Bmi-1 previously has been shown transiently co-localized with the centromeres [24]. Furthermore, Bmi-12chromatin association has been proven by immunostaining [25–26]. However, mecha- nisms regulating Bmi-1 chromatin association are still unclear. We performed biochemical fractionation of Bmi-1 to see differential chromatin association and found that more Bmi-1 chromatin association is seen in Atm+/+ NSCs than in Atm-/- NSCs (data not shown). In normal condition, Bmi-1 chromatin association is strictly regulated by its phosphorylation status in the cell cycle [26– 27]. Therefore, our data might be explained by the fact that Atm-/- NSCs show abnormal Akt and p38. We have proven that abnormal p38-Akt signaling contributes to defective NSCs proliferation and self-renewal [6]. Another interesting question to be addressed is whether or not Akt/p38-dependent Bmi-1 phosphorylation is critical for its chromatin association. It is unclear if Bmi-1 is a p38 substrate from our study, but it has been reported that Bmi-1 could be phosphorylated by 3pk (MAPKAP kinase 3), which is a downstream effector of p38 [26]. Phosphorylation of Bmi-1 by 3pk reduces Bmi-1’s ability to bind to chromatin, thus reducing its suppressive effect on p21. Another way in which p38 may regulate Bmi-1 levels is via down- regulation of Akt because Bmi-1 restoration by the p38 inhibitor, SB203580, is accompanied by an increase in Akt activation levels under oxidative stress conditions (Figure 5). This result is supported by another report that describes the oxidative stress- induced activation of p38, which attenuates insulin-like growth factor stimulation of Akt [28]. Our studies also show that Bmi-1 is a substrate of Akt, and up-regulation of p-Akt coincides with up- regulation of p-Bmi-1. Until now, no one has shown that Akt can phosphorylate and up-regulate Bmi-1. Therefore, this will be the first time a direct functional link between Akt and Bmi-1 is made. Further study is under way to make their direct links. The connection between the p38/Akt signaling and Bmi-1 stability in A-T is a newly described angle of ATM signaling that may be relevant to clinical aspects of the this disease. Protein kinase activities are redox-sensitive because key cysteine residues in these proteins can undergo posttranslational modifica- tions by oxidants [5]. It has recently been shown that H2O2 is directly responsible for disulfide bond formation, which results in a conformational alteration and activation of ATM [29]. This suggests that ATM can be activated in response to increased ROS levels to regulate the redox status of the cells, independently of its response to DNA damage [30]. Our findings demonstrate that in the absence of ATM, chronic oxidative stress results in activation of the p38-Bmi-1-p21 pathway in NSCs. Taken together, we propose a possible mechanism regulating Bmi-1 stability by Akt or p38 signaling (Figure 7). We hypothesize that a function of ATM is to act as a redox modulator. As a consequence of increased ROS levels p38 is activated, which may block the Akt-dependent Bmi-1 stabilizing process. This event accelerates proteasome-mediated degradation of Bmi-1, which in turn upregulates p21 and suppresses proliferation (red arrow). During normal condition, EGF-mediated Akt phosphorylation leads to Bmi-1 stabilization and neural stem cell proliferation (green arrows). By controlling p38 or Akt signaling, which has yin and yang effects on Bmi-1 stability, we may be able to control Atm-/- NSC proliferation as described in the above mechanism. NSCs possess a range of actions that can be potentially used for therapy, because they are capable of self-renewal and can differentiate into cells of glial and neuronal lineages in the CNS [31–32]. Bmi-1 is also important for maintenance of NSC multipotency, which is required for generating neurons [11], [33], and Bmi-1 overexpression increases neurogenic capacity of NSCs [14]. Previous studies demonstrated that normal p38 ROS Accelerates Proteasomal Degradation of Bmi-1 Figure 7. Proposed mechanism underlying defective prolifer- ation of Atm-/- NSCs. Bmi-1 expression is regulated mainly through posttranscriptional mechanism by proteasomal degradation. EGF- mediated Akt phosphorylation renders Bmi-1 resistant to the degrada- tion process, leading to its stabilization and neural stem cell proliferation (green arrows). However, chronic oxidative stress, in the absence of ATM, activates p38 signaling blocks Akt-dependent Bmi-1 stabilizing, resulting in accelerated proteasome-mediated degradation of Bmi-1 and p21 up-regulation, which in turn may suppress proliferation (red arrow). doi:10.1371/journal.pone.0016615.g007 signaling is essential for NSC differentiation [34] and activated p38 leads to abnormal differentiation in NPC1-/- (The Niemann- Pick type C1) NSCs [35]. We show here that Atm-/- NSCs have abnormally activated p38 and lower level of Bmi-1 compared to Atm+/+ NSCs. We also observed that Atm-/- NSCs are capable of undergoing multilineage differentiation, but they show different frequencies of differentiated cells when compared to Atm+/+ NSCs. (data not shown). Therefore, we hypothesize that abnormal p38– Bmi-1 signaling in Atm-/- NSCs may affect their neurogenic capacity, which would change the fate of the progenitor cells. Continuing our investigation, it will be still interesting to examine the fate of the progenitor cells to astroglia, oligodendroglia, and neurons by assessing the frequency of multilineage cells from Atm+/+ and Atm-/- NSCs, and to determine whether p38 signaling inhibition affects the neurogenic capacity of Atm-/- NSCs. In recent years, NSC transplantation has proven to be a more tractable therapeutic strategy for neurodegenerative disorders than the conventional placement of differentiated neurons, since NSCs can replace many cell types, not just one, and can synergize with other therapies, including drug treatments [31–32], [36]. When NSCs are defective, as they are in Atm-/- mice, transplanting normal Atm+/+ NSCs may rescue impaired NSCs by restoring homeostasis to the NSCs themselves, or by stabilizing normal metabolism and redox balance in the supporting cell microenvi- ronment. The imposition of ‘‘molecular homeostasis’’ may be the basis for this NSC rescue. Here we show that Atm-/- NSCs have abnormally reduced levels of Bmi-1, and we know that Bmi-1 is necessary for improved NSC survival and proliferation. Thus, our PLoS ONE | www.plosone.org 9 January 2011 | Volume 6 | Issue 1 | e16615