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Background: The mitochondrial unfolded protein response (UPR mt ) is an evolutionarily conserved mitochondrial response that is critical for maintaining mitochondrial and energetic homeostasis under cellular stress after tissue injury and disease. Here, we ask whether UPR mt may be a potential therapeutic target for ischemic stroke., Methods: We performed the middle cerebral artery occlusion and oxygen-glucose deprivation models to mimic ischemic stroke in vivo and in vitro, respectively. Oligomycin and meclizine were used to trigger the UPR mt . We used 2,3,5-triphenyltetrazolium chloride staining, behavioral tests, and Nissl staining to evaluate cerebral injury in vivo. The Cell Counting Kit-8 assay and the Calcein AM Assay Kit were conducted to test cerebral injury in vitro., Results: Inducing UPR mt with oligomycin protected neuronal cultures against oxygen-glucose deprivation. UPR mt could also be triggered with meclizine, and this Food and Drug Administration-approved drug also protected neurons against oxygen-glucose deprivation. Blocking UPR mt with siRNA against activating transcription factor 5 eliminated the neuroprotective effects of meclizine. In a mouse model of focal cerebral ischemia, pretreatment with meclizine was able to induce UPR mt in vivo, which reduced infarction and improved neurological outcomes., Conclusions: These findings suggest that the UPR mt is important in maintaining the survival of neurons facing ischemic/hypoxic stress. The UPR mt mechanism may provide a new therapeutic avenue for ischemic stroke., Competing Interests: Disclosures None.

Previous work demonstrated that hyperthermia (43°C for 2 h) results in delayed, apoptotic-like death in striatal neuronal cultures. We investigated early changes in mitochondrial function induced by this heat stress. Partial depolarization of the mitochondrial membrane potential (ΔΨ(m)) began about 1 h after the onset of hyperthermia and increased as the stress continued. When the heat stress ended, there was a partial recovery of ΔΨ(m), followed hours later by a progressive, irreversible depolarization of ΔΨ(m). During the heat stress, O(2) consumption initially increased but after 20-30 min began a progressive, irreversible decline to about one-half the initial rate by the end of the stress. The percentage of oligomycin-insensitive respiration increased during the heat stress, suggesting an increased mitochondrial leak conductance. Analysis using inhibitors and substrates for specific respiratory chain complexes indicated hyperthermia-induced dysfunction at or upstream of complex I. ATP levels remained near normal for ∼4 h after the heat stress. Mitochondrial movement along neurites was markedly slowed during and just after the heat stress. The early, persisting mitochondrial dysfunction described here likely contributes to the later (>10 h) caspase activation and neuronal death produced by this heat stress. Consistent with this idea, proton carrier-induced ΔΨ(m) depolarizations comparable in duration to those produced by the heat stress also reduced neuronal viability. Post-stress ΔΨ(m) depolarization and/or delayed neuronal death were modestly reduced/postponed by nicotinamide adenine dinucleotide, a calpain inhibitor, and increased expression of Bcl-xL.

The terminal step of ATP synthesis in intact mitochondria is catalyzed by the ATP synthase (F(0)F(1)) that works in close synchrony with the P(i) and ADP/ATP carriers. Each carrier consists of only a single polypeptide chain in dimeric form, while the ATP synthase is highly complex consisting in animals of 17 known subunit types and more than 30 total subunits. Although structures at high resolution have been obtained for the water-soluble F(1) part of the ATP synthase consisting of only five subunit types, such structures have not been obtained for either the complete ATP synthase or the P(i) and ADP/ATP carriers. Here, we report that all three proteins are localized in highly purified cristae-like vesicles obtained by extensive subfractionation of the mitochondrial inner membrane. Moreover, using a multiwell detergent screening assay, 4 nonionic detergents out of 80 tested were found to disperse these cristae-like vesicles into single soluble complexes or "ATP synthasomes" that contain the ATP synthase in association with the P(i) and ADP/ATP carriers. These studies offer new mechanistic insights into the terminal steps of oxidative phosphorylation in mitochondria and set the stage for future structural efforts designed to visualize in atomic detail the entire complex involved. They also provide evidence that the cristae are a subcompartment of the inner membrane.

Calphostin C-mediated apoptosis in glioma cells was reported previously to be associated with down-regulation of Bcl-2 and Bcl-xL. In this study, we report that 100 nM calphostin C also induces translocation and integration of monomeric Bax into mitochondrial membrane, followed by cytochrome c release into cytosol and subsequent decrease of mitochondrial inner membrane potential (DeltaPsim) before activation of caspase-3. The integration of monomeric Bax was associated with acquirement of alkali-resistance. The translocated monomeric Bax was partly homodimerized after cytochrome c release and decrease of DeltaPsim. The translocation and homodimerization of Bax, cytochrome c release, and decrease of DeltaPsim were not blocked by 100 microM z-VAD.fmk, a pan-caspase inhibitor, but the homodimerization of Bax and decrease of DeltaPsim were inhibited by 10 microM oligomycin, a mitochondrial F0F1-ATPase inhibitor. Therefore, it would be assumed that mitochondrial release of cytochrome c results from translocation and integration of Bax and is independent of permeability transition of mitochondria and caspase activation, representing a critical step in calphostin C-induced cell death.

Four cDNAs for the import precursors of oligomycin sensitivity conferring protein (OSCP), ATPase inhibitor protein (IF1) and subunit cs (encoded by P1 and P2 genes) of rat mitochondrial H(+)-ATP synthase have been cloned from a rat cDNA library. The import precursors and the mature polypeptides of rat OSCP, IF1, subunit c (P1) and subunit c (P2) consisted of 23/190, 25/82, 61/75 and 66/75 amino acids, respectively.

The presence of mitochondrial sex factor, omega, was demonstrated in haploid strains of yeast Saccharomyces cerevisiae which came from our laboratory. Transmission and recombination of the mitochondrial genes (CR/CS, ER/ES and OR/OS), conferring the resistance/sensitivity to chloramphenicol, erythromycin and oligomycin, respectively, were non-polar in homosexual crosses and highly polar in heterosexual crosses. Different results were obtained in crosses involving an erythromycin resistant mutant G706E11 (CSEROS) which was found to contain cellular DNA of diploid level. This strain was omega- and showed no alleles from G706E11 (CS, ER and OS) were transmitted to the zygote progeny in preference to the CR, ES and OR alleles. When crossed to omega+ haploid strains, there was a highly polar recombination, but no transmission was seen for the E and O alleles. Polar transmission of markers from omega+ haploid parental strain, characteristic of heterosexual crosses, was noticed only for the C allele. The crosses of G706E11 to omega+ haploids featured an increase in the recombination frequency. The values of % suppressiveness of sigma- petite mutants were relatively low when determined by crossing to G706E11 or to sigma+ diploid strain M2-8C rather than by crossing to sigma+ haploid strains, indicating that there is a positive correlation between the polar transmission of drug resistance markers and the suppressiveness degrees. Genetic mechanism of the anomalous behaviors if mitochondrial genes in crosses involving G706E11 was discussed and interpreted as due to an unbalanced supply of mitochondrial genomes from parental strains.

1. The excessive accumulation of Ca(2+) by mitochondria suspended in an iso-osmotic buffered potassium chloride medium containing oxidizable substrate and phosphate led to extensive swelling and release of accumulated Ca(2+) from the mitochondria. When the Ca(2+) was removed from the medium by chelation with ethylene glycol bis(aminoethyl)tetra-acetate, the swelling was reversed in a respiration-dependent contraction. The contracted mitochondria were shown to have regained some degree of respiratory control. 2. The respiration-dependent contraction could be supported by electron transport through a restricted portion of the respiratory chain, and by substrates donating electrons at different levels in the respiratory chain. 3. Respiratory inhibitors appropriate to the substrate present completely inhibited the contraction. Uncoupling agents, and the inhibitors oligomycin and atractyloside, were without effect. 4. When the reversal of swelling had been prevented by respiratory inhibitors, the addition of ATP induced a contraction of the mitochondria. In the absence of added chelating agent the contraction was very slow. The ATP-induced contraction was completely inhibited by oligomycin and atractyloside, was incomplete in the presence of uncoupling agents and was unaffected by respiratory inhibitors. 5. The relationship between the energy requirements of respiration-dependent contraction and the requirements of ion transport and other contractile systems are discussed.

1. Investigation of a number of reactions involving both internal and externally added adenine nucleotides of isolated liver mitochondria has revealed that atractylate and oligomycin differ markedly in the site of their inhibitory action. 2. Both atractylate and oligomycin inhibited the respiratory-chain-level phosphorylation of added ADP. Neither compound inhibited the substrate-level phosphorylation of internal (endogenous) ADP or the respiration-dependent accumulation of bivalent metal ions (Ca(2+), Sr(2+) or Mn(2+)). 3. Atractylate, but not oligomycin, inhibited the substrate-level phosphorylation of externally added ADP, the ATP- and carnitine-dependent reduction of nicotinamide nucleotide by palmitate and the ATP-induced activation of succinate oxidation. 4. Oligomycin, but not atractylate, inhibited the respiratory-chain-linked phosphorylation of internal ADP, and the dephosphorylation of internal ATP that occurred on the addition of antimycin. 5. The enhancement of arsenate-stimulated respiration by ADP was prevented by atractylate added either before or after the ADP. Oligomycin abolished both the arsenate and ADP stimulation. 6. It is suggested that atractylate prevents the passage of adenine nucleotides across the mitochondrial membrane, whereas oligomycin interferes with the formation of a ;high-energy' phosphorylated intermediate.