Brain contains a highly diversified complement of molecular species of a mitochondria-specific phospholipid, cardiolipin (CL), which - due to its polyunsaturation - can readily undergo oxygenation. Here, we used global lipidomics analysis in experimental traumatic brain injury (TBI) and showed that TBI was accompanied by oxidative consumption of polyunsaturated CL and accumulation of more than 150 new oxygenated molecular species in CL. RNAi-based manipulations of CL-synthase and CL levels conferred resistance of primary rat cortical neurons to mechanical stretch - an in vitro model of traumatic neuronal injury. By applying the novel brain permeable mitochondria-targeted electron-scavenger, we prevented CL oxygenation 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 CL oxygenation generates neuronal death signals and that its prevention by mitochondria-targeted small molecule inhibitors represents a new target for neuro-drug discovery.

BackgroundAging results in a progressive loss of skeletal muscle, a condition known as sarcopenia. Mitochondrial DNA (mtDNA) mutations accumulate with aging in skeletal muscle and correlate with muscle loss, although no causal relationship has been established.Methodology/Principal FindingsWe investigated the relationship between mtDNA mutations and sarcopenia at the gene expression and biochemical levels using a mouse model that expresses a proofreading-deficient version (D257A) of the mitochondrial DNA Polymerase γ, resulting in increased spontaneous mtDNA mutation rates. Gene expression profiling of D257A mice followed by Parametric Analysis of Gene Set Enrichment (PAGE) indicates that the D257A mutation is associated with a profound downregulation of gene sets associated with mitochondrial function. At the biochemical level, sarcopenia in D257A mice is associated with a marked reduction (35–50%) in the content of electron transport chain (ETC) complexes I, III and IV, all of which are partly encoded by mtDNA. D257A mice display impaired mitochondrial bioenergetics associated with compromised state-3 respiration, lower ATP content and a resulting decrease in mitochondrial membrane potential (Δψm). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage.Conclusions/SignificanceThese findings demonstrate that mutations in mtDNA can be causal in sarcopenia by affecting the assembly of functional ETC complexes, the lack of which provokes a decrease in oxidative phosphorylation, without an increase in oxidative stress, and ultimately, skeletal muscle apoptosis and sarcopenia.

Mammalian cytochrome c (Cytc) transfers electrons from the bc(1) complex to cytochrome c oxidase (CcO) as part of the mitochondrial electron transport chain, and it also participates in type II apoptosis. Our recent discovery of two tyrosine phosphorylation sites in Cytc, Tyr97 in bovine heart and Tyr48 in bovine liver, indicates that Cytc functions are regulated through cell signaling. To characterize the role of Cytc tyrosine phosphorylation in detail using an independent approach, we here overexpressed and purified a Tyr48Glu mutant Cytc, mimicking the in vivo Tyr48 phosphorylation found in cow liver, along with wild-type and Tyr48Phe variants as controls. The midpoint redox potential of the phosphomimetic mutant was decreased by 45 mV compared to control (192 vs 237 mV). Similar to Tyr48 in vivo phosphorylated Cytc, direct kinetic analysis of the Cytc reaction with isolated CcO revealed decreased V(max) for the Tyr48Glu mutant by 30% compared to wild type or the Tyr48Phe variants. Moreover, the phosphomimetic substitution resulted in major changes of Cytc functions related to apoptosis. The binding affinity of Tyr48Glu Cytc to cardiolipin was decreased by about 30% compared to wild type or the Tyr48Phe variants, and Cytc peroxidase activity of the Tyr48Glu mutant was cardiolipin-inducible only at high cardiolipin concentration, unlike controls. Importantly, the Tyr48Glu Cytc failed to induce any detectable downstream activation of caspase-3. Our data suggest that in vivo Tyr48 phosphorylation might serve as an antiapoptotic switch and highlight the strategic position and role of the conserved Cytc residue Tyr48 in regulating multiple functions of Cytc.

Hydrogen sulfide (H(2)S) concentration can be maintained in cell cultures within the range reported for rat brain by repetitive pulses of sodium hydrogen sulfide. Less than 2 h exposure to H(2)S concentrations within 50 and 120 microM (i.e., within the upper segment of the reported physiological range of H(2)S in rat brain), produces a large shift of the intracellular calcium homeostasis in cerebellar granule neurons (CGN) in culture, leading to a large and sustained increase of cytosolic calcium concentration. Only 1 h exposure to H(2)S concentrations within 100 and 300 microM raises intracellular calcium to the neurotoxic range, with nearly 50% cell death after 2 h. L-type Ca(2+) channels antagonists nimodipine and nifedipine block both the H(2)S-induced rise of cytosolic calcium and cell death. The N-methyl-D-aspartate receptor antagonists (+)-MK-801 and DL-2-amino-5-phosphonovaleric acid afforded a nearly complete protection against H(2)S-induced CGN death and largely attenuated the rise of cytosolic calcium. Thus, H(2)S-induced rise of cytosolic calcium eventually reaches the neurotoxic cytosolic calcium range, leading to glutamate-induced excitotoxic CGN death. The authors conclude that H(2)S is a major modulator of calcium homeostasis in neurons as it induces activation of Ca(2+) entry through L-type Ca(2+) channels, and thereby of neuronal activity.

The risk of radionuclide release in terrorist acts or exposure of healthy tissue during radiotherapy demand potent radioprotectants/radiomitigators. Ionizing radiation induces cell death by initiating the selective peroxidation of cardiolipin in mitochondria by the peroxidase activity of its complex with cytochrome c leading to release of hemoprotein into the cytosol and commitment to the apoptotic program. Here we design and synthesize mitochondria-targeted triphenylphosphonium-conjugated imidazole-substituted oleic and stearic acids which blocked peroxidase activity of cytochrome c/cardiolipin complex by specifically binding to its heme-iron. We show that both compounds inhibit pro-apoptotic oxidative events, suppress cyt c release, prevent cell death, and protect mice against lethal doses of irradiation. Significant radioprotective/radiomitigative effects of imidazole-substituted oleic acid are observed after pretreatment of mice from 1 hr before through 24 hrs after the irradiation.

Mitophagy is a selective autophagic process, essential for cellular homeostasis, that eliminates dysfunctional mitochondria. Activated by inner membrane depolarization, it plays an important role during development and is fundamental in highly differentiated post-mitotic cells that are highly dependent on aerobic metabolism, such as neurons, muscle cells, and hepatocytes. Both defective and excessive mitophagy have been proposed to contribute to age-related neurodegenerative diseases, such as Parkinson’s and Alzheimer’s diseases, metabolic diseases, vascular complications of diabetes, myocardial injury, muscle dystrophy, and liver disease, among others. Pharmacological or dietary interventions that restore mitophagy homeostasis and facilitate the elimination of irreversibly damaged mitochondria, thus, could serve as potential therapies in several chronic diseases. However, despite extraordinary advances in this field, mainly derived from in vitro and preclinical animal models, human applications based on the regulation of mitochondrial quality in patients have not yet been approved. In this review, we summarize the key selective mitochondrial autophagy pathways and their role in prevalent chronic human diseases and highlight the potential use of specific interventions.

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Cytochrome c (cyt c), a mitochondrial intermembrane electron shuttle between complexes III and IV, can, upon binding with an anionic phospholipid, cardiolipin (CL), act as a peroxidase that catalyzes cardiolipin oxidation. H(2)O(2) was considered as a source of oxidative equivalents for this reaction, which is essential for programmed cell death. Here we report that peroxidase cyt c/CL complexes can utilize free fatty acid hydroperoxides (FFA-OOH) at exceptionally high rates that are approximately 3 orders of magnitude higher than for H(2)O(2). Similarly, peroxidase activity of murine liver mitochondria was high with FFA-OOH. Using EPR spin trapping and LC-MS techniques, we have demonstrated that cyt c/CL complexes split FFA-OOH predominantly via a heterolytic mechanism, yielding hydroxy-fatty acids, whereas H(2)O(2) (and tert-butyl hydroperoxide, t-BuOOH) undergo homolytic cleavage. Computer simulations have revealed that Arg(38) and His(33) are important for the heterolytic mechanism at potential FFA-OOH binding sites of cyt c (but not for H(2)O(2) or t-BuOOH). Regulation of FFA-OOH metabolism may be an important function of cyt c that is associated with elimination of toxic FFA-OOH and synthesis of physiologically active hydroxy-fatty acids in mitochondria.