Until now, the identity of upstream effectors that control the level or the function of Bmi-1 remains unclear. Here we show that Bmi-1 may be a substrate of Akt and up-regulation of Akt signaling coincides with up-regulation of Bmi-1 phosphorylation and that phosphorylated Bmi-1 is more stable. In contrast, activated oxidative stress-dependent p38 signaling causes Bmi-1 to degrade, and lose its chromatin modifying ability. Furthermore, we also show that posttranscriptional regulation of Bmi-1 via proteasomal degradation causes defective proliferation in Atm-/- NSCs as a result of p21 up-regulation. Collectively, these observations strongly support the notion that down-regulation of Bmi-1 is associated with oxidative stress-dependent pathways in Atm-/- NSCs with p38 activation, Akt deactivation, and p21 up- regulation. Our findings have identified these players as potential targets for A-T treatment. Results Bmi-1 is posttranscriptionally downregulated in Atm-/- NSCs We previously reported that Atm-/- NSCs display highly decreased self-renewal and proliferation, which is correlated with increased levels of CDK inhibitors [6]. The polycomb protein Bmi-1 promotes NSC self-renewal and proliferation through the repression of CDK inhibitor genes [8], [10–11], therefore we investigated Bmi-1 levels in Atm-/- NSCs. Atm-/- NSCs show decreased levels of Bmi-1 and Akt activation, which are essential for NSC proliferation. These results are accompanied by elevated levels of activated-p38 and p21 (Figure 1A). To investigate whether the loss of ATM affected bmi-1 expression in NSCs, quantitative RT-PCR analysis was performed to measure mRNA levels. No significant decrease of bmi-1 mRNA were seen in Atm-/- NSCs (Figure 1B). We also examined the mRNA levels of the CDK inhibitors p21, p16, and p19, which are regulated by Bmi-1, and observed elevated levels of these transcripts in Atm-/- NSCs. This observation is consistent with studies by others that p21, p16, and p19 expression levels are increased in bmi-1-/- NSCs [8], [10] or in bmi-1-knockdown NSCs [11]. The magnitude of increase in p21 mRNA was higher than the increase seen in the mRNA levels of p16 and p19 (Figure 1B). Using fluorescence microscopy, we ROS Accelerates Proteasomal Degradation of Bmi-1 confirmed that levels of activated p38 and p21 are up-regulated, whereas levels of activated Akt and Bmi-1 are downregulated in Atm-/- NSCs (Figure S1). These results indicate that the loss of ATM causes a down-regulation in Bmi-1 protein levels in NSCs posttranscriptionally. Down-regulation of Bmi-1 causes defective proliferation in Atm-/- NSCs Atm-/- NSCs exhibit significantly reduced proliferation and Bmi- 1 is downregulated in these cells. Therefore, we decided to investigate whether down-regulation of Bmi-1 contributes to defective proliferation of Atm-/- NSCs. To address this question, we generated Bmi-1 over-expressing Atm+/+ and Atm-/- NSCs by infecting cells with a retrovirus bearing Bmi-1. A retrovirus bearing Bmi-1 was made using a mouse stem cell virus (MSCV) vector, which contains an internal ribosome entry site (IRES) followed by GFP (Figure 2A) [14]. To compare the proliferative capacity of control vs. Bmi-1 over- expressing NSCs, the number of new neurospheres, total number of cells per well, and the size of neurospheres generated 2 days after subculture were determined. The number of newly generated neurosphere is indicative of NSC self-renewing activity, total cell number in a well represents mitotic division of daughter cells in neurosphere, and the size of newly generated neurosphere represents proliferation within each neurosphere. As expected and confirming the observation from our previous study [6], Atm-/- NSCs formed significantly lower neurosphere numbers, total cell numbers per well, and smaller size of neurospheres compared to Atm+/+ NSCs. However, Bmi-1 over-expression by infecting Atm-/- NSCs with a retrovirus bearing Bmi-1 greatly improved newly formed neurosphere numbers (Figure 2B), total cell numbers per well (Figure 2C), and neurosphere size (Figure 2D). Neurosphere number of Atm-/- NSCs over-expressing Bmi-1 was comparable to untreated Atm+/+ NSCs, while neurosphere size and total cell number were partially recovered. We also observed that Bmi-1 over-expression increased proliferation in Atm+/+ NSCs by increase in neurosphere numbers. In figure 2E, photomicrographs of both Atm+/+ and Atm-/- neurospheres expressing either MSCV-GFP or MSCV-GFP-Bmi- 1 are shown. The restorative action of Bmi-1 over-expression was Figure 1. Bmi-1 is posttranscriptionally downregulated in Atm-/- NSCs. (A) Protein samples (30 mg) from Atm+/+ and Atm-/- NSCs were analyzed by Western blotting for phospho-p38, p38, phospho-Akt, Akt, Bmi-1, and p21 expression. (B) Total RNA was purified from Atm+/+ and Atm-/- NSCs and subjected to quantitative RT-PCR analysis for bmi-1, p21, p16, and p19 expression. Gapdh was used as an internal control. doi:10.1371/journal.pone.0016615.g001 PLoS ONE | www.plosone.org 2 January 2011 | Volume 6 | Issue 1 | e16615