ROS Accelerates Proteasomal Degradation of Bmi-1 Figure 6. p38 signaling leads to proteasomal degradation of Bmi-1 in Atm-/- NSCs. (A) Protein samples (30 mg) from Atm+/+ or Atm-/- NSCs were treated in vitro with l phosphatase for 1.5 hours and analyzed by Western blotting for changes in migration of Bmi-1 in SDS-PAGE. (B) Atm+/+ or Atm-/- NSCs were treated for 4 hours with 10 mM MG132 and phospho-Akt, Akt, and Bmi-1 levels were determined by Western blotting. (C) Atm-/- NSCs were pretreated with 10 mM SB203580. Atm+/+ and Atm2/2 NSCs were then treated for times indicated with cycloheximide (50 mg/mL) and Bmi- 1 levels were determined by Western blotting. Probing with an antibody to b-actin was used as a loading control. The values represent the Bmi-1 signal intensity compared to the 0 h time point. (D) Atm+/+ or Atm-/- NSCs treated for 24 hours with either cultured in simple medium (middle panel) or 10 mM SB203580 (right panel) were analyzed by Immunofluorescence for Bmi-1, phospho-p38, and ubiqutinated-histone H2A (H2A-Ub). Cells were counterstained by DAPI (49-6-Diamidino-2-phenylindole), which identifies the nuclei of the NSCs. doi:10.1371/journal.pone.0016615.g006 astrocytes. In the present study we focused on NSCs. NSC-specific targeting holds the prospect to provide a cure for many neurodegenerative diseases, as NSCs possess the potential to differentiate into neurons, oligodendrocytes, and astrocytes. Because of their pluripotency, impairment of NSCs likely results in neuronal cell loss in Atm-/- mice. We previously demonstrated that NSCs from SVZ of Atm-/- mouse brain are abnormal, in terms of decreased proliferative capacity and lower Bmi-1 expression levels compared to Atm+/+ NSCs [6]. Here we show that down-regulation of Bmi-1 contributes to defective self-renewal and proliferation in Atm-/- NSCs because over-expression of Bmi-1 restores Atm-/- NSC proliferation to normal (Figure 2). Additionally, we identified upstream effectors that control Bmi-1 levels and function in chromatin modifica- tion. Our data suggests that p38 is a central player in the defective proliferation of Atm-/- NSCs induced by oxidative stress. Previous report demonstrated that p38 participates in p21 up-regulation, subsequently resulting in cell growth inhibition [23]. Here, we show that Bmi-1 levels are regulated post- transcriptionally by proteasome-mediated degradation (Figure 3). In addition, EGF-mediated Akt activation is associated with Bmi-1 stabilization (Figure 4), and the p38 inhibitor, SB203580, also stabilizes Bmi-1 in H2O2-treated NSCs and Atm-/- NSCs (Figure 3, 5, and 6). We conclude that in the absence of ATM, chronic oxidative stress results in the activation of p38, leading to inhibition of the Akt-dependent Bmi-1 stabilizing process, thus causing Bmi-1 to degrade more rapidly. It is also possible that p38 downregulates Bmi-1’s ability to bind to chromatin, thus Bmi-1 loses its suppressive effects on p21, which is supported by our findings that the p38 inhibitor, SB203580, restores H2A ubiquitination (Figure 6C) and restores p21 levels in Atm-/- NSCs [6]. PLoS ONE | www.plosone.org 8 January 2011 | Volume 6 | Issue 1 | e16615