Cyclin-C-dependent cell-cycle entry is required for activation of non-homologous end joining DNA repair in postmitotic neurons

Abstract

It is commonly believed that neurons remain in G(0) phase of the cell cycle indefinitely. Cell-cycle re-entry, however, is known to contribute to neuronal apoptosis. Moreover, recent evidence demonstrates the expression of cell-cycle proteins in differentiated neurons under physiological conditions. The functional roles of such expression remain unclear. Since DNA repair is generally attenuated by differentiation in most cell types, the cell-cycle-associated events in postmitotic cells may reflect the need to re-enter the cell cycle to activate DNA repair. We show that cyclin-C-directed, pRb-dependent G(0) exit activates the non-homologous end joining pathway of DNA repair (NHEJ) in postmitotic neurons. Using RNA interference, we found that abrogation of cyclin-C-mediated exit from G(0) compromised DNA repair but did not initiate apoptosis. Forced G(1) entry combined with prevention of G(1) --> S progression triggered NHEJ activation even in the absence of DNA lesions, but did not induce apoptosis in contrast to unrestricted progression through G(1) --> S. We conclude that G(0) --> G(1) transition is functionally significant for NHEJ repair in postmitotic neurons. These findings reveal the importance of cell-cycle activation for controlling both DNA repair and apoptosis in postmitotic neurons, and underline the particular role of G(1) --> S progression in apoptotic signaling, providing new insights into the mechanisms of DNA damage response (DDR) in postmitotic neurons.

Figure 1. DSB induction is associated with cell cycle entry of postmitotic cortical neurons

(A, B) Immunoblot analysis of γH2AX and apoptotic caspase-3 cleavage in neurons treated with 5 μM H₂O₂. Negative control (NC), untreated cultures, positive control (PC), extract from staurosporine-treated Jurkat cells. The values are the means and SD (n = 5); *p < 0.002; **p < 0.001. (C) Immunoblot analysis of the expression of cell cycle-related proteins in neurons exposed to 5 μM H₂O₂. Negative control (NC), untreated neurons, positive control (PC), extracts from proliferating HeLa cells. The values are the means and SD (n = 4); *p < 0.002; **p < 0.001 compared with corresponding NC. (D) Lysates from cortical neurons were prepared at the indicated times and subjected to immune precipitation (IP) using anticyclin C, anticyclin D1, and anticyclin E. Precipitates were tested for in vitro kinase activity using Rb 769 as substrate (IPK). Negative control (NC), untreated neurons; positive control (PC), proliferating HeLa cells. Isospecific control (IgG), normal rabbit or mouse IgG. (E) Lysates from cortical neurons treated with 5 μM H₂O₂ were subjected to immune precipitation followed by immune blot analysis. Anti-cyclin C precipitates were analyzed by anti-CDK3 antibody and anti-CDK3 precipitates were analyzed by anticyclin C antibody. Normal rabbit IgG (IgG) was used as a control for both anti-CDK3 and cyclin C antibodies.

Figure 2. The co-expression of cell cycle and neuronal markers in neurons at 1 h of H₂O₂ exposure

Co-expression of neuronal marker NeuN and a marker of active cell cycle, Ki-67 were detected by immunocytochemistry. Scale bars are 50 μm.

Figure 3. NHEJ activation in postmitotic neurons exposed to 5 μM H₂O₂

(A) Ku-DNA binding analysis of cortical neurons exposed to 5 μM H₂O₂. Negative control (NC), untreated cultures; positive control (PC), the Raji nuclear extract. The values are the means and SD (n = 4); *p < 0.02; **p < 0.005. (B) The NHEJ analysis of cortical neurons exposed to 5 μM H₂O₂. DNA substrate was prepared by linearizing pGL3 basic was added to the NHEJ reaction. Products were subjected to electrophoresis (0.8% agarose gel) and Southern blot, hybridized with the ³²P-labeled plasmid probe and visualized by autoradiography. Negative control (NC), untreated cultures; positive control (PC), T4 ligase treated linearized plasmid DNA (pGL3 basic/XhoI). The values are the means and SD (n = 4); *p < 0.05; **p < 0.002. (C). Co-expression of neuronal marker NeuN and phosphorylated at Thr 2609 DNA-PKcs as a marker of activated NHEJ repair was detected by immunofluorescence. Scale bars are 50 μm.

Figure 4. Blockade of cell cycle entry attenuates NHEJ activation in postmitotic neurons

(A, B) Cortical neurons transfected either with cyclin C (cycC), Cdc25A or control (Con) siRNA were exposed to 5 μM H₂O₂ for the indicated durations and analyzed by immunoblotting for the expression of cyclin C and Cdc25A or by immune precipitation (IP) with anticyclin C antibody and following in vitro kinase activity assay using Rb679 as substrate (IPK). Positive control (PC), lysates from proliferating HeLa cells. Isospefic control for anticyclin C (IgG), normal rabbit IgG. (C, D) Immunoblot analysis of DSBs (γH2AX) and apoptotic caspase-3 cleavage in neurons transfected either with cyclin C (cycC), Cdc25A or control (Con) siRNA and exposed to 5 μM H₂O₂ for indicated durations. Positive control (PC), extracts from staurosporine-treated apoptotic Jurkat cells. (E) Ku-DNA binding of cortical neurons transfected with either cyclin C (cycC), Cdc25A or control (Con) siRNA and exposed to 5 μM H₂O₂ (1 h). Negative control (NC), untreated untransfected cultures; positive control (PC), the nuclear extract of Raji cells. The values are the means and SD (n = 4); *p < 0.01; **p < 0.001. (F) The NHEJ analysis of cortical neurons transfected with either cyclin C (cycC), Cdc25A or control (Con) siRNA and exposed to 5 μM H₂O₂. Negative control (NC), untreated cultures; positive control (PC), T4 ligase treated linearized plasmid DNA (pGL3 basic/XhoI). The values are the means and SD (n = 4); *p < 0.005; **p < 0.001.

Figure 5. p21 silencing leads to S phase entry, while co-targeting p21 and CDK2 reactivates only G1

(A) Cortical neurons were transfected with either cyclin p21 or control (Con) siRNA or co-transfected with p21 and CDK2 (p21/CDK2) siRNA. Expression of p21 and CDK3 was analyzed 1 and 4h after transfection. (B) Cortical neurons were transfected either with p21, p21/CDK2 or control (Con) siRNA. In 1 and 4 h after transfection, lysates were subjected to immune precipitation (IP) using anti-cyclin C, anti-cyclin D1 or anti-cyclin E. Precipitates were tested for in vitro kinase activity using Rb679 as substrate (IPK). Positive control (PC), lysates from proliferating HeLa cells. Isospefic controls for anti-cyclin C, anti-cyclin D1 and anti-cyclin E (IgG), normal rabbit and mouse IgG. (C) Immunoblot analysis of the expression of E2F1 in neurons transfection with p21/CDK2 or control (Con) siRNA. Positive control (PC), proliferating HeLa cells. (D, E) Immunoblot analysis of γH2AX- or cleaved caspase-3 expression in neurons transfected with p21, p21/CDK2 or control (Con) siRNA. Positive control (PC), extract from staurosporine-treated apoptotic Jurkat cells. Note the appearance of γH2AX and the expression of cleaved caspase-3 only in cells transfected with p21, in contrast to neurons transfected with p21/CDK2 and control siRNA.

Figure 6. Forcing neurons into the cell cycle activates NHEJ in postmitotic neurons

(A) Ku-DNA binding analysis of cortical neurons transfected with p21/CDK2 or control (Con) siRNA. Positive control (PC), the nuclear extract of Raji cells. The values are the means and SD (n = 4); *p < 0.01; **p < 0.001. (B) Cortical neurons were transfected with p21/CDK2 or control (Con) siRNA and after indicated time after transfection, DNA-PK activity was measured by kinase activity assay of whole DNA-PK complex. Positive control (PC), HeLa cells exposed to etoposide. (C) The NHEJ analysis of cortical neurons transfected with either p21/CDK2 or control (Con) siRNA. Positive control (PC), T4 ligase treated linearized plasmid DNA (pGL3 basic/XhoI). The values are the means and SD (n = 4); *p <0.002; **p <0.001.

Figure 7. Hypothesized role of cell cycle activation in response to DNA damage in postmitotic neurons

DNA damage generated by a genotoxic insult results in activation of the DNA damage response (DDR). Cell cycle reentry which is an essential element of the DDR, plays a role in both DNA repair and apoptosis. G1→S transition is critical for apoptotic signaling.