Vladimir A. Tyurin

Cardiolipin externalization to the outer mitochondrial membrane acts as an elimination signal for mitophagy in neuronal cells

Recognition of injured mitochondria for degradation by macroautophagy is essential for cellular health, but the mechanisms remain poorly understood. Cardiolipin is an inner mitochondrial membrane phospholipid. We found that rotenone, staurosporine, 6-hydroxydopamine and other pro-mitophagy stimuli caused externalization of cardiolipin to the mitochondrial surface in primary cortical neurons and SH-SY5Y cells. RNAi knockdown of cardiolipin synthase or of phospholipid scramblase-3, which transports cardiolipin to the outer mitochondrial membrane, decreased the delivery of mitochondria to autophagosomes. Furthermore, we found that the autophagy protein microtubule-associated-protein-1 light chain 3 (LC3), which mediates both autophagosome formation and cargo recognition, contains cardiolipin-binding sites important for the engulfment of mitochondria by the autophagic system. Mutation of LC3 residues predicted as cardiolipin-interaction sites by computational modelling inhibited its participation in mitophagy. These data indicate that redistribution of cardiolipin serves as an ‘eat-me’ signal for the elimination of damaged mitochondria from neuronal cells.

Cytochrome c/cardiolipin relations in mitochondria: a kiss of death

Recently, phospholipid peroxidation products gained a reputation as key regulatory molecules and participants in oxidative signaling pathways. During apoptosis, a mitochondria-specific phospholipid, cardiolipin (CL), interacts with cytochrome c (cyt c) to form a peroxidase complex that catalyzes CL oxidation; this process plays a pivotal role in the mitochondrial stage of the execution of the cell death program. This review is focused on redox mechanisms and essential structural features of cyt cs conversion into a CL-specific peroxidase that represent an interesting and maybe still unique example of a functionally significant ligand change in hemoproteins. Furthermore, specific characteristics of CL in mitochondria--its asymmetric transmembrane distribution and mechanisms of collapse, the regulation of its synthesis, remodeling, and fatty acid composition--are given significant consideration. Finally, new concepts in drug discovery based on the design of mitochondria-targeted inhibitors of cyt c/CL peroxidase and CL peroxidation with antiapoptotic effects are presented.

Inactivation of the ferroptosis regulator Gpx4 triggers acute renal failure in mice

Ferroptosis is a non-apoptotic form of cell death induced by small molecules in specific tumour types, and in engineered cells overexpressing oncogenic RAS. Yet, its relevance in non-transformed cells and tissues is unexplored and remains enigmatic. Here, we provide direct genetic evidence that the knockout of glutathione peroxidase 4 (Gpx4) causes cell death in a pathologically relevant form of ferroptosis. Using inducible Gpx4−/− mice, we elucidate an essential role for the glutathione/Gpx4 axis in preventing lipid-oxidation-induced acute renal failure and associated death. We furthermore systematically evaluated a library of small molecules for possible ferroptosis inhibitors, leading to the discovery of a potent spiroquinoxalinamine derivative called Liproxstatin-1, which is able to suppress ferroptosis in cells, in Gpx4−/− mice, and in a pre-clinical model of ischaemia/reperfusion-induced hepatic damage. In sum, we demonstrate that ferroptosis is a pervasive and dynamic form of cell death, which, when impeded, promises substantial cytoprotection.

Lipid accumulation and dendritic cell dysfunction in cancer

Dendritic cells (DCs), a type of professional antigen-presenting cells, are responsible for initiation and maintenance of immune responses. Here we report that a substantial proportion of DCs in tumor-bearing mice and people with cancer have high amounts of triglycerides as compared with DCs from tumor-free mice and healthy individuals. In our studies, lipid accumulation in DCs was caused by increased uptake of extracellular lipids due to upregulation of scavenger receptor A. DCs with high lipid content were not able to effectively stimulate allogeneic T cells or present tumor-associated antigens. DCs with high and normal lipid levels did not differ in expression of major histocompatibility complex and co-stimulatory molecules. However, lipid-laden DCs had a reduced capacity to process antigens. Pharmacological normalization of lipid abundance in DCs with an inhibitor of acetyl-CoA carboxylase restored the functional activity of DCs and substantially enhanced the effects of cancer vaccines. These findings suggest that immune responses in cancer can be improved by manipulating the lipid levels in DCs.

Oxidative stress following traumatic brain injury in rats: Quantitation of biomarkers and detection of free radical intermediates

Abstract: Oxidative stress may contribute to many pathophysiologic changes that occur after traumatic brain injury. In the current study, contemporary methods of detecting oxidative stress were used in a rodent model of traumatic brain injury. The level of the stable product derived from peroxidation of arachidonyl residues in phospholipids, 8‐epi‐prostaglandin F2α, was increased at 6 and 24 h after traumatic brain injury. Furthermore, relative amounts of fluorescent end products of lipid peroxidation in brain extracts were increased at 6 and 24 h after trauma compared with sham‐operated controls. The total antioxidant reserves of brain homogenates and water‐soluble antioxidant reserves as well as tissue concentrations of ascorbate, GSH, and protein sulfhydryls were reduced after traumatic brain injury. A selective inhibitor of cyclooxygenase‐2, SC 58125, prevented depletion of ascorbate and thiols, the two major water‐soluble antioxidants in traumatized brain. Electron paramagnetic resonance (EPR) spectroscopy of rat cortex homogenates failed to detect any radical adducts with a spin trap, 5,5‐dimethyl‐1‐pyrroline N‐oxide, but did detect ascorbate radical signals. The ascorbate radical EPR signals increased in brain homogenates derived from traumatized brain samples compared with sham‐operated controls. These results along with detailed model experiments in vitro indicate that ascorbate is a major antioxidant in brain and that the EPR assay of ascorbate radicals may be used to monitor production of free radicals in brain tissue after traumatic brain injury.

Assessment of Antioxidant Reserves and Oxidative Stress in Cerebrospinal Fluid after Severe Traumatic Brain Injury in Infants and Children

Studies in experimental traumatic brain injury (TBI) support a key role for oxidative stress. The degree of oxidative injury in clinical TBI, however, remains to be defined. We assessed antioxidant defenses and oxidative stress in pediatric TBI by applying a comprehensive battery of assays to cerebrospinal fluid samples. Using a protocol approved by our institutional review board, 87 cerebrospinal fluid samples from 11 infants and children with severe TBI (Glasgow Coma Scale score ≤8) and 8 controls were studied. Cerebrospinal fluid was drained as standard care after TBI. CSF was assessed on d 1, 2, and 5–7 after ventricular drain placement. Biochemical markers of oxidative stress included F2-isoprostane and protein sulfhydryl (detected by ELISA and fluorescence assay, respectively). Antioxidant defenses were measured by determination of total antioxidant reserve (via chemiluminescence assay), and ascorbate (via HPLC) and glutathione (via fluorescence assay) concentrations. Free radical production (ascorbate radical) was assessed by electron paramagnetic resonance spectroscopy. F2-isoprostane was markedly increased versus control, maximal on d 1 (93.8 ± 30.8 pg/mL versus 7.6 ± 5.1 pg/mL, p < 0.05). Total antioxidant reserve was reduced versus control. Reduction was maximal on d 5–7 (81.8 ± 3.7 μM versus 178.9 ± 2.2 μM, p < 0.05). Ascorbate was remarkably reduced (53.8 ± 8 μM versus 163.8 ± 21 μM on d 1, p < 0.05). Ascorbate depletion was likely associated with its free radical oxidation, as evidenced by electron paramagnetic resonance spectroscopy. Glutathione levels increased on d 1, then decreased versus control (0.19 ± 0.05 μM versus 1.2 ± 0.16 μM, p < 0.05). This is the first comprehensive study of antioxidant reserve and oxidative injury in clinical TBI. Progressive compromise of antioxidant defenses and evidence of free radical–mediated lipid peroxidation are noted. These markers could be used to monitor antioxidant strategies in clinical trials.

Lipidomics identifies cardiolipin oxidation as a mitochondrial target for redox therapy of brain injury

The brain contains a highly diversified complement of molecular species of a mitochondria-specific phospholipid, cardiolipin, which, because of its polyunsaturation, can readily undergo oxygenation. Using global lipidomics analysis in experimental traumatic brain injury (TBI), we found that TBI was accompanied by oxidative consumption of polyunsaturated cardiolipin and the accumulation of more than 150 new oxygenated molecular species of cardiolipin. RNAi-based manipulations of cardiolipin synthase and cardiolipin levels conferred resistance to mechanical stretch, an in vitro model of traumatic neuronal injury, in primary rat cortical neurons. By applying a brain-permeable mitochondria-targeted electron scavenger, we prevented cardiolipin oxidation in the brain, achieved a substantial reduction in neuronal death both in vitro and in vivo, and markedly reduced behavioral deficits and cortical lesion volume. We conclude that cardiolipin oxygenation generates neuronal death signals and that prevention of it by mitochondria-targeted small molecule inhibitors represents a new target for neuro-drug discovery.

Oxidative signaling pathway for externalization of plasma membrane phosphatidylserine during apoptosis

Active maintenance of membrane phospholipid asymmetry is universal in normal cell membranes and its disruption with subsequent externalization of phosphatidylserine is a hallmark of apoptosis. Externalized phosphatidylserine appears to serve as an important signal for targeting recognition and elimination of apoptotic cells by macrophages, however, the molecular mechanisms responsible for phosphatidylserine translocation during apoptosis remain unresolved. Studies have focused on the function of aminophospholipid translocase and phospholipid scramblase as mediators of this process. Here we present evidence that unique oxidative events, represented by selective oxidation of phosphatidylserine, occur during apoptosis that could promote phosphatidylserine externalization. We speculate that selective phosphatidylserine oxidation could affect phosphatidylserine recognition by aminophospholipid translocase and/or directly result in enzyme inhibition. The potential interactions between the anionic phospholipid phosphatidylserine and the redox‐active cationic protein effector of apoptosis, cytochrome c, are presented as a potential mechanism to account for selective oxidation of phosphatidylserine during apoptosis. Thus, cytochrome c‐mediated phosphatidylserine oxidation may represent an important component of the apoptotic pathway.

Selective early cardiolipin peroxidation after traumatic brain injury: an oxidative lipidomics analysis

Enhanced lipid peroxidation is well established in traumatic brain injury. However, its molecular targets, identity of peroxidized phospholipid species, and their signaling role have not been deciphered.

Antioxidant action of ubiquinol homologues with different isoprenoid chain length in biomembranes

Ubiquinones (CoQn) are intrinsic lipid components of many membranes. Besides their role in electron-transfer reactions they may act as free radical scavengers, yet their antioxidant function has received relatively little study. The efficiency of ubiquinols of varying isoprenoid chain length (from Q0 to Q10) in preventing (Fe2+ + ascorbate)-dependent or (Fe2+ + NADPH)-dependent lipid peroxidation was investigated in rat liver microsomes and brain synaptosomes and mitochondria. Ubiquinols, the reduced forms of CoQn, possess much greater antioxidant activity than the oxidized ubiquinone forms. In homogenous solution the radical scavenging activity of ubiquinol homologues does not depend on the length of their isoprenoid chain. However in membranes ubiquinols with short isoprenoid chains (Q1-Q4) are much more potent inhibitors of lipid peroxidation than the longer chain homologues (Q5-Q10). It is found that: i) the inhibitory action, that is, antioxidant efficiency of short-chain ubiquinols decreases in order Q1 greater than Q2 greater than Q3 greater than Q4; ii) the antioxidant efficiency of long-chain ubiquinols is only slightly dependent on their concentrations in the order Q5 greater than Q6 greater than Q7 greater than Q8 greater than Q9 greater than Q10 and iii) the antioxidant efficiency of Q0 is markedly less than that of other homologues. Interaction of ubiquinols with oxygen radicals was followed by their effects on luminol-activated chemiluminescence. Ubiquinols Q1-Q4 at 0.1 mM completely inhibit the luminol-activated NADPH-dependent chemiluminescent response of microsomes, while homologues Q6-Q10 exert no effect. In contrast to ubiquinol Q10 (ubiquinone Q10) ubiquinone Q1 synergistically enhances NADPH-dependent regeneration of endogenous vitamin E in microsomes thus providing for higher antioxidant protection against lipid peroxidation. The differences in the antioxidant potency of ubiquinols in membranes are suggested to result from differences in partitioning into membranes, intramembrane mobility and non-uniform distribution of ubiquinols resulting in differing efficiency of interaction with oxygen and lipid radicals as well as different efficiency of ubiquinols in regeneration of endogenous vitamin E.