amide analogues induce apoptosis in many different cell types including neurons (Hannun, 1996; Mathias et al., 1998). These observations are consistent with ceramide acting as a lipid second messenger that functions to link cell-surface receptors and environmental stresses to the cellular apoptotic machinery. However, the cellular mech- anisms for ceramide-induced apoptosis remain unclear. Our current study as well as work by others suggests that caspase activation is required for ceramide-induced ap- optosis and that ceramide-induced apoptosis can be suppressed by either constitutively active Akt or Bcl-2, probably by different cellular mechanisms. Embryonic fibro- blasts lacking Apaf-1 are less susceptible to C6-ceramide– induced apoptosis than wide-type embryonic fibroblasts, suggesting that Apaf-1 is involved in ceramide-induced ap- optosis (Cecconi et al., 1998). Further studies are needed to determine whether members of the Bcl-2 family, such as Bax are involved in ceramide-induced apoptosis. Cytochrome c release from mitochondria represents an important control point during apoptosis, although the mechanisms causing cytochrome c release remain unclear (Green and Reed, 1998). It has been proposed that at least two different mechanisms exist for cytochrome c release from mitochondria, one that is caspase dependent and the other that is caspase independent. In Fas-induced apopto- sis, oligomerization of the Fas receptor leads to recruit- ment of FADD and subsequent caspase-8 activation (Na- gata, 1997). Activated caspase-8 cleaves BID, a BH3 domain-only proapoptotic member of the BCL-2 family. The cleaved p15 BID fragment translocates to mitochon- dria, where it causes release of cytochrome c, probably by changing the conformation of Bax (Li et al., 1998; Luo et al., 1998; Desagher et al., 1999). Bid-deficient hepatocytes do not release cytochrome c in response to agonistic anti– Fas antibody, which confirms the importance for cleaved Bid to induce cytochrome c release in this model (Yin et al., 1999). In our study, C2-ceramide–induced cytochrome c release appears to be caspase independent since a broad caspase inhibitor, zVAD-fmk, inhibits caspase activation but not cytochrome c translocation. The mechanism for caspase-independent release of cytochrome c remains un- clear. A recent study (Kennedy et al., 1999) indicates that Akt inhibits the release of cytochrome c from mitochon- dria after UV irradiation, which may involve activation of cell-surface Fas receptor (Rehemtulla et al., 1997; Aragane et al., 1998). In contrast, our data indicate that Akt fails to inhibit cytochrome c release induced by cer- amide via a caspase-independent mechanism. Therefore, the function for Akt to regulate cytochrome c release may vary in response to different apoptotic stimuli or within a different cellular context. In mammalian cells, the release of cytochrome c from mi- tochondria has been proposed as the critical event for cells to initiate the apoptotic cascade. In the cytosol, cytochrome c binds to Apaf-1 and triggers Apaf-1–mediated caspase-9 activation. Activated caspase-9 propagates the death signal by activating caspase-3 and other caspases (Slee et al., 1999; Zou et al., 1999). In cell-free assays of apoptosis, exoge- nously added cytochrome c initiates rapid apoptotic events, including caspase-3 activation and DNA fragmentation (Liu et al., 1996; Li et al., 1997a; Slee et al., 1999). Data from our cell fractionation study and cell-free assay are consistent with Bcl-2 acting to inhibit apoptosis mainly at the mitochondrial level to suppress cytochrome c release, since Bcl-2 inhibits the release of cytochrome c but fails to inhibit caspase activation induced by added cytochrome c in the cell-free assay. Akt phosphorylates and inactivates BAD, which may maintain Bcl-2 and Bcl-xL function, and it has been suggested that Akt inhibits cell death by pre- venting cytochrome c release from mitochondria (Kennedy et al., 1999). In contrast, our data indicate that Akt protects neural cells from apoptosis at a postmitochondrial stage to inhibit caspase activation. First, active Akt protects cells from apoptosis without blocking cytochrome c release into the cytosol. Second, addition of cytochrome c failed to in- duce caspase-9 activation, caspase-3 processing, or PARP cleavage in extracts from cells expressing active Akt. Third, addition of active Akt to lysates from control cells blocked cytochrome c–induced caspase activation. Fourth, cells ex- pressing active Akt are resistant to apoptosis induced by microinjection of cytochrome c. Microinjection of purified cytochrome c into the cyto- plasm induces apoptosis in a variety of cell types, whereas microinjection of anti–cytochrome c antibody into sympa- thetic neurons inhibits NGF withdrawal-induced apopto- sis, indicating that cytochrome c is both sufficient and nec- essary for apoptosis to progress in vivo (Li et al., 1997b; Duckett et al., 1998; Neame et al., 1998; Zhivotovsky et al., 1998). However, recent studies show that certain cell types, including neurons, have postmitochondrial mecha- nisms to inhibit the cell death machinery after cytochrome c release, suggesting a more complicated mechanism for regulating apoptosis than previously thought (for review, see Newmeyer and Green, 1998; Reed and Paternostro, 1999). For example, cytochrome c release induced by NGF withdrawal in sympathetic neurons is a reversible event, indicating that redistribution of cytochrome c into the cy- tosol does not always result in irreversible cell death in neurons (Martinou et al., 1999). Also, microinjection of cytochrome c into sympathetic neurons is not lethal in the presence of NGF. Therefore, NGF signaling appears to block cytochrome c–induced apoptosis in sympathetic neurons. Sympathetic neurons only commit to apoptosis in response to cytochrome c after NGF is removed for a pro- longed period. It is proposed that neurons must undergo an unidentified event to develop “competence to die” in addition to the presence of cytochrome c (Deshmukh and Johnson, 1998). Our data demonstrate that expressing ac- tive Akt inhibits cytochrome c–induced caspase activation in a cell-free assay and also suppresses apoptosis after cy- toplasmic injection of cytochrome c, indicating that Akt blocks apoptosis in the presence of cytochrome c. In this regard, loss of Akt signaling may be related to developing neuronal “competence-to-die” in response to cytochrome c since prolonged NGF withdrawal may progressively de- crease PI 3-kinase/ Akt activity and lower the threshold for cytochrome c to initiate caspase activation. Recently, cytochrome c was implicated in the pathogene- sis of Parkinson’s disease since purified cytochrome c stim- ulates a-synuclein aggregation in vitro and cytochrome c colocalizes with a-synuclein in Lewy bodies of patients with Parkinson’s disease (Hashimoto et al., 1999). Our findings here indicate that Akt protects neural cells from acute apop- tosis caused by cytochrome c release and may allow cyto- The Journal of Cell Biology, Volume 151, 2000 492