A graphic of the experimental procedure is shown. (B) Immunohistochemical staining of TH (left panel), Nissl (middle panel) on the midbrain sections and TH staining on striatal sections (right panel) from saline- and MPTP-injected control and mice with gradient dosages of Tideglusib (50 mg/kg, 200 mg/kg, and 500 mg/kg). Scale bar: 100 μm (Midbrain), 500 μm (Striatum). n = 6–8 per group. (C–E) The bar graph shows the cell counts of TH-positive neurons of the whole SNpc (C), Nissl-positive cells of the whole SNpc (D), and measured IOD of striatal TH immunostaining (E). The data shows the mean ± SEM (n = 6–8 per group). ***p < 0.001 vs. saline-treated mice with vehicle administration. ^#p < 0.05, ^##p < 0.01 vs. MPTP mice with vehicle administration as indicated.

Figure 5

Tideglusib administration improved motor dysfunction caused by MPTP lesions. (A–C) The bar graphs show the results of challenging beam tests: traverse time (A), Numbers of error steps (B), and numbers of total steps (C), respectively. The data shows the mean ± SEM (n = 10–12 per group). **p < 0.01, ***p < 0.001 vs. saline-treated mice with vehicle administration. ^#p < 0.05, ^##p < 0.01 vs. MPTP mice with vehicle administration as indicated. (D) The bar graph shows the numbers of rearings in the cylinder test. The data shows the mean ± SEM (n = 10–12 per group). **p < 0.01 vs. saline-treated mice with vehicle administration. ^#p < 0.05 vs. MPTP mice with vehicle administration as indicated.