Postnatal developmental regulation of Bcl-2 family proteins in brain mitochondria.

Developmental differences in energy metabolism, glutamate excitotoxicity, response to oxidative stress, and susceptibility to apoptosis distinguish the immature brain response to injury from that of the adult (Vannucci and Hagberg, 2004; Blomgren and Hagberg, 2006; Robertson et al., 2006). Extensive experimental evidence indicates that the involvement of apoptosis in acute neurodegeneration is age dependent (Johnston et al., 2001, 2002; Vannucci and Hagberg, 2004; Northington et al., 2005). More prominent activation of caspase-3 and induction of apoptosis are reported for the immature relative to the adult brain following both traumatic brain injury (Bittigau et al., 1999; Pohl et al., 1999) and ischemia/reperfusion (Liu et al., 2004; Zhu et al., 2005). A potential mechanism is indicated by reports of increased level of expression of the apoptosome component Apaf-1 and of caspase-3 in the immature relative to adult brain (Yakovlev et al., 2001). Developmental regulation of the mitochondrial apoptotic pathway at other levels is also reported. The release of apoptogenic proteins cytochrome c (Cyt C) and AIF from mitochondria following hypoxia/ischemia is also age dependent, being more pronounced in the immature than in the adult brain (Zhu et al., 2005). We have also shown previously that mitochondria isolated from immature but not adult brain are particularly sensitive to Bax-BH3-peptide-induced Cyt C release and demonstrated that one mechanism involved in this effect is the increased association of Bax with immature brain mitochondria (Polster et al., 2003). These studies, suggest that developmental changes that occur upstream in the apoptotic pathway, particularly expression of mitochondria-localized Bcl-2 family proteins, also play a critical role in regulating the susceptibility to apoptosis in the immature brain through modulating the release of apoptogenic proteins that activate both caspase-dependent (i.e., Cyt C) and caspase-independent death mechanisms (e.g., AIF; Polster et al., 2003; Zhu et al., 2005). The Bcl-2 family consists of pro- and antiapoptotic proteins characterized by the presence of at least one of the four Bcl-2 homology (BH) domains. Proapoptotic proteins are subdivided into multidomain (BH1–3; Bax, Bak, and Bok) and BH3-only proteins (Bid, Bim, Bad, Bmf, Dp5/Hrk, Puma, Noxa, Bik, Bnip3) sharing only the amphipathic α-helical BH3 region (Adams and Cory, 1998; Gross et al., 1999; Huang and Strasser, 2000). Bcl-2 proteins regulate the permeability of the outer mitochondrial membrane (OMM) and the release of apoptogenic proteins (e.g., Cyt C, AIF, EndoG, Smac/Diablo) from the mitochondrial intermembrane space through mechanisms involving mitochondrial relocalization of proapoptotic members (e.g., Bax, BH3-only proteins) and a number of protein–protein interactions at the OMM. The OMM permeabilization is triggered by Bax/Bak in cooperation with BH3-only proteins and is inhibited by antiapoptotic members (e.g., Bcl-2, Bcl-xL, Mcl-1, Bcl-w). Two models are proposed to explain the interactions among Bcl-2 family proteins that lead to Bax/Bak oligomerization and OMM permeabilization. The hierarchical activation model based on the model proposed by Letai et al. (2002) indicates that binding of derepressor BH3-only proteins (e.g., Bad, Noxa) to antiapoptotic Bcl-2 proteins results in displacement of direct activator BH3-only (Bid, Bim, and Puma), the only proteins that can directly interact with Bax/Bak and induce their oligomerization in the OMM (Letai et al., 2002; Kuwana et al., 2005; Kim et al., 2006). A second model holds that any BH3-only protein or combination that is sufficient to bind and neutralize all antiapoptotic Bcl-2-like proteins present in mitochondria will displace a “primed” or preactivated Bax/Bak protein from antiapoptotic members and will induce OMM permeabilization (Chen et al., 2005). In this model, the strong apoptotic activity of Bid, Bim, and Puma correlates with their ability to bind all antiapoptotic Bcl-2 family members and not direct interaction with Bax/Bak. Although it has been long recognized that the relative balance of pro- vs. antiapoptotic Bcl-2 proteins at the mitochondria is critical for OMM permeabilization and commitment to apoptosis, only a few studies have examined the relative levels of Bcl-2 proteins present in mitochondria during postnatal brain development. y using mitochondria isolated at various postnatal ages and from the adult brain, we examined in this study the developmental regulation of mitochondrial Bcl-2 family proteins. Our results support the concept that developmental differences in the level of mitochondrial Bcl-2 proteins regulate the susceptibility of brain cells to initiate apoptosis following injury.