Recruitment of pro-IL-1α to mitochondrial cardiolipin, via shared LC3 binding domain, inhibits mitophagy and drives maximal NLRP3 activation.

The balance between NLRP3 inflammasome activation and mitophagy is essential for homeostasis and cellular health, but this relationship remains poorly understood. Here we found that interleukin-1α (IL-1α)-deficient macrophages have reduced caspase-1 activity and diminished IL-1ß release, concurrent with reduced mitochondrial damage, suggesting a role for IL-1α in regulating this balance. LPS priming of macrophages induced pro-IL-1α translocation to mitochondria, where it directly interacted with mitochondrial cardiolipin (CL). Computational modeling revealed a likely CL binding motif in pro-IL-1α, similar to that found in LC3b. Thus, binding of pro-IL-1α to CL in activated macrophages may interrupt CL-LC3b-dependent mitophagy, leading to enhanced Nlrp3 inflammasome activation and more robust IL-1ß production. Mutation of pro-IL-1α residues predicted to be involved in CL binding resulted in reduced pro-IL-1α-CL interaction, a reduction in NLRP3 inflammasome activity, and increased mitophagy. These data identify a function for pro-IL-1α in regulating mitophagy and the potency of NLRP3 inflammasome activation.

Extracellular cardiolipin regulates select immune functions of microglia and microglia-like cells.

Cardiolipin is a mitochondrial membrane phospholipid with several well-defined metabolic roles. Cardiolipin can be released extracellularly by damaged cells and has been shown to affect peripheral immune functions. We hypothesized that extracellular cardiolipin can also regulate functions of microglia, the resident immune cells of the central nervous system (CNS). We demonstrate that extracellular cardiolipin increases microglial phagocytosis and neurotrophic factor expression, as well as decreases the release of inflammatory mediators and cytotoxins by activated microglia-like cells. These results identify extracellular cardiolipin as a potential CNS intercellular signaling molecule that can regulate key microglial immune functions associated with neurodegenerative diseases.

The mito-DAMP cardiolipin blocks IL-10 production causing persistent inflammation during bacterial pneumonia.

Bacterial pneumonia is a significant healthcare burden worldwide. Failure to resolve inflammation after infection precipitates lung injury and an increase in morbidity and mortality. Gram-negative bacteria are common in pneumonia and increased levels of the mito-damage-associated molecular pattern (DAMP) cardiolipin can be detected in the lungs. Here we show that mice infected with Klebsiella pneumoniae develop lung injury with accumulation of cardiolipin. Cardiolipin inhibits resolution of inflammation by suppressing production of anti-inflammatory IL-10 by lung CD11b+Ly6GintLy6CloF4/80+ cells. Cardiolipin induces PPARγ SUMOylation, which causes recruitment of a repressive NCOR/HDAC3 complex to the IL-10 promoter, but not the TNF promoter, thereby tipping the balance towards inflammation rather than resolution. Inhibition of HDAC activity by sodium butyrate enhances recruitment of acetylated histone 3 to the IL-10 promoter and increases the concentration of IL-10 in the lungs. These findings identify a mechanism of persistent inflammation during pneumonia and indicate the potential of HDAC inhibition as a therapy.

Reduction in cardiolipin decreases mitochondrial spare respiratory capacity and increases glucose transport into and across human brain cerebral microvascular endothelial cells.

Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. Cardiolipin is a mitochondrial phospholipid required for function of the electron transport chain and ATP generation. We examined the role of cardiolipin in maintaining mitochondrial function necessary to support barrier properties of brain microvessel endothelial cells. Knockdown of the terminal enzyme of cardiolipin synthesis, cardiolipin synthase, in hCMEC/D3 cells resulted in decreased cellular cardiolipin levels compared to controls. The reduction in cardiolipin resulted in decreased mitochondrial spare respiratory capacity, increased pyruvate kinase activity, and increased 2-deoxy-[(3) H]glucose uptake and glucose transporter-1 expression and localization to membranes in hCMEC/D3 cells compared to controls. The mechanism for the increase in glucose uptake was an increase in adenosine-5'-monophosphate kinase and protein kinase B activity and decreased glycogen synthase kinase 3 beta activity. Knockdown of cardiolipin synthase did not affect permeability of fluorescent dextran across confluent hCMEC/D3 monolayers grown on Transwell(®) inserts. In contrast, knockdown of cardiolipin synthase resulted in an increase in 2-deoxy-[(3) H]glucose transport across these monolayers compared to controls. The data indicate that in hCMEC/D3 cells, spare respiratory capacity is dependent on cardiolipin. In addition, reduction in cardiolipin in these cells alters their cellular energy status and this results in increased glucose transport into and across hCMEC/D3 monolayers. Microvessel endothelial cells form part of the blood-brain barrier, a restrictively permeable interface that allows transport of only specific compounds into the brain. In human adult brain endothelial cell hCMEC/D3 monolayers cultured on Transwell(®) plates, knockdown of cardiolipin synthase results in decrease in mitochondrial cardiolipin and decreased mitochondrial spare respiratory capacity. The reduced cardiolipin results in an increased activity of adenosine monophosphate kinase (pAMPK) and protein kinase B (pAKT) and decreased activity of glycogen synthase kinase 3 beta (pGSK3ß) which results in elevated glucose transporter-1 (GLUT-1) expression and association with membranes. This in turn increases 2-dexoyglucose uptake from the apical medium into the cells with a resultant 2-deoxyglucose movement into the basolateral medium.

Cell cycle arrest and cell survival induce reverse trends of cardiolipin remodeling.

Cell survival from the arrested state can be a cause of the cancer recurrence. Transition from the arrest state to the growth state is highly regulated by mitochondrial activity, which is related to the lipid compositions of the mitochondrial membrane. Cardiolipin is a critical phospholipid for the mitochondrial integrity and functions. We examined the changes of cardiolipin species by LC-MS in the transition between cell cycle arrest and cell reviving in HT1080 fibrosarcoma cells. We have identified 41 cardiolipin species by MS/MS and semi-quantitated them to analyze the detailed changes of cardiolipin species. The mass spectra of cardiolipin with the same carbon number form an envelope, and the C64, C66, C68, C70 C72 and C74 envelopes in HT1080 cells show a normal distribution in the full scan mass spectrum. The cardiolipin quantity in a cell decreases while entering the cell cycle arrest, but maintains at a similar level through cell survival. While cells awakening from the arrested state and preparing itself for replication, the groups with short acyl chains, such as C64, C66 and C68 show a decrease of cardiolipin percentage, but the groups with long acyl chains, such as C70 and C72 display an increase of cardiolipin percentage. Interestingly, the trends of the cardiolipin species changes during the arresting state are completely opposite to cell growing state. Our results indicate that the cardiolipin species shift from the short chain to long chain cardiolipin during the transition from cell cycle arrest to cell progression.

Serendipity and the discovery of novel compounds that restore mitochondrial plasticity.

The mitochondrial electron transport chain (ETC) plays a central role in energy generation in the cell. Mitochondrial dysfunctions diminish adenosine triphosphate (ATP) production and result in insufficient energy to maintain cell function. As energy output declines, the most energetic tissues are preferentially affected. To satisfy cellular energy demands, the mitochondrial ETC needs to be able to elevate its capacity to produce ATP at times of increased metabolic demand or decreased fuel supply. This mitochondrial plasticity is reduced in many age-associated diseases. In this review, we describe the serendipitous discovery of a novel class of compounds that selectively target cardiolipin on the inner mitochondrial membrane to optimize efficiency of the ETC and thereby restore cellular bioenergetics in aging and diverse disease models, without any effect on the normal healthy organism. The first of these compounds, SS-31, is currently in multiple clinical trials.

Emerging roles of lipids in BCL-2 family-regulated apoptosis.

Apoptosis is an intricately regulated process required for the health and homeostasis of living systems. The mitochondrial apoptotic pathway depends on the BCL-2 family of pro- and anti-apoptotic members whose interactions form a complex network of checks and balances in regulating cell fate. A diverse set of signals recruits distinct BH3-domain only BCL-2 proteins to trigger activation of the executioner proteins BAX and BAK. In addition to protein components of the apoptotic machinery, literature of the past several decades supports crucial functions for lipids in apoptosis and cooperation between lipid metabolism and BCL-2 proteins. In this review we present the two key examples of ceramide and cardiolipin in apoptosis, focusing particularly on BCL-2 family-regulated pathways at the mitochondrial level. This article is part of a Special Issue entitled Lipid Metabolism in Cancer.

The stability and activity of respiratory Complex II is cardiolipin-dependent.

Respiratory Complex II of the mitochondrial inner membrane serves as a link between the tricarboxylic acid cycle and the electron transport chain. Complex II dysfunction has been implicated in a wide range of heritable mitochondrial diseases, including cancer, by a mechanism that likely involves the production of reactive oxygen species (ROS). Using Complex II enzymes reconstituted into nanoscale lipid bilayers (nanodiscs) with varying lipid composition, we demonstrate for the first time that the phospholipid environment, specifically the presence of cardiolipin, is critical for the assembly and enzymatic activity of the complex, as well as in the curtailment of ROS production.

Dynamic regulation of cardiolipin by the lipid pump Atp8b1 determines the severity of lung injury in experimental pneumonia.

Pneumonia remains the leading cause of death from infection in the US, yet fundamentally new conceptual models underlying its pathogenesis have not emerged. We show that humans and mice with bacterial pneumonia have markedly elevated amounts of cardiolipin, a rare, mitochondrial-specific phospholipid, in lung fluid and find that it potently disrupts surfactant function. Intratracheal cardiolipin administration in mice recapitulates the clinical phenotype of pneumonia, including impaired lung mechanics, modulation of cell survival and cytokine networks and lung consolidation. We have identified and characterized the activity of a unique cardiolipin transporter, the P-type ATPase transmembrane lipid pump Atp8b1, a mutant version of which is associated with severe pneumonia in humans and mice. Atp8b1 bound and internalized cardiolipin from extracellular fluid via a basic residue-enriched motif. Administration of a peptide encompassing the cardiolipin binding motif or Atp8b1 gene transfer in mice lessened bacteria-induced lung injury and improved survival. The results unveil a new paradigm whereby Atp8b1 is a cardiolipin importer whose capacity to remove cardiolipin from lung fluid is exceeded during inflammation or when Atp8b1 is defective. This discovery opens the door for new therapeutic strategies directed at modulating the abundance or molecular interactions of cardiolipin in pneumonia.

Peroxidase mechanism of lipid-dependent cross-linking of synuclein with cytochrome C: protection against apoptosis versus delayed oxidative stress in Parkinson disease.

Damage of presynaptic mitochondria could result in release of proapoptotic factors that threaten the integrity of the entire neuron. We discovered that alpha-synuclein (Syn) forms a triple complex with anionic lipids (such as cardiolipin) and cytochrome c, which exerts a peroxidase activity. The latter catalyzes covalent hetero-oligomerization of Syn with cytochrome c into high molecular weight aggregates. Syn is a preferred substrate of this reaction and is oxidized more readily than cardiolipin, dopamine, and other phenolic substrates. Co-localization of Syn with cytochrome c was detected in aggregates formed upon proapoptotic stimulation of SH-SY5Y and HeLa cells and in dopaminergic substantia nigra neurons of rotenone-treated rats. Syn-cardiolipin exerted protection against cytochrome c-induced caspase-3 activation in a cell-free system, particularly in the presence of H(2)O(2). Direct delivery of Syn into mouse embryonic cells conferred resistance to proapoptotic caspase-3 activation. Conversely, small interfering RNA depletion of Syn in HeLa cells made them more sensitive to dopamine-induced apoptosis. In human Parkinson disease substantia nigra neurons, two-thirds of co-localized Syn-cytochrome c complexes occurred in Lewy neurites. Taken together, these results indicate that Syn may prevent execution of apoptosis in neurons through covalent hetero-oligomerization of cytochrome c. This immediate protective function of Syn is associated with the formation of the peroxidase complex representing a source of oxidative stress and postponed damage.

Mitochondria-targeted disruptors and inhibitors of cytochrome c/cardiolipin peroxidase complexes: a new strategy in anti-apoptotic drug discovery.

The critical role of mitochondria in programmed cell death leads to the design of mitochondriotropic agents as a strategy in regulating apoptosis. For anticancer therapy, stimulation of proapoptotic mitochondrial events in tumor cells and their suppression in surrounding normal cells represents a promising paradigm for new therapies. Different approaches targeting regulation of components of mitochondrial antioxidant system such as Mn-SOD demonstrated significant antitumor efficiency, particularly in combination therapy. This review is focused on a newly discovered early stage of mitochondria-dependent apoptosis - oxidative lipid signaling involving a mitochondria-specific phospholipid cardiolipin (CL). Cytochrome c (cyt c) acts as a CL-specific peroxidase very early in apoptosis. At this stage, the hostile events are still secluded within the mitochondria and do not reach the cytosolic targets. CL oxidation process is required for the release of pro-apoptotic factors into the cytosol. Manipulation of cyt c interactions with CL, inhibition of peroxidase activity, and prevention of CL peroxidation are prime targets for the discovery of anti-apoptotic drugs acting before the "point-of-no-return" in the fulfillment of the cell death program. Therefore, mitochondria-targeted disruptors and inhibitors of cyt c/CL peroxidase complexes and suppression of CL peroxidation represent new strategies in anti-apoptotic drug discovery.

Cardiolipin deficiency leads to decreased cardiolipin peroxidation and increased resistance of cells to apoptosis.

Cardiolipin (CL), a unique mitochondrial phospholipid synthesized by CL synthase (CLS), plays important, yet not fully understood, roles in mitochondria-dependent apoptosis. We manipulated CL levels in HeLa cells by knocking down CLS using RNA interference and selected a clone of CL-deficient cells with approximately 45% of its normal content. ESI-MS analysis showed that the CL molecular species were the same in CL-deficient and CL-sufficient cells. CL deficiency did not change mitochondrial functions (membrane potential, reactive oxygen species generation, cellular ATP levels) but conferred resistance to apoptosis induced by actinomycin D (ActD), rotenone, or gamma-irradiation. During ActD-induced apoptosis, decreased CL peroxidation along with suppressed cytochrome (cyt) c release was observed in CL-deficient cells, whereas Bax translocation to mitochondria remained similar to that in CL-sufficient HeLa cells. The amounts of loosely bound cyt c (releasable under high ionic strength conditions) were the same in CL-deficient and CL-sufficient cells. Given that CL peroxidation during apoptosis is catalyzed by CL/cyt c complexes and CL oxidation products are essential for cyt c release from mitochondria, our results suggest that CL deficiency prevents adequate assembly of productive CL/cyt c complexes and CL peroxidation, resulting in increased resistance to apoptosis.

Cardiolipin clusters and membrane domain formation induced by mitochondrial proteins.

We show in this study that mitochondrial creatine kinase promotes segregation and clustering of cardiolipin in mixed membranes, a phenomenon that has been proposed to occur at contact sites in the mitochondria. This property of mitochondrial creatine kinase is dependent on the native octameric structure of the protein and does not occur after heat-denaturation or with the native dimeric form of the protein. Cardiolipin segregation was demonstrated by differential scanning calorimetry using membranes containing cardiolipin and either dipalmitoylphosphatidylethanolamine or 1-palmitoyl-2-oleoylphosphatidylethanolamine. Addition of the ubiquitous form of mitochondrial creatine kinase leads to the formation of a phosphatidylethanolamine-rich domain as a result of the protein binding preferentially to the cardiolipin. Such phase separation does not occur if cardiolipin is replaced with dioleoyl phosphatidylglycerol. Lipid phase separation is observed with other cardiolipin-binding proteins, including cytochrome c and, to a very small extent, with truncated Bid (t-Bid), as well as with the cationic polypeptide poly-L-lysine, but among these proteins the octameric form of mitochondrial creatine kinase is by far the most effective in causing segregation and clustering of cardiolipin. The proteins included in this study are found at mitochondrial contact sites where they are known to associate with cardiolipin. Domains in mitochondria enriched in cardiolipin play an important role in apoptosis and in energy flux processes.

[The role of ion channels in apoptosis]. / Rola kanalów jonowych w procesie apoptozy.

The plasma membrane as well as the mitochondrial outer and inner membranes contain a number of ion channels that are responsible not only for existence of cells under physiological conditions but they also participate directly in apoptosis. In the apoptotic cells the activated K+, Cl- channels of plasma membrane control the cell volume and mediate the regulation of protease and nuclease activities. The mitochondrial channels are involved in the ionic movements and leakage of apoptogenic factors from the intermembrane space to cytosol. During apoptosis, an important role in the permeabilization of the outer mitochondrial membrane play Bcl-2 family proteins. In this review the recent findings on the function of ion channels in apoptotic cells and the role played by Bcl-2 proteins in the control of apoptosis are discussed.

Newcomers in the process of mitochondrial permeabilization.

Under stress conditions, apoptogenic factors normally sequestered in the mitochondrial intermembrane space are released into the cytosol, caspases are activated and cells die by apoptosis. Although the precise mechanism that leads to the permeabilization of mitochondria is still unclear, the activation of multidomain pro-apoptotic proteins of the Bcl-2 family, such as Bax and Bak, is evidently crucial. Regulation of Bax and Bak by other members of the family has been known for a long time, but recent evidence suggests that additional unrelated proteins participate in the process, both as inhibitors and activators. The important rearrangements mitochondrial lipids undergo during apoptosis play a role in the permeabilization process and this role is probably more central than first envisioned.

Anticancer drug development based on modulation of the Bcl-2 family core apoptosis mechanism.

Cancer cells generally maintain their survival by suppressing apoptosis. Mitochondrial mechanisms are involved in most forms of apoptosis (referred to as mitochondrial apoptosis), and the Bcl-2 family controls apoptosis at the mitochondrion via a balance of the effects of pro- and antiapoptotic members. Antiapoptotic molecules such as Bcl-2 and Bcl-x(L) are often overexpressed in cancer cells and their inhibition is an attractive target for selective killing of tumor cells via induction of apoptosis. Reduction of the levels of these proteins with antisense molecules has shown encouraging experimental and clinical results and there has been some success in developing small-molecule inhibitors. These are likely to be the most productive drug development approaches in the near future. However, growing understanding of the molecular mechanisms involved has identified other potential targets. Cardiolipin is important for the proapoptotic activity of Bcl-2 family members such as Bid, Bax and Bak, and modulation of its metabolism and translocation in mitochondrial membranes could potentially have a strong influence on apoptosis. Post-translational modifications strongly influence the activity of Bcl-2 family members. Several molecules have been identified that bind to Bcl-2 family members and could be intracellular control mechanisms. These mechanisms may yield several drug development targets for the induction of apoptosis. Further research will qualify these targets and, in the longer term, could lead to a more specific means of inducing apoptosis in cancer cells.

Cell membranes and apoptosis: role of cardiolipin, phosphatidylcholine, and anticancer lipid analogues.

The apoptotic program utilizes cellular membranes to transduce and generate operative signals. Lipids are major components of cellular membranes and have the potential to control the effectiveness of the signal by directing it to the proper location, being a source of new signals or as mediators in the response. These possible lipid functions are illustrated in the present review, focussing on the role that two different phospholipids, cardiolipin and phosphatidyl choline, play in apoptosis. Mitochondria have a central role in apoptosis, and many important aspects of the process mediated by this organelle converge through its distinctive lipid cardiolipin. Specifically, changes in cardiolipin metabolism have been detected in early steps of the death program and it is postulated (i) to mediate recruitment of pro apoptotic proteins like Bid to the mitochondria surface and (ii) to actively participate in the release of proteins relevant for the execution phase of apoptosis, like cytochrome c. Unlike the organelle specific distribution of cardiolipin, phosphatidylcholine is widely distributed among all organelles of the cell. The importance of phosphatidylcholine in apoptosis has been approached mainly through the study of the mode of action of (i) phosphatidylcholine anticancer analogues such as edelfosine and (ii) molecules that alter phosphatidylcholine metabolism, such as farnesol. The contribution of phosphatidylcholine metabolism to the apoptotic program is discussed, analyzing the experimental evidence available and pointing out some controversies in the proposed mechanisms of action.

Cardiolipin is not required for Bax-mediated cytochrome c release from yeast mitochondria.

Cardiolipin (CL) is an inner mitochondrial membrane phospholipid that contributes to optimal mitochondrial function and is gaining widespread attention in studies of mitochondria-mediated apoptosis. Divergent hypotheses describing the role of CL in cytochrome c release and apoptosis have evolved. We addressed this controversy directly by comparing the spontaneous- and Bax-mediated cytochrome c release from mitochondria isolated from two strains of Saccharomyces cerevisiae: one lacking CL-synthase and therefore CL (DeltaCRD1) and the other, its corresponding wild type (WT). We demonstrated by liquid chromatography-mass spectrometry that the main yeast CL species [(16:1)2(18:1)2] differs in fatty acid composition from mammalian CL [(18:2)4], and we verified the absence of the yeast CL species in the DeltaCRD1 strain. We also demonstrated that the mitochondrial association of Bax and the resulting cytochrome c release is not dependent on the CL content of the yeast mitochondrial membranes. Bax inserted equally into both WT and DeltaCRD1 mitochondrial membranes under conditions that lead to the release of cytochrome c from both strains of yeast mitochondria. Furthermore, using models of synthetic liposomes and isolated yeast mitochondria, we found that cytochrome c was bound more "loosely" to the CL-deficient systems compared with when CL is present. These data challenge recent studies implicating that CL is required for Bax-mediated pore formation leading to the release of proteins from the mitochondrial intermembrane space. In contrast, they support our recently proposed two-step mechanism of cytochrome c release, which suggests that CL is required for binding cytochrome c to the inner mitochondrial membrane.