ABSTRACT
Objective:To investigate the effects and significance of α-synuclein(α-syn)on the expression level of β-arrestin 2 in Parkinson's disease(PD)in a mouse model.Methods:Twenty-eight C57BL/6J mice with similar motor skills were randomly divided into a model group and a control group, with 14 mice in each group.A PD model was established by injecting preformed fibrils of α-syn into the striatum of the brain, and behavioral changes were monitored after 4 weeks.The expression levels of α-syn, the dopamine receptor(DR), tyrosine hydroxylase(TH), inflammatory factors, β-arrestin 2 and the nuclear transcription factor-κB(NF-κB)signaling pathway-related proteins were determined by Western blotting.The interaction between α-syn and β-arrestin 2 was detected by fluorescence resonance energy transfer(FRET), and the regulation of α-syn on β-arrestin 2 transcriptional activation was detected by the dual luciferase report assay.Results:After 4 weeks of modeling, compared with the control group, the average movement speed of mice in the model group was significantly reduced( t=9.415, P<0.001), the movement track was sparse and concentrated around the open field, and the time needed to climb the pole was significantly prolonged( t=16.412, P<0.001). Compared with the control group, the relative expression of α-synin in astrocytes in the model group increased significantly, the relative expressions of D1DR and TH decreased significantly[(1.14±0.18) vs.(0.53±0.16), (0.67±0.13) vs.(1.15±0.11), (0.46±0.05) vs.(0.81±0.06)]( t=9.810, 10.917 and 17.356, all P<0.001), the relative expression of tumor necrosis factor-α, interleukin-1β, interleukin-6 and NF-κB signaling pathway-related proteins increased significantly( t=3.583, 4.284, 5.396, 11.747, 16.375 and 18.294, all P<0.001), and the relative expression of β-arrestin 2 protein[(0.42±0.11) vs.(1.33±0.14)]in astrocytes decreased significantly( t=19.795, P<0.001). The FRET results suggested a possible direct interaction between α-syn and β-arrestin 2.The results of the dual luciferase report assay showed that the transcription activity of β-arrestin 2 was significantly increased after α-syn gene knockout. Conclusions:The α-syn may induce inflammation in astrocytes by activating the NF-κB signaling pathway and participate in the pathogenesis of PD by reducing dopamine biosynthesis and inhibiting its physiological function through negative regulation of β-arrestin 2.
ABSTRACT
Objective:To observe the expression level of β-arrestin 1/2 in mice with Parkinson's disease(PD)and its relationship with pathogenesis of PD.Methods:PD model was prepared by using 1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine hydrochloride(MPTP). The mice were killed at 3 days after the last administration and the brain tissue was taken for observing brain histopathological changes.The colocalization of β-arrestin1/2 with microglia was detected by using immunofluorescence double-labeling of β-arrestin1/2 and microglia.Tyrosine hydroxylase(TH)and Iba-1 were used to label cells, and then the loss of dopaminergic neurons and the activation of microglia were observed by immunohistochemistry.Results:As compared with the blank control group, the relative expression level of β-arrestin1 protein in brain tissue of PD mice was increased significantly, while the relative expression level of β-arrestin2 protein was decreased significantly( t=11.535, 9.948, both P=0.000), and β-arrestin1/2 shared cell localization with microglia.After MPTP induced PD, the number of Th + neurons in SNc area of midbrain was decreased significantly in β-arrestin1 + /+ group and β-arrestin1 -/- group( t=4.098, 3.571, P=0.000, 0.001), while the number of Iba-1 + cells in SNc area of midbrain was increased significantly( t=10.097、6.448, both P=0.000). After MPTP induced PD, the number of Th + neurons in SNc area of midbrain was decreased significantly in β-arrestin2 + /+ group and β-arrestin2 -/- group( t=3.512, 5.237, P=0.001, 0.000), while the number of Iba-1 + cells in SNc area of midbrain was increased significantly( t=5.816、8.402, P=0.000). Compared with β-arrestin1 + /+ group, the expressions of TRAF6, NF-κB and COX-2 in mouse microglia were significantly increased in β-arrestin1 -/- group( t=5.324, 5.837, 9.350, all P=0.0000). Compared with β-arrestin2 + /+ group, the expressions of TRAF6, NF-κB and COX-2 in mouse microglia were significantly down-regulated in β-arrestin2 -/- group( t=5.094, 6.318, 9.466, all P=0.000). Conclusions:The expression of β-arrestin1 is up-regulated and β-arrestin2 is down-regulated in brain tissue of PD mice.β-arrestin1/2 may affect the proliferation and activation of microglia and the loss of dopaminergic neurons through TRAF6/NF-κB/COX-2 pathway, and participate in the pathological process of PD.