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Mitochondrial isocitrate dehydrogenase 2 (IDH2) converts NADP + to NADPH and promotes regeneration of reduced glutathione (GSH) by supplying NADPH to glutathione reductase or thioredoxin reductase. We have previously shown that under calorie restriction, mitochondrial deacetylase Sirt3 deacetylates and activates IDH2, thereby regulating the mitochondrial glutathione antioxidant defense system in mice. To investigate the regulatory mechanism of mIDH2 (mouse mitochondrial IDH2), we used lysine-to-glutamine (KQ) mutants to mimic acetylated lysines and screened 15 KQ mutants. Among these mutants, the activities of the K256Q and K413Q proteins were less than 50% of the wild-type value. We then solved the crystal structures of the wild-type mIDH2 and the K256Q mutant proteins, revealing conformational changes in the substrate-binding pocket. Structural data suggested that positively charged Lys256 was important in stabilizing the pocket because it repelled a lysine cluster on the other side. Glutamine (or acetylated lysine) was neutral and thus caused the pocket size to decrease, which might be the main reason for the lower activity of the K256Q mutant. Together, our data provide the first structure of an acetylation mimic of mIDH2 and new insights into the regulatory mechanism of acetylation of mIDH2.

Age-related hearing loss (AHL), known as presbycusis, is a universal feature of mammalian aging and is the most common sensory disorder in the elderly population. The molecular mechanisms underlying AHL are unknown, and currently there is no treatment for the disorder. Here we report that C57BL/6J mice with a deletion of the mitochondrial pro-apoptotic gene Bak exhibit reduced age-related apoptotic cell death of spiral ganglion neurons and hair cells in the cochlea, and prevention of AHL. Oxidative stress induces Bak expression in primary cochlear cells, and Bak deficiency prevents apoptotic cell death. Furthermore, a mitochondrially targeted catalase transgene suppresses Bak expression in the cochlea, reduces cochlear cell death, and prevents AHL. Oral supplementation with the mitochondrial antioxidants α-lipoic acid and coenzyme Q₁₀ also suppresses Bak expression in the cochlea, reduces cochlear cell death, and prevents AHL. Thus, induction of a Bak-dependent mitochondrial apoptosis program in response to oxidative stress is a key mechanism of AHL in C57BL/6J mice.

Cisplatin is one of the most widely used chemotherapeutic drugs for the treatment of cancer. Unfortunately, one of its major side effects is permanent hearing loss. Here, we show that glutathione transferase α4 (GSTA4), a member of the Phase II detoxifying enzyme superfamily, mediates reduction of cisplatin ototoxicity by removing 4-hydroxynonenal (4-HNE) in the inner ears of female mice. Under cisplatin treatment, loss of Gsta4 results in more profound hearing loss in female mice compared to male mice. Cisplatin stimulates GSTA4 activity in the inner ear of female wild-type, but not male wild-type mice. In female Gsta4 −/− mice, cisplatin treatment results in increased levels of 4-HNE in cochlear neurons compared to male Gsta4 −/− mice. In CBA/CaJ mice, ovariectomy decreases mRNA expression of Gsta4 , and the levels of GSTA4 protein in the inner ears. Thus, our findings suggest that GSTA4-dependent detoxification may play a role in estrogen-mediated neuroprotection. A common complication of cisplatin-based chemotherapy is hearing loss. Here, Park et al. show that glutathione transferase α4 (GSTA4) contributes to reducing cisplatin toxicity in the inner ear of female mice by removing 4-hydroxynonenal (4-HNE).

Rapamycin, an mTOR inhibitor, has been shown to extend lifespan in a range of model organisms. It has been reported to extend lifespan in multiple strains of mice, administered chronically or acutely early or late in life. The ability of rapamycin to extend health (healthspan) as opposed to life is less well documented. To assess the effects chronic rapamycin treatment on healthspan, enteric rapamycin was given to male and female C57BL/6J mice starting at 4 months of age and continued throughout life. Repeated, longitudinal assessments of health in individual animals were made starting at 16 months of age (=12 months of treatment) until death. A number of health parameters were improved (female grip strength, female body mass and reduced sleep fragmentation in both sexes), others showed no significant difference, while at least one (male rotarod performance) was negatively affected. Rapamycin treatment affected many measures of health in a highly sex-specific manner. While sex-specific phenotypic effects of rapamycin treatment have been widely reported, in this study we document sex differences in the direction of phenotypic change. Rapamycin-fed males and females were both significantly different from controls; however the differences were in the opposite direction in measures of body mass, percent fat and resting metabolic rate, a pattern not previously reported.

Isocitrate dehydrogenase (IDH) 2 participates in the TCA cycle and catalyzes the conversion of isocitrate to α-ketoglutarate and NADP + to NADPH. In the mitochondria, IDH2 also plays a key role in protecting mitochondrial components from oxidative stress by supplying NADPH to both glutathione reductase (GSR) and thioredoxin reductase 2 (TXNRD2). Here, we report that loss of Idh2 accelerates age-related hearing loss, the most common form of hearing impairment, in male mice. This was accompanied by increased oxidative DNA damage, increased apoptotic cell death, and profound loss of spiral ganglion neurons and hair cells in the cochlea of 24-month-old Idh2 −/− mice. In young male mice, loss of Idh2 resulted in decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the inner ear. In HEI-OC1 mouse inner ear cell lines, knockdown of Idh2 resulted in a decline in cell viability and mitochondrial oxygen consumption. This was accompanied by decreased NADPH redox state and decreased activity of TXNRD2 in the mitochondria of the HEI-OC1 cells. Therefore, IDH2 functions as the principal source of NADPH for the mitochondrial thioredoxin antioxidant defense and plays an essential role in protecting hair cells and neurons against oxidative stress in the cochlea of male mice.

Glutathione reductase (GSR), a key member of the glutathione antioxidant defense system, converts oxidized glutathione (GSSG) to reduced glutathione (GSH) and maintains the intracellular glutathione redox state to protect the cells from oxidative damage. Previous reports have shown that Gsr deficiency results in defects in host defense against bacterial infection, while diquat induces renal injury in Gsr hypomorphic mice. In flies, overexpression of GSR extended lifespan under hyperoxia. In the current study, we investigated the roles of GSR in cochlear antioxidant defense using Gsr homozygous knockout mice that were backcrossed onto the CBA/CaJ mouse strain, a normal-hearing strain that does not carry a specific Cdh23 mutation that causes progressive hair cell degeneration and early onset of hearing loss. Gsr-/- mice displayed a significant decrease in GSR activity and GSH/GSSG ratios in the cytosol of the inner ears. However, Gsr deficiency did not affect ABR (auditory brainstem response) hearing thresholds, wave I amplitudes or wave I latencies in young mice. No histological abnormalities were observed in the cochlea of Gsr-/- mice. Furthermore, there were no differences in the activities of cytosolic glutathione-related enzymes, including glutathione peroxidase and glutamate-cysteine ligase, or the levels of oxidative damage markers in the inner ears between WT and Gsr-/- mice. In contrast, Gsr deficiency resulted in increased activities of cytosolic thioredoxin and thioredoxin reductase in the inner ears. Therefore, under normal physiological conditions, GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea. Given that the thioredoxin system is known to reduce GSSG to GSH in multiple species, our findings suggest that the thioredoxin system can support GSSG reduction in the mouse peripheral auditory system.

Aging 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. We 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 gamma, 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 (Deltapsim). Surprisingly, mitochondrial dysfunction was not accompanied by an increase in mitochondrial reactive oxygen species (ROS) production or oxidative damage. These 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.

Non-alcoholic fatty liver disease (NAFLD) is a common metabolic disorder in obese individuals. Adenine nucleotide translocase (ANT) exchanges ADP/ATP through the mitochondrial inner membrane, and Ant2 is the predominant isoform expressed in the liver. Here we demonstrate that targeted disruption of Ant2 in mouse liver enhances uncoupled respiration without damaging mitochondrial integrity and liver functions. Interestingly, liver specific Ant2 knockout mice are leaner and resistant to hepatic steatosis, obesity and insulin resistance under a lipogenic diet. Protection against fatty liver is partially recapitulated by the systemic administration of low-dose carboxyatractyloside, a specific inhibitor of ANT. Targeted manipulation of hepatic mitochondrial metabolism, particularly through inhibition of ANT, may represent an alternative approach in NAFLD and obesity treatment. Adenine nucleotide translocase (ANT) 2 promotes ADP/ATP exchange across the mitochondrial inner membrane. Cho et al . show that liver specific Ant2 deletion increases uncoupled respiration and protects mice against fatty liver and obesity-induced insulin resistance.

Usher's syndrome is the most common combined blindness-deafness disorder with USH1B, caused by mutations in , resulting in the most severe phenotype. The existence of numerous, naturally occurring mice harboring variable mutations on different genetic backgrounds has complicated the characterization of MYO7A knockout (KO) and heterozygote mice. We generated a novel MYO7A KO mouse ( ) that is easily genotyped, maintained, and confirmed to be null for MYO7A in both the eye and inner ear. Like USH1B patients, mice are profoundly deaf, and display near complete loss of inner and outer cochlear hair cells (HCs). No gross structural changes were observed in vestibular HCs. mice exhibited modest declines in retinal function but, unlike patients, no loss of retinal structure. We attribute the latter to differential expression of MYO7A in mouse vs. primate retina. Interestingly, heterozygous mice had reduced numbers of cochlear HCs and concomitant reductions in auditory function relative to controls. Notably, this is the first report that loss of a single allele significantly alters auditory structure and function and suggests that audiological characterization of USH1B carriers is warranted. Maintenance of vestibular HCs in mice suggests that gene replacement could be used to correct the vestibular dysfunction in USH1B patients. While mice do not exhibit sufficiently robust retinal phenotypes to be used as a therapeutic outcome measure, they can be used to assess expression of vectored on a null background and generate valuable pre-clinical data toward the treatment of USH1B.

Background Mechanical ventilation (MV) during a cardio-thoracic surgery contributes to diaphragm muscle dysfunction that impairs weaning and can lead to the ventilator- induced diaphragm dysfunction. Especially, it is critical in older adults who have lower muscle reparative capacity following MV. Reports have shown that the intraoperative intermittent hemidiaphragm electrical stimulation can maintain and/or improve post-surgery diaphragm function. In particular, from a molecular point of view, intermittent electrical stimulation (ES) may reduce oxidative stress and increase regulatory autophagy levels, and therefore improve diaphragm function in animal studies. We have recently shown in humans that intraoperative ES attenuates mitochondrial dysfunction and force decline in single diaphragm muscle fibers. The aim of this study was to investigate an effect of ES on oxidative stress, antioxidant status and autophagy biomarker levels in the human diaphragm during surgery. Methods One phrenic nerve was simulated with an external cardiac pacer in operated older subjects (62.4 ± 12.9 years) (n = 8) during the surgery. The patients received 30 pulses per min every 30 min. The muscle biopsy was collected from both hemidiaphragms and frozen for further analyses. 4-hydroxynonenal (4-HNE), an oxidative stress marker, and autophagy marker levels (Beclin-1 and the ratio of microtubule-associated protein light chain 3, I and II-LC3 II/I) protein concentrations were detected by the Western Blot technique. Antioxidant enzymatic activity copper-zinc (CuZnSOD) and manganese (MnSOD) superoxide dismutase were analyzed. Results Levels of lipid peroxidation (4-HNE) were significantly lower in the stimulated side (p < 0.05). The antioxidant enzyme activities (CuZnSOD and MnSOD) in the stimulated side of the diaphragm were not different than in the unstimulated side (p > 0.05). Additionally, the protein concentrations of Beclin-1 and the LC3 II/I ratio were higher in the stimulated side (p < 0.05). Conclusion These results suggest that the intraoperative electrical stimulation decreases oxidative stress levels and upregulates autophagy levels in the stimulated hemidiaphragm. These results may contribute future studies and clinical applications on reducing post-operative diaphragm dysfunction.