Maturation of neuronal synapses is thought to involve mitochondria. Bcl-xL protein inhibits mitochondria-mediated apoptosis but may have other functions in healthy adult neurons in which Bcl-xL is abundant. Here, we report that overexpression of Bcl-xL postsynaptically increases frequency and amplitude of spontaneous miniature synaptic currents in rat hippocampal neurons in culture. Bcl-xL, overexpressed either pre or postsynaptically, increases synapse number, the number and size of synaptic vesicle clusters, and mitochondrial localization to vesicle clusters and synapses, likely accounting for the changes in miniature synaptic currents. Conversely, knockdown of Bcl-xL or inhibiting it with ABT-737 decreases these morphological parameters. The mitochondrial fission protein, dynamin-related protein 1 (Drp1), is a GTPase known to localize to synapses and affect synaptic function and structure. The effects of Bcl-xL appear mediated through Drp1 because overexpression of Drp1 increases synaptic markers, and overexpression of the dominant-negative dnDrp1-K38A decreases them. Furthermore, Bcl-xL coimmunoprecipitates with Drp1 in tissue lysates, and in a recombinant system, Bcl-xL protein stimulates GTPase activity of Drp1. These findings suggest that Bcl-xL positively regulates Drp1 to alter mitochondrial function in a manner that stimulates synapse formation.Bcl-2 ͉ synaptic transmission ͉ mitochondria ͉ cell death ͉ ABT-737

Early in mitochondria-mediated apoptosis, the mitochondrial outer membrane becomes permeable to proteins that, when released into the cytosol, initiate the execution phase of apoptosis. Proteins in the Bcl-2 family regulate this permeabilization, but the molecular composition of the mitochondrial outer membrane pore is under debate. We reported previously that at physiologically relevant levels, ceramides form stable channels in mitochondrial outer membranes capable of passing the largest proteins known to exit mitochondria during apoptosis (Siskind, L. J., Kolesnick, R. N., and Colombini, M. (2006) Mitochondrion 6, 118 -125). Here we show that Bcl-2 proteins are not required for ceramide to form protein-permeable channels in mitochondrial outer membranes. However, both recombinant human Bcl-x L and CED-9, the Caenorhabditis elegans Bcl-2 homologue, disassemble ceramide channels in the mitochondrial outer membranes of isolated mitochondria from rat liver and yeast. Importantly, Bcl-x L and CED-9 disassemble ceramide channels in the defined system of solvent-free planar phospholipid membranes. Thus, ceramide channel disassembly likely results from direct interaction with these anti-apoptotic proteins. Mutants of Bcl-x L act on ceramide channels as expected from their ability to be anti-apoptotic. Thus, ceramide channels may be one mechanism for releasing pro-apoptotic proteins from mitochondria during the induction phase of apoptosis.Apoptosis is required for normal development and tissue homeostasis in multicellular organisms. Deregulation of apoptosis is fundamental to many diseases, such as cancer, stroke, heart disease, neurodegenerative disorders, autoimmune disorders, and viral diseases. During apoptosis, DNA fragments and other contents of the cell are packaged into apoptotic bodies that are consumed by phagocytosis. There are two main pathways for apoptosis, namely the extrinsic receptor-mediated pathway and an intrinsic mitochondria-mediated one. There is also cross-talk between these two pathways. The intrinsic pathway is initiated when one or more of a multitude of signals converge on mitochondria that ultimately result in an increase in the permeability of the mitochondrial outer membrane (MOM).2 This permeabilization leads to the release of intermembrane space proteins, including cytochrome c, procaspases, apoptosis-inducing factor, heat shock proteins, Smac/ Diablo, and endonuclease G (1). In the cytosol, these proteins activate caspases and DNases that carry out the execution phase of apoptosis.The mechanism for the increased permeability of the MOM during the induction phase of apoptosis is currently highly debated. Several mechanisms have been proposed to explain how mitochondrial intermembrane space proteins are released into the cytosol to facilitate apoptotic cell death. Some involve direct pore formation in the MOM. Several candidate pores exist, but most involve activated multidomain pro-apoptotic Bcl-2 family proteins, Bax and Bak (for example see Refs. 2-5). Another model for MOM permeabil...

Caspase-dependent apoptotic cell death has been extensively studied in cultured cells and during embryonic development, but the existence of analogous molecular pathways in single-cell species is uncertain. This has reduced enthusiasm for applying the advanced genetic tools available for yeast to study cell death regulation. However, partial characterization in mammals of additional genetically encoded cell death mechanisms, which lead to a range of dying cell morphologies and necrosis, suggests potential applications for yeast genetics. In this light, we revisited the topic of gene-dependent cell death in yeast to determine the prevalence of yeast genes with the capacity to contribute to cell-autonomous death. We developed a rigorous strategy by allowing sufficient time for gene-dependent events to occur, but insufficient time to evolve new populations, and applied this strategy to the Saccharomyces cerevisiae gene knockout collection. Unlike sudden heat shock, a ramped heat stimulus delivered over several minutes with a thermocycler, coupled with assessment of viability by automated counting of microscopic colonies revealed highly reproducible gene-specific survival phenotypes, which typically persist under alternative conditions. Unexpectedly, we identified over 800 yeast knockout strains that exhibit significantly increased survival following insult, implying that these genes can contribute to cell death. Although these death mechanisms are yet uncharacterized, this study facilitates further exploration.