Cardiolipin is a mitochondrion-specific phospholipid that stabilizes the assembly of respiratory chain complexes, favoring full-yield operation. It also mediates key steps in apoptosis. In Barth syndrome, an X chromosome-linked cardiomyopathy caused by tafazzin mutations, cardiolipins display acyl chain modifications and are present at abnormally low concentrations, whereas monolysocardiolipin accumulates. Using immortalized lymphoblasts from Barth syndrome patients, we showed that the production of abnormal cardiolipin led to mitochondrial alterations. Indeed, the lack of normal cardiolipin led to changes in electron transport chain stability, resulting in cellular defects. We found a destabilization of the supercomplex (respirasome) I+III2+IVn but also decreased amounts of individual complexes I and IV and supercomplexes I+III and III+IV. No changes were observed in the amounts of individual complex III and complex II. We also found decreased levels of complex V. This complex is not part of the supercomplex suggesting that cardiolipin is required not only for the association/stabilization of the complexes into supercomplexes but also for the modulation of the amount of individual respiratory chain complexes. However, these alterations were compensated by an increase in mitochondrial mass, as demonstrated by electron microscopy and measurements of citrate synthase activity. We suggest that this compensatory increase in mitochondrial content prevents a decrease in mitochondrial respiration and ATP synthesis in the cells. We also show, by extensive flow cytometry analysis, that the type II apoptosis pathway was blocked at the mitochondrial level and that the mitochondria of patients with Barth syndrome cannot bind active caspase-8. Signal transduction is thus blocked before any mitochondrial event can occur. Remarkably, basal levels of superoxide anion production were slightly higher in patients cells than in control cells as previously evidenced via an increased protein carbonylation in the taz1? mutant in the yeast. This may be deleterious to cells in the long term. The consequences of mitochondrial dysfunction and alterations to apoptosis signal transduction are considered in light of the potential for the development of future treatments.
Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria.
The pro-apoptotic effector Bid induces mitochondrial apoptosis in synergy with Bax and Bak. In response to death receptors activation, Bid is cleaved by caspase-8 into its active form, tBid (truncated Bid), which then translocates to the mitochondria to trigger cytochrome c release and subsequent apoptosis. Accumulating evidence now indicate that the binding of tBid initiates an ordered sequences of events that prime mitochondria from the action of Bax and Bak: (1) tBid interacts with mitochondria via a specific binding to cardiolipin (CL) and immediately disturbs mitochondrial structure and function idependently of its BH3 domain; (2) Then, tBid activates through its BH3 domain Bax and/or Bak and induces their subsequent oligomerization in mitochondrial membranes. To date, the underlying mechanism responsible for targeting tBid to mitochondria and disrupting mitochondrial bioenergetics has yet be elucidated.
BH3 interacting domain death agonist (Bid), a pro-apoptotic member of the Bcl-2 family of proteins, is activated through cleavage by caspase-8. The active C-terminal fragment of Bid (tBid) translocates to the mitochondria where it interacts with cardiolipins at contact sites and induces the release of cytochrome c by a mechanism that is not yet fully understood. It has been shown that the alpha-helices alphaH6 and alphaH7 (which create the hairpin-forming domain of tBid) mediate the insertion of Bid into mitochondrial membranes and are essential for the cytochrome c-releasing activity. In the present study, we focused on the interaction between the alphaH6 and the mitochondrial membrane. By the use of single-cell electropermeabilization associated with flow cytometric analysis of intact cells, we demonstrated that H6 is able to induce cell death when used in the micromolar range. We also studied the interactions of the alphaH6 with artificial monolayers. We showed that the presence of negatively charged cardiolipins greatly enhances the insertion of alphaH6 into the phospholipid monolayer. The modification of two charged amino acid residues in alphaH6 abolished its insertion and also its in vivo effects. Furthermore, the negative values of the excess areas of mixing indicate that attractive interactions between cardiolipins and alphaH6 occur in the mixed monolayers. Fluorescence microscopy observations revealed that alphaH6 significantly disrupts cardiolipin packing and stabilizes the fluid lipid phase. These results suggest that cardiolipins at the contact sites between the two mitochondrial membranes could mediate the binding of tBid via alphaH6.
Oxysterols are oxidized forms of cholesterol. They have been shown to be implicated in cholesterol turnover, inflammation and in neurodegenerative diseases such as Alzheimers disease and multiple sclerosis. Glial cells are targets of oxysterols: they inhibit astrocyte proliferation after brain injury, and we have previously shown that 25-hydroxycholesterol (25OH) provokes oligodendrocyte apoptosis and stimulates the expression of sPLA2 type IIA (sPLA2-IIA), which has a protective effect.
In several neurodegenerative diseases of the CNS, oligodendrocytes are implicated in an inflammatory process associated with altered levels of oxysterols and inflammatory enzymes such as secreted phospholipase A2 (sPLA2). In view of the scarce literature related to this topic, we investigated oxysterol effects on these myelinating glial cells. Natural oxysterol 25-hydroxycholesterol (25-OH; 1 and 10 microM) altered oligodendrocyte cell line (158N) morphology and triggered apoptosis (75% of apoptosis after 72 h). These effects were mimicked by 22(S)-OH (1 and 10 microM) which does not activate liver X receptor (LXR) but not by a synthetic LXR ligand (T0901317). Therefore, oxysterol-induced apoptosis appears to be independent of LXR. Interestingly, sPLA2 type IIA (sPLA2-IIA) over-expression partially rescued 158N cells from oxysterol-induced apoptosis. In fact, 25-OH, 24(S)-OH, and T0901317 stimulated sPLA2-IIA promoter and sPLA2 activity in oligodendrocyte cell line. Accordingly, administration of T0901317 to mice enhanced sPLA2 activity in brain extracts by twofold. Short interfering RNA strategy allowed to establish that stimulation of sPLA2-IIA is mediated by pregnane X receptor (PXR) at high oxysterol concentration (10 microM) and by LXR beta at basal oxysterol concentration. Finally, GC coupled to mass spectrometry established that oligodendrocytes contain oxysterols and express their biosynthetic enzymes, suggesting that they may act through autocrine/paracrine mechanism. Our results show the diversity of oxysterol signalling in the CNS and highlight the positive effects of the LXR/PXR pathway which may open new perspectives in the treatment of demyelinating and neurodegenerative diseases.
The control of the functional pancreatic ?-cell mass serves the key homeostatic function of releasing the right amount of insulin to keep blood sugar in the normal range. It is not fully understood though how ?-cell mass is determined.
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