Cell Death in BAX/BAK DKO Cells is Independent of CypD Expression BAX/BAK DKO cells exhibit decreased steady state ER calcium content due to enhanced calcium leak, with concomitant resistance to PTP-induced cell death [27]. The ectopic expression of the ER-calcium pump ATPase SERCA in BAX/BAK DKO cells restores normal ER calcium levels, enhancing PTP-mediated apoptosis [27]. We did not observe any effects of SERCA overexpression in the occurrence of cell death after ER stress and serum withdrawal treatment in the same cells used in the previous study [27] (Figure 6A). To test the role of CypD in the occurrence of BAX/BAK- independent apoptosis, we reduced the levels of cypD expression in BAX/BAK DKO cells with an efficient shRNA construct we recently described and validated [50] (Figure 6B, left panel). Stable knockdown of cypD mRNA in BAX/BAK DKO cells did not alter the occurrence of cell death after ER stress stimulation together with mild serum withdrawal (Figure 6B, right panel; and Figure S4A). Finally, to rule out the participation of CypD in this BAX/ BAK-independent cell death pathway, we employed triple knockout (TKO) cells for bax, bak, and cypD (Figure 6C) previously described [22]. As expected, TKO cells were highly resistant to ER stress-induced cell death when compared with WT cells (Figure 6D and Figure S4B). Remarkably, this phenotype was completely reverted when ER stress stimulation was performed under conditions of mild serum withdrawal (Figure 6D and Figure S4B). In addition, the pharmacological inhibitor of cyclophilin D, cyclosporine A, was also used in our experimental system. Although DKO cells where not protected against ER stress/ serum withdrawal, WT cells were partially protected against ER stress when pretreated with cyclosporine A (Figure S5). In addition, We also tested the impact of the VDAC inhibitor 4,49- diisothiocyanatostilbene-2,29-disulfonic acid (DIDS) [51,52] on the death of TKO cells. 500 mM DIDS treatment did not block the induction of cell death of TKO cells by Tm and serum withdrawal co-treatments (not shown). Cell death of TKO cells occurred through apoptosis as measured by evident cell shrinkage, chromatin condensation and fragmentation as observed by phase contrast (Figure 6D) or EM analysis (Figure 6E). Visualization of cells by EM did not reveal clear signs of mitochondrial swelling in TKO cells undergoing ER stress and serum withdrawal (Figure 6E). His observation was confirmed by measurements of mitochondrial area, perimeter or circularity (Fig. 6F and not shown). Since cytochrome c release and the engagement of intrinsic apoptosis of BAX/BAK DKO cells by arachidonic acid and A23187 co- treatments was reported to be fully blocked by the serine protease inhibitor 4-(2-Aminoethyl)-benzenesulfonyl fluoride hydrochloride (AEBSF) [22], we monitored the impact of AEBSF in our experimental system. Cell death induced by co-treatment of Tm with mild serum withdrawal was insensitive to 100 mg/ml AEBSF (Figure 6G). We confirmed the protection of AEBSF in the induction of cell death by arachidonic acid plus A23187 in BAX and BAK DKO cells (Figure 6H). Higher AEBSF concentrations were toxic to the cells (not shown). Finally, reactive oxygen species are well known mediators of cell death. Treatment of TKO cells with 1 mM Trolox or 5 mM N- acetylcysteine (NAC), two potent antioxidants, did not attenuate the death of TKO cells after Tm and serum withdrawal co- treatments (Figure 6G). These results suggest that BAX/BAK/ CypD-independent cell death observed in our experimental system is not regulated by serine proteases. A BAX/BAK/CypD-Independent Intrinsic Death Pathway Discussion In this article we have identified experimental conditions where classical intrinsic death stimuli engage the mitochondrial apoptosis machinery in the absence of BAX, BAK and CypD. BAX and BAK are fundamental components of the core apoptosis pathway upon which multiple death signals converge through activation/upregulation of specific BH3-only proteins to trigger cytochrome c release [2,3,53,54]. Bax and bak double deficiency in mice is embryonic lethal due to failure of developmental programs that depend on apoptosis [4]. Howev- er, a small percentage of bax and bak DKO mice (,10%) are viable [4], indicating that proper development can occur in the absence of these pro-apoptotic proteins. Whether developmental cell death in the absence of BAX and BAK is dependent on the CypD and mitochondrial PTP is unknown. We speculate that this may not be the case since most stimuli that trigger CypD- induced cell death rely on mitochondrial calcium uptake and production of reactive oxygen species, conditions more prompt to undergo necrotic cell death due to a rapid drop in ATP production. In fact, genetic ablation of cypD does not generate evident developmental defects, and does not alter the suscep- tibility to apoptosis of cells to a large number of known intrinsic death stimuli, but it modulated cell death after exposure to H2O2 (oxidative stress) in vitro and protected mice against brain and heart ischemia/reperfusion injury [16,19,55]. Our results, together with another report [22], suggest the existence of an alternative mitochondrial regulatory mechanism to instigate under certain circumstances the release of cytochrome c and apoptosome assembly in the absence of BAX/BAK and CypD. Similarly to Tsujimoto’s study [22], we can not exclude the possibility that Cyclosporin A-insensitive PTP (previously described in vitro [55]) operates in our experimental system to trigger cytochrome c release and apoptosis. However we did not detect any evidence for mitochondrial swelling in our EM analysis, a key feature of PRP-mediated cell death. A recent study described the activation of BAX/BAK-independent apoptosis when cell where deprived of glucose [56]. Apoptosis induction required RIPK1 and caspase-8 activation, but was independent of caspase-9 [57]. Irreversible ER stress leads to apoptosis, which requires the expression of BAX and/or BAK at the mitochondria [30]. Here we describe conditions in which mild serum withdrawal reestab- lishes the susceptibility of BAX/BAK DKO MEFs to undergo ER stress-dependent cell death in an apoptosome-dependent manner. Cell death was insensitive to BCL-XL overexpression. Similar results were observed in cells double deficient for the upstream BH3-only proteins BIM and PUMA, where their resistance to ER stress was reverted by mild serum withdrawal. Unexpectedly, these effects were not observed when cell death was triggered with other intrinsic death stimuli such as DNA damage, inhibition of protein synthesis, or perturbations of microtubule dynamics, suggesting that a specific signal emerging from the ER may activate this alternative intrinsic death pathway. Since removal of serum from the cell culture media renders BAX/BAK/CypD TKO cells susceptible to ER stress-induced apoptosis, we speculate that trophic growth factor signaling may mask the activity of an alternative pathway to trigger apoptosis in a BAX/BAK/CypD-independent manner (model in Figure 7). Insulin/IGF1 signaling has been shown to inhibit cytochrome c release through activation of AKT, inducing the translocation of hexokinases to the mitochondrial membrane. Of note, this pathway controlled the release of cytochrome c even in the absence of BAX and BAK after exposition to UV light together PLoS ONE | www.plosone.org 10 June 2012 | Volume 7 | Issue 6 | e37782