Roles Of Dickkopf-1and The Inhibition Of The Canonical Wnt Signaling Pathway In Parkinson’s Disease Models

Aims:Wnt signaling pathways have an important role in the development of the dopaminergic neurons and loss of dopaminergic neurons of Parkinson’s Disease(PD). The present study was designed to study whether the pivotal components of the canonical Wnt signaling pathway and Dkk1(an antagonist of the canonical Wnt signaling pathway) were changed in MPP+-induced PC12cells.Methods:PC12cells were exposed to MPP+at various concentrations (1-1000μM) for different time periods. LiCl (1mM) was added1h prior to MPP+. Cell viability was determined by MTT assay and the expressions of Dkk1, β-catenin and p-Ser9-GSK-3β were examined at different time points. Apoptosis was determined by Annexin Ⅴ-FITC/PI study and TUNEL assay.Results:Exposure of PC12cells for24h to MPP+(up to10000μM) couldn’t induce a significant decrease in cell viability compared with the control cells. Treatment with higher concentrations of MPP+for48h and72h resulted in the significant decrease of cell viability, and the minimal viability was45.0%at1000μM MPP+for72h. MPP+induced a significant increase of Dkk1from6h until24h in PC12cells, preceding the cell loss, but Dkk1reduced to the normal level at48h. The induction of Dkk1was associated with the inhibition of the canonical Wnt pathway, including activation of GSK-3β and inhibition of β-catenin, which was reversed by GSK-3β inhibitor LiCl. LiCl also rescued the decrease of PC12cell viability and apoptosis induced by MPP+Conclusion:These data suggest that the induction of Dkk1might contribute to the MPP+-induced neurotoxicity in PC12cells via inhibition of the canonical Wnt pathway. Aims:To examine the special role of Dkk1in the neurotoxicity of MPP+-induced PC12cells.Methods:PC12cells were exposed to MPP+at various concentrations (1-1000μM) for different time periods. LiCl (1mM) was added1h prior to MPP+. RhDkk1(100ng/ml) was added with MPP+simultaneously. Cell viability was determined by MTT assay and the expressions of Dkk1, β-catenin and p-Ser9-GSK-3β were examined. PC12cells were transfected with siRNA duplexes against Dkk1. Then apoptosis was determined by TUNEL assay and the expressions of Dkk1, β-catenin and p-Ser9-GSK-3β were examined.Results:There was a significant decrease of cell viability in PC12cells treated with rhDkk1in combination of MPP+compared with those treated with MPP+only, while LiCl couldn’t rescue the cell loss induced by MPP+combination with rhDkk1. The level of TH was decreased in MPP+-induced PC12cells and the decrease of TH was exacerbated by rhDkkl. Exogenous application of rhDkkl led to an obvious decrease of β-catenin and a slight decrease of p-Ser9-GSK-3β in MPP+-induced PC12cells. The apoptosis of Dkkl siRNA-transfected cells was lower than that of the negative siRNA-transfected cells following MPP+treatment, but higher than that of the negative siRNA-transfected PC12cells. Dkkl siRNA efficiently increased the expression of β-catenin and p-Ser9-GSK-3β and markedly enhanced the level of TH in MPP+-induced PC12cells.Conclusion:These data suggest that Dkkl might contribute to the MPP+-induced neurotoxicity in PC12cells via inhibition of the canonical Wnt pathway. Dkkl antagonists which could rescue the canonical Wnt pathway might be neuroprotective in PD. Aims:To examine the role of Dkk1in the neurotoxicity of dopaminergic neurons in6-OHDA-lesioned rats.Methods:Forty male Wistar rats were randomly divided into4groups (n=10per group): sham,6-OHDA, LiCl+6-OHDA, rhDkk1+6-OHDA. The sham-operated rats were injected with the vehicle alone into the medial forebrain bundle (MFB). Rats in Group LiCl+6-OHDA were pretreated with LiCl (1mEq/Kg, twice a day, i.p.) for7days before the operation, and then given6μl of2μg/μl6-OHDA into the MFB on the right side. Rats in group rhDkk1+6-OHDA were also injected rhDkk1(1μg) into the substantia nigra after they were subjected to6-OHDA. Rats were anesthetized and perfused transcardially with saline followed by4%paraformaldehyde at35d after the surgery. Sections (4μm) were obtained from the brains of each rat and examined by Tyrosine hydroxylase (TH) immunohistochemistry. Western blot was performed in protein lysates obtained from the ventral midbrain of rats at35d after the surgery and was analyzed for Dkkl, TH, β-catenin, and p-Ser9-GSK-3p.Results:The number of TH positive neurons was less in6-OHDA-lesioned rats as compared to those in the sham group, while the TH expression in LiCl-pretreated rats was increased as compared to those in6-OHDA-lesioned rats. And the number of TH positive neurons in rats subjected to rhDkkl combination with6-OHDA was much less than those lesioned by6-OHDA only. The expression of Dkkl was increased in the ventral midbrain of6-OHDA-lesioned rats at35d after surgery by western blot. Western blot analysis also indicated that inhibition of the canonical Wnt signaling pathway, including the decrease of β-catenin and p-Ser9-GSK-3β, was found in the6-OHDA-lesioned rats, which was reversed by LiCl. Application of rhDkkl exacerbated the decrease of β-catenin and p-Ser9-GSK-3β in6-OHDA-lesioned rats.Conclusion:These data suggest that Dkkl plays an important role in the etiology of PD models and it contributes to the neurodegeneration in6-OHDA-lesioned rats via inhibition of the canonical Wnt pathway.