HSCs are sensitive to ROS excessive ROS
HSCs are sensitive to ROS; excessive ROS level following mTOR activation has been reported to be the underlying cause of HSC loss (Chen et al., 2008), although not always (Lee et al., 2010). Since OXPHOS is a major source of cellular ROS, it is likely that activation of mTOR in HSCs is modulated to prevent significant buildup of ROS in HSCs. In support of this argument, I found that the expression of REDD1, a crucial inhibitor of mTOR activation (Brugarolas et al., 2004; Reiling and Hafen, 2004), in HSCs correlated with mitochondrial ETC of HSCs. Interestingly, the findings of the current study show that relatively higher apoptosis in WT HSCs was not due to the higher level of ROS in these cells, as NAC treatment initiated soon after 5-FU challenge did not improve HSC numbers in WT mice (data not shown). Rather, higher apoptosis of WT HSPCs during early recovery phase was due to lower mitochondrial ETC capacity, and increased mTOR activation. Although HSCs at steady-state mainly undergo glycolytic metabolism (Simsek et al., 2010; Takubo et al., 2010), findings of the current study demonstrate that expansion of HSCs following 5-FU treatment is dependent on mitochondrial metabolism. The critical role of mitochondria in HSCs\' maintenance is further supported by the findings that deficiency of Lkb1 leads to HSC depletion (Nakada et al., 2010; Gurumurthy et al., 2010). Depletion of HSCs in Lkb1 deficient mice is associated with mitochondrial dysfunction and energy production (Gan et al., 2010). Furthermore, I found lowering mTOR activation during early phase by rapamycin treatment preserved WT HSCs and led to their improved numbers during later stage, likely by reducing energy stress. Energy stress can lead to p53 activation resulting in senescence (Lee et al., 2010) or apoptosis or reduction in the ability to undergo symmetric division—critical for stem cell expansion, as seen in mammary stem eph receptor (Cicalese et al., 2009). The findings of this study show that mTORC1 activation can lead to increased HSC proliferation provided the HSCs have the capacity to boost ETC to meet the increased demand for energy created by mTORC1 activation. An earlier study had shown that mTOR activation leads to the loss of HSCs due to increased mitochondrial metabolism (Chen et al., 2008). Although the current findings appear to contradict the finding of the previous study (Chen et al., 2008), differences in hematopoiesis models—stress versus steady-state—analyzed may account for the difference. Unlike steady-state, a majority of HSCs undergo proliferation (Harrison and Lerner, 1991) post-5-FU treatment to replenish hematopoiesis. 5-FU treatment leads to myeloablation and loss of a majority of sinusoidal microvasculature in BM (Kopp et al., 2005). It is likely that the loss of BM microvasculature severely restricts the availability of nutrients. Although ATP can be derived from glycolysis, OXPHOS yields maximum ATP molecules per molecule of glucose. It is therefore likely that post-5-FU treatment HSCs boost mitochondrial metabolism to meet the increased energy demand required to support rapid proliferation of HSCs. Indeed the difference in proportion of IL-7R-KSL cells undergoing mitochondrial metabolism post-5-FU treatment compared to that of untreated cells (as shown by NADH fluorescence pattern in the presence and absence of antimycin A) supports the conjecture and demonstrates an increase in mitochondrial metabolism in HSCs during initial recovery of hematopoietic phase following 5-FU treatment, unlike steady-state hematopoiesis, during which glycolysis is required for the maintenance of HSCs (Simsek et al., 2010). However, if the HSCs lack the ability to enhance ETC activity in tune with increased mTORC1 activity, mTORC1 activation leads to the loss of HSCs, as observed in the case of WT mice. Thus, a fine tuning between mTORC1 activation and mitochondrial biogenesis is important for efficient expansion of HSPCs following 5-FU treatment. The following are the Supplementary data to this article.