Figure 9. Addition of active Akt to vector cell extracts inhibits cytochrome c–induced PARP cleavage in a cell-free assay. Cell extracts from the vector line were incubated with either control mouse immunoglobulin immunoprecipitation (2 myr-Akt) or immunoprecipitated active Akt [1 myr-Akt (A7) or (B10)] from either the A7 or B10 line that expresses constitutively active Akt. Cytochrome c and dATP were then added to initiate caspase ac- tivation and PARP cleavage. Data are from a representative ex- periment performed three times. c and dATP was inhibited in extracts prepared from Akt- stable lines compared with those in vector-control cell extracts, indicating that expressing active Akt effectively inhibits the cytochrome c–initiated caspase activation cascade. Constitutively Active Akt Inhibits Apoptosis After Microinjection of Cytochrome c To determine whether Akt inhibits cytochrome c–induced apoptosis in vivo, purified cytochrome c was microinjected into the cytoplasm of Akt-expressing and vector-trans- fected cells. Control injections did not induce apoptosis, whereas microinjection of cytochrome c was sufficient to induce rapid apoptosis in HMN1 cells. Cytochrome c but not control-injected cells exhibited condensed and/or frag- mented nuclei (Fig. 8 A). Moreover, constitutively active Akt inhibited and delayed cytochrome c–induced apopto- sis (Fig. 8 B), which is consistent with results obtained from the cell-free system in which expressing active Akt- inhibited apoptotic events such as PARP cleavage and ac- tivation of caspase-9 and caspase-3 in response to cyto- chrome c (Fig. 7, A, C, and D). Immunoprecipitated Active Akt Added to Vector Cell Extracts Inhibits Caspase Activation Induced by Cytochrome c To test the role of posttranslational modification of pro- teins by active Akt in inhibiting cytochrome c–induced caspase activation, active Akt was immunoprecipitated from cells expressing active Akt and incubated with vector cell extracts. Subsequently, cytochrome c and dATP were added to initiate caspase activation. PARP cleavage was markedly inhibited and delayed in vector-cell extracts pre- incubated with active Akt (Fig. 9). In contrast, PARP cleavage was not altered in vector cell extracts preincu- bated with control mouse immunoglobulin immunoprecip- itate (Fig. 9). These results indicate that active Akt has a direct effect on inhibiting cytochrome c–induced caspase activation, and suggest that the inhibition by Akt is through posttranslational modification of cytosolic fac- tor(s) involved in activating caspases. Discussion Apoptotic death of neurons occurs at many different de- velopmental stages. The most frequently studied and mod- eled neuronal death is that occurring during the period of synaptogenesis (Oppenheim, 1991). Neurons are pro- duced in excess, and then compete for specific neu- rotrophic factors produced by their target neurons or or- gans. Neurons that do not get sufficient neurotrophic factor die; this appears to be important for matching the extent of innervation with the size of the target field. Com- pelling evidence supporting this hypothesis comes from a number of studies showing that neurotrophic factors regu- late target-dependent apoptotic neuronal death during de- velopment in vivo and in vitro (Snider, 1994). The two ma- jor pathways implicated in supporting neuronal survival by neurotrophic factors are the PI 3-kinase and MAP kinase pathways (Pettmann and Henderson, 1998). Activation of PI 3-kinase generates the lipid products phosphatidylinositol 3,4-biphosphate (PIP2) and phos- phatidylinositol 3,4,5-triphosphate (PIP3). These products subsequently activate Akt, a serine-threonine protein ki- nase believed to be the major effector for PI 3-kinase to promote cell survival (Franke et al., 1997). Pharmacologi- cal studies with PI 3-kinase and/or MEK inhibitors indi- cate that only the PI 3-kinase pathway is essential for HMN1 cell survival (Fig. 1). We previously showed that C2-ceramide inhibited insulin-stimulated Akt activation but did not affect the activity of a membrane-targeted, constitutively active form of Akt, indicating that mem- brane association activates Akt and bypasses the inhibi- tory effect of ceramide (Zhou et al., 1998). In this study, we generated stable HMN1 cell lines expressing constitu- tively active Akt to explore the cellular mechanisms through which Akt regulates cell survival. Overexpression of active Akt inhibits apoptosis, as exemplified by its in- hibitory effects on caspase-3 activation, PARP cleavage, and DNA fragmentation. However, unlike the observa- tion that Akt upregulates Bcl-2 expression in BAF/3 cells (Ahmed et al., 1997), Akt promotes HMN1 cell survival in the absence of altering Bcl-2, Bcl-X, and Bax expression, which is consistent with data from hippocampal H19-7 neuronal cells and epithelial BRK cells (Eves et al., 1998; Sabbatini and McCormick, 1999). Nevertheless, given the pivotal role of BCL-2 family proteins in regulating apopto- sis and a growing number of signaling pathways shown to regulate apoptosis by modulating expression of BCL-2 family members (Gross et al., 1999), these results do not rule out the possibility that Akt may regulate the expres- sion of other members of the BCL-2 family to inhibit apop- tosis. Apoptosis induced by a variety of initiators such as acti- vation of p75 low affinity NGF receptor and growth factor withdrawal is associated with increased ceramide produc- tion. In addition, a strong correlation exists between pro- duction of ceramide and subsequent cell death (Hannun, 1996; Mathias et al., 1998). Moreover, cell-permeable cer- Zhou et al. Akt Inhibits Apoptosis after Cytochrome c Release 491