Monitoring of Single Vesicle Cytochrome-c Release Illuminates BAK as a Novel Target of Aβ Oligomers

Intrinsic apoptosis is initiated by the BAX- or BAK-dependent permeabilization of the mitochondrial outer membrane and subsequent release of cytochrome c. In Alzheimer’s disease (AD), significant death of hippocampal and cortical neuronal populations occurs through multiple processes including activation of the intrinsic apoptosis pathway (1). In particular, the expression of BAK is upregulated in AD hippocampal and cortical tissues when compared to age-matched control tissues (2,3). Prior studies have demonstrated that amyloid beta peptide 1–42 (Aβ1–42) oligomers induce cytochrome-c release and neuronal apoptosis via a BAX-dependent mechanism (4). In this issue of the Biophysical Journal, Kim et al. (5) identify a significant role for BAK in cytochrome-c release induced by Aβ1–42 oligomers. The authors demonstrate that Aβ1–42 oligomer-induced HT22 hippocampal cell death is significantly reduced by siRNA knockdown of BAK. Moreover, mitochondria isolated from BAK knockdown HT22 cells display reduced cytochrome-c release in vitro in response to either Aβ1–42 oligomers or truncated BID (tBID).

Most impressively, the authors have developed a cell-free, liposome-based, fluorescence cytochrome-c release assay by which single vesicle release events can be accurately recorded and monitored over time. They carefully validate this assay using BAKΔC-term and tBID recombinant proteins and convincingly show that Aβ1–42 oligomers are capable of inducing cytochrome-c release from BAKΔC-term-containing vesicles as well as enhancing release in the presence of tBID. Finally, they show that Aβ1–42 oligomers bind directly to BAKΔC-term-containing vesicles but do not bind as significantly to vesicles containing tBID or protein-free liposomes. In regard to this latter point, although the authors suggest that the relatively weak binding of Aβ1–42 oligomers to protein-free liposomes represents nonspecific binding, this may in fact not be the case.

Many previous studies have demonstrated that Aβ1–42 (or Aβ1–40) oligomers interact directly with lipid membranes and that these interactions are significantly influenced by a number of factors including oligomer concentration, Aβ1–40/Aβ1–42 ratio, cholesterol content, and charge on both the lipid surface and the peptide (6–10). It would be interesting to examine how these different factors influence the Aβ1–42 oligomer interaction with BAK because the oligomerization process can be significantly modulated by the specific interactions of the Aβ1–42 peptide with the lipid membrane.

We believe the results presented by Kim et al. (5) are novel and clearly demonstrate a central role for BAK in cytochrome-c release triggered by Aβ1–42 oligomers (Fig. 1). This is in contrast to previous studies suggesting that Aβ1–42 (or Aβ1–40) oligomers induce direct permeabilization of mitochondrial or other lipid membranes in the absence of any membrane-associated proteins (11,12). However, these prior studies used much higher concentrations of Aβ oligomers, which might account for the differences observed. Furthermore, the development of a liposome-based, fluorescence cytochrome-c release assay by which single vesicle release events can be monitored over time represents a significant technological advance that will allow the probing of additional pathways which regulate this process. Future studies that examine, in more detail, the mechanism by which Aβ1–42 oligomers activate BAK will be informative. Finally, the findings of Kim et al. (5) suggest that BAK may be a new therapeutic target for AD, and should prompt investigation into the effects of crossing BAK knockout mice with transgenic mouse models of high Aβ1–42 content such as the APPswe/PSEN1dE9 (APP/PS1) mouse.

Figure 1.

A central role for BAK in cytochrome-c release triggered by Aβ1–42 oligomers. BAK oligomerization and pore formation in the mitochondrial outer membrane are triggered by interactions with either tBID or Aβ1–42 oligomers. Cytochrome c released through the BAK pore becomes an integral part of the apoptosome complex, which also includes APAF-1 and pro-caspase-9 (not shown). The apoptosome activates the intrinsic initiator caspase-9, which, in turn, cleaves and activates the executioner caspase-3, leading to apoptotic cell death. To see this figure in color, go online.