Figure 1.
PCNA and DNA pol-β are required for neuronal death induced by MPP+.
(A) Cell viability assay. The neurons were exposed to 200 µM MPP+ for 6 h, 12 h, and 24 h, respectively. Cell viability was determined using the MTT method. Data are expressed as a percentage of the value in untreated control cells. (B) Apoptosis rata determined by TUNEL staining. After the neurons were exposed to 200 µM MPP+ for 6 h, 12 h, or 24 h, they were immunostained with neuronal marker MAP2. Neuronal apoptosis was detected by TUNEL staining. (C) Apoptotic rate determined by Hoechst staining. (D) Representative images of cells after Hoechst staining. Arrows indicate the apoptotic cells. (E) Immunoblotting analysis of the expression of PCNA, DNA pol-β, DNA pol-λ, DNA pol-η, and active caspase-3 after treatment with MPP+. The protein levels of PCNA, DNA pol-β and active caspase-3 increased significantly after the neurons were exposed to 200 µM MPP+ for 12 h or 24 h. (F) Immunoblotting experiments showing the efficiency of PCNA and DNA pol-β shRNA. The neurons were infected with PCNA and/or DNA pol-β shRNA lentivirus for 24 h, and then exposed to 200 µM MPP+ for 12 h. The expression of PCNA and DNA pol-β was knocked down by their specific shRNAs. (G) Effect of PCNA and DNA pol-β knockdown on neuronal apoptosis was detected by TUNEL staining. Both shRNAs attenuated neuronal apoptosis induced by MPP+. Date represent the mean ± SEM from four independent experiments. *p<0.05, **p<0.01 compared with control group.
Figure 2.
PCNA binds DNA pol-β in MPP+-treated neurons and in post-mortem brain tissue of PD patients.
(A–B) Coimmunoprecipitation of PCNA and DNA pol-β in vehicle- and MPP+-treated neurons. Coimmunoprecipitation studies were carried out with lysates prepared from neurons treated with vehicle (A) or 200 µM MPP+ for 12 h (B). The lysates were immunoprecipitated with an anti-DNA pol-β antibody and then immunoblotted using an anti-PCNA antibody, or immunoprecipitated with an anti-PCNA antibody and then immunoblotted using an anti-DNA pol-β antibody. Mouse IgG was used as a negative control. (C) Coimmunoprecipitation of PCNA and DNA pol-β in post-mortem brain tissue of PD patients. Human brain lysates from PD patients and control were immunoprecipitated with an anti-PCNA antibody and then immunoblotted using anti-DNA pol-β and anti-PCNA antibody. (D) GST pull-down assay. The HEK293 cells were transfected with plasmids expressing GST-DNA pol-β full length, GST-DNA pol-β DNA binding domain, and GST-DNA pol-β catalytic domain, respectively. 48 h after transfection, GST-tagged proteins were pulled down with Glutathione Sepharose 4B beads, and then immunoblotted using an anti-PCNA antibody. PCNA binds to the catalytic domain but not the DNA binding domain of DNA pol-β.
Figure 3.
Loading of PCNA and DNA pol-β into DNA replication forks.
(A) The neurons were treated with 200 µM MPP+ or vehicle for 12 h. The nucleoprotein fragments were immunoprecipitated using an anti-Cdc45 antibody and immunoblotted using anti-DNA pol-β and anti-Cdc 45 antibodies. (B) The nucleoprotein fragments were immunoprecipitated using a mouse anti-p49 antibody and immunoblotted using anti-DNA pol-β and rabbit anti-p49 antibodies. (C) The neurons were treated with 200 µM MPP+ or vehicle for 12 h. The nucleoprotein fragments were immunoprecipitated using anti-DNA pol-β antibody and immunoblotted using anti-Cdc45, anti-p49, and anti-DNA pol-β antibodies. (D) The nucleoprotein fragments of PD brain tissue or control brain tissue were immunoprecipitated using anti-DNA pol-β antibody and immunoblotted using anti-Cdc45, anti-p49, and anti-DNA pol-β antibodies.
Figure 4.
The interaction of PCNA and DNA pol-β is required for neuronal DNA replication.
(A) Expression of DNA pol-β, PCNA and cell proliferation marker ki67 in neurons infected with DNA pol-β-expressing retroviruses, PCNA-expressing retroviruses, or both. (B) DNA replication in neurons expressing DNA pol-β and/or PCNA. 24 hours after infection, the neurons were incubated with 10 µM BrdU for 4 h, followed by immunostaining with anti-BrdU antibody. The percentage of BrdU positive nuclei increased only when the neurons express PCNA and DNA pol-β at the same time. (C) Expression of DNA pol-β, PCNA and ki67 in neurons infected with PCNA and DNA pol-β specific shRNA lentivirus. (D) The neurons were exposed to 200 uM MPP+ in the presence or absence of PCNA and DNA pol-β specific shRNA lentivirus. The percentage of cells with DNA replication was determined by BrdU incorporation assay. Date represent the mean ± SEM from four independent experiments. *p<0.05, **p<0.01.
Figure 5.
The interaction of PCNA and DNA pol-β induces p53-dependent neuronal apoptosis.
(A) The expression of p53 in neurons infected with retroviruses expressing DNA pol-β, PCNA in the presence or absence of p53 shRNA lentiviral particles. Overexpression of DNA pol-β and PCNA increased the protein level of p53, which was abolished by p53 shRNA lentivirus. (B) Apoptotic rate determined by Hoechst staining. The apoptotic rate increased significantly when the neurons overexpress DNA pol-β and PCNA at the same time. Knockdown of p53 attenuated neuronal apoptosis induced by DNA pol-β and PCNA. (C) Immunoblot analysis of p53 in MPP+-treated neurons. The protein level of p53 increased after the neurons were incubated with MPP+. P53 shRNA lentivirus effectively knocked down the expression of p53. (D) Effect of p53 knockdown on neuronal apoptosis. P53 shRNA significantly attenuated neuronal death induced by MPP+. Date represent the mean ± SEM from four independent experiments. *p<0.05, **p<0.01. (E) The interaction of PCNA and DNA pol-β was abolished by H222A/F223A mutation. Neurons were infected with retrovirus encoding PCNA together with retrovirus encoding wild-type DNA pol-β or H222A/F223A mutant DNA pol-β. The lysates were immunoprecipitated with an anti-PCNA antibody and then immunoblotted using an anti-DNA pol-β antibody. (F) H222A/F223A mutant attenuate the toxic effect of DNA pol-β. The neurons were infected with retrovirus encoding PCNA together with retrovirus encoding wild-type DNA pol-β or H222A/F223A mutant DNA pol-β. Neuronal apoptosis was assessed by TUNEL staining. (G) Western blot analysis of PCNA and DNA pol-β expression.