No abstract

The localization and control of Bcl-2 proteins on mitochondria is essential for the intrinsic pathway of apoptosis. Anti-apoptotic Bcl-2 proteins reside on the outer mitochondrial membrane (OMM) and prevent apoptosis by inhibiting the activation of the pro-apoptotic family members Bax and Bak. The Bcl-2 subfamily of BH3-only proteins can either inhibit the anti-apoptotic proteins or directly activate Bax or Bak. How these proteins interact with each other, the mitochondrial surface and within the OMM are complex processes we are only beginning to understand. However, these interactions are fundamental for the transduction of apoptotic signals to mitochondria and the subsequent release of caspase activating factors into the cytosol. In this review we will discuss our knowledge of how Bcl-2 proteins are directed to mitochondria in the first place, a crucial but poorly understood aspect of their regulation. This article is part of a Special Issue entitled Mitochondria: the deadly organelle.

Natural products from the plants of the family Annonaceae, collectively called Annonaceous acetogenins, are very potent inhibitors of the NADH-ubiquinone reductase (Complex I) activity of mammalian mitochondria. The properties of five of such acetogenins are compared with those of rotenone and piericidin, classical potent inhibitors of Complex I. Rolliniastatin-1 and rolliniastatin-2 are more powerful than piericidin in terms of both their inhibitory constant and the protein-dependence of their titre in bovine submitochondrial particles. These acetogenins could be considered therefore the most potent inhibitors of mammalian Complex I. Squamocin and otivarin also have an inhibitory constant lower than that of piericidin, but display a larger protein-dependence of the titre. Squamocin and otivarin, contrary to the other acetogenins, behave qualitatively like rotenone. Rolliniastatin-2 shows unique properties as its interaction, although mutually exclusive to that of piericidin, appears to be mutually non-exclusive to that of rotenone. It is the first time that a potent inhibitor of Complex I is found not to overlap the active site of rotenone.

Cellular stress responses can be activated following functional defects in organelles such as mitochondria and the endoplasmic reticulum. Mitochondrial dysfunction caused by loss of the serine protease HtrA2 leads to a progressive movement disorder in mice and has been linked to parkinsonian neurodegeneration in humans. Here, we demonstrate that loss of HtrA2 results in transcriptional upregulation of nuclear genes characteristic of the integrated stress response, including the transcription factor CHOP, selectively in the brain. We also show that loss of HtrA2 results in the accumulation of unfolded proteins in the mitochondria, defective mitochondrial respiration and enhanced production of reactive oxygen species that contribute to the induction of CHOP expression and to neuronal cell death. CHOP expression is also significantly increased in Parkinson's disease patients' brain tissue. We therefore propose that this brain-specific transcriptional response to stress may be important in the advance of neurodegenerative diseases.

Bid is an abundant proapoptotic protein of the Bcl-2 family that is crucial for the induction of death receptor-mediated apoptosis in primary tissues such as liver. Bid action has been proposed to involve the relocation of its truncated form, tBid, to mitochondria to facilitate the release of apoptogenic cytochrome c. The mechanism of Bid relocation to mitochondria was unclear. We report here novel biochemical evidence indicating that Bid has lipid transfer activity between mitochondria and other intracellular membranes, thereby explaining its dynamic relocation to mitochondria. First, physiological concentrations of phospholipids such as phosphatidic acid and phosphatidylgycerol induced an accumulation of full-length Bid in mitochondria when incubated with light membranes enriched in endoplasmic reticulum. Secondly, native and recombinant Bid, as well as tBid, displayed lipid transfer activity under the same conditions and at the same nanomolar concentrations leading to mitochondrial relocation and release of cytochrome c. Thus, Bid is likely to be involved in the transport and recycling of mitochondrial phospholipids. We discuss how this new role of Bid may relate to its proapoptotic action.

The Fas/CD95 surface receptor mediates rapid death of various cell types, including autoreactive T cells with the potential for triggering autoimmunity. Here, we present novel aspects of Fas signalling that define a ‘social’ dimension to receptor-induced apoptosis. Fas stimulation rapidly induces extensive membrane nanotube formation between neighbouring T cells. This is critically dependent on Rho GTPases but not on caspase activation. Bidirectional transfer of membrane and cytosolic elements including active caspases can be observed to occur via these nanotubes. Nanotube formation and intercellular exchanges of death signals are defective in T lymphocytes from patients with autoimmune lymphoproliferative syndrome harbouring mutations in the Fas receptor. We conclude that nanotube-mediated exchanges constitute a novel form of intercellular communication that augments the propagation of death signalling between neighbouring T cells.

The vacuolating cytotoxin (VacA) is an important virulence factor of Helicobacter pylori with pleiotropic effects on mammalian cells, including the ability to trigger mitochondria-dependent apoptosis. However, the mechanism by which VacA exerts its apoptotic function is unclear. Using a genetic approach, in this study we show that killing by VacA requires the proapoptotic Bcl-2 family members BAX and BAK at the mitochondrial level, but not adequate endoplasmic reticulum Ca 2 þ levels, similarly controlled by BAX and BAK. A combination of subcellular fractionation and imaging shows that wild-type VacA, but not mutants in its channel-forming region, induces the accumulation of BAX on endosomes and endosome-mitochondria juxtaposition that precedes the retrieval of active BAX on mitochondria. It is noteworthy that in Bax-and Bak-deficient cells, VacA is unable to cause endosome-mitochondria juxtaposition and is not retrieved in mitochondria. Thus, VacA causes BAX/BAK-dependent juxtaposition of endosomes and mitochondria early in the process of cell death, revealing a new function for these proapoptotic proteins in the regulation of relative position of organelles.

Human periapical cyst mesenchymal stem cells (hPCy-MSCs) are a newly discovered cell population innovatively collected from inflammatory periapical cysts. The use of this biological waste guarantees a source of stem cells without any impact on the surrounding healthy tissues, presenting a valuable potential in tissue engineering and regenerative medicine applications. In the present study, hPCy-MSCs were collected, isolated, and seeded on three experimental mineral-doped porous scaffolds produced by the thermally-induced phase-separation (TIPS) technique. Mineral-doped scaffolds, composed of polylactic acid (PLA), dicalcium phosphate dihydrate (DCPD), and/or hydraulic calcium silicate (CaSi), were produced by TIPS (PLA-10CaSi, PLA-5CaSi-5DCPD, PLA-10CaSi-10DCPD). Micro-CT analysis evaluated scaffolds micromorphology. Collected hPCy-MSCs, characterized by cytofluorimetry, were seeded on the scaffolds and tested for cell proliferation, cells viability, and gene expression for osteogenic and odontogenic differentiation (DMP-1, OSC, RUNX-2, HPRT). Micro-CT revealed an interconnected highly porous structure for all the scaffolds, similar total porosity with 99% open pores. Pore wall thickness increased with the percentage of CaSi and DCPD. Cells seeded on mineral-doped scaffolds showed a superior proliferation compared to pure PLA scaffolds (control), particularly on PLA-10CaSi-10DCPD at day 12. A higher number of non-viable (red stained) cells was observable on PLA scaffolds at days 14 and 21. DMP-1 expression increased in hPCy-MSCs cultured on all mineral-doped scaffolds, in particular on PLA-5CaSi-5DCPD and PLA-10CaSi-10DCPD. In conclusion, the innovative combination of experimental scaffolds colonized with autologous stem cells from periapical cyst represent a promising strategy for regenerative healing of periapical and alveolar bone.