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.

Sestrin 2 (SESN2) is a stress-inducible protein that protects tissues from oxidative stress and delays the aging process. However, its role in maintaining the functional and structural integrity of the cochlea is largely unknown. Here, we report the expression of SESN2 protein in the sensory epithelium, particularly in hair cells. Using C57BL/6J mice, a mouse model of age-related cochlear degeneration, we observed a significant age-related reduction in SESN2 expression in cochlear tissues that was associated with early onset hearing loss and accelerated age-related sensory cell degeneration that progressed from the base toward the apex of the cochlea. Hair cell death occurred by caspase-8 mediated apoptosis. Compared to C57BL/6J control mice, Sesn2 KO mice displayed enhanced expression of proinflammatory genes and activation of basilar membrane macrophages, suggesting that loss of SESN2 function provokes the immune response. Together, these results suggest that Sesn2 plays an important role in cochlear homeostasis and immune responses to stress.

Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP+ to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. We investigated the roles of G6PD in the cytosolic antioxidant defense in the cochlea of G6pd hypomorphic mice that were backcrossed onto normal-hearing CBA/CaJ mice. Young G6pd-deficient mice displayed a significant decrease in cytosolic G6PD protein levels and activities in the inner ears. However, G6pd deficiency did not affect the cytosolic NADPH redox state, or glutathione or thioredoxin antioxidant defense in the inner ears. No histological abnormalities or oxidative damage was observed in the cochlea of G6pd hemizygous males or homozygous females. Furthermore, G6pd deficiency did not affect auditory brainstem response hearing thresholds, wave I amplitudes or wave I latencies in young males or females. In contrast, G6pd deficiency resulted in increased activities and protein levels of cytosolic isocitrate dehydrogenase 1, an enzyme that catalyzes the conversion of isocitrate to α-ketoglutarate and NADP+ to NADPH, in the inner ear. In a mouse inner ear cell line, knockdown of Idh1, but not G6pd, decreased cell growth rates, cytosolic NADPH levels, and thioredoxin reductase activities. Therefore, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in mouse cochlea. Under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea.SIGNIFICANCE STATEMENT Glucose-6-phosphate dehydrogenase (G6PD) is the first and rate-limiting enzyme of the pentose phosphate pathway; it catalyzes the conversion of glucose-6-phosphate to 6-phosphogluconate and NADP+ to NADPH and is thought to be the principal source of NADPH for the cytosolic glutathione and thioredoxin antioxidant defense systems. In the current study, we show that, under normal physiological conditions, G6pd deficiency does not affect the cytosolic glutathione or thioredoxin antioxidant defense in the mouse cochlea. However, under G6pd deficiency conditions, isocitrate dehydrogenase 1 likely functions as the principal source of NADPH for cytosolic antioxidant defense in the cochlea.

Usher's syndrome is the most common combined blindness-deafness disorder with USH1B, caused by mutations in MYO7A, resulting in the most severe phenotype. The existence of numerous, naturally occurring shaker1 mice harboring variable MYO7A mutations on different genetic backgrounds has complicated the characterization of MYO7A knockout (KO) and heterozygote mice. We generated a novel MYO7A KO mouse (Myo7a - / -) that is easily genotyped, maintained, and confirmed to be null for MYO7A in both the eye and inner ear. Like USH1B patients, Myo7a - / - 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. Myo7a - / - 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 Myo7a + / - mice had reduced numbers of cochlear HCs and concomitant reductions in auditory function relative to Myo7a +/+ controls. Notably, this is the first report that loss of a single Myo7a allele significantly alters auditory structure and function and suggests that audiological characterization of USH1B carriers is warranted. Maintenance of vestibular HCs in Myo7a - / - mice suggests that gene replacement could be used to correct the vestibular dysfunction in USH1B patients. While Myo7a - / - 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 MYO7A on a null background and generate valuable pre-clinical data toward the treatment of USH1B.

Mitochondrial DNA (mtDNA) mutations are thought to have a causal role in a variety of age-related neurodegenerative diseases, including age-related hearing loss (AHL). In the current study, we investigated the roles of mtDNA deletions and point mutations in AHL in mitochondrial mutator mice (Polgmut/mut) that were backcrossed onto CBA/CaJ mice, a well-established model of late-onset AHL. mtDNA deletions accumulated significantly with age in the inner ears of Polgmut/mut mice, while there were no differences in mtDNA deletion frequencies in the inner ears between 5 and 17 months old Polg+/+ mice or 5 months old Polg+/+ and Polgmut/mut mice. mtDNA deletions also accumulated significantly in the inner ears of CBA/CaJ mice during normal aging. In contrast, 5 months old Polgmut/mut mice displayed a 238-fold increase in mtDNA point mutation frequencies in the inner ears compared to age-matched Polg+/+ mice, but there were no differences in mtDNA point mutation frequencies in the inner ears between 5 and 17 months old Polgmut/mut mice. Seventeen-month-old Polgmut/mut mice also displayed early-onset severe hearing loss associated with a significant reduction in neural output of the cochlea, while age-matched Polg+/+ mice displayed little or no hearing impairment. Consistent with the physiological and mtDNA deletion test result, 17-month-old Polgmut/mut mice displayed a profound loss of spiral ganglion neurons in the cochlea. Thus, our data suggest that a higher burden of mtDNA point mutations from a young age and age-related accumulation of mtDNA deletions likely contribute to early-onset AHL in mitochondrial mutator mice.

Mefloquine is widely used for the treatment of malaria. However, this drug is known to induce neurological side effects including depression, anxiety, balance disorder, and sensorineural hearing loss. Yet, there is currently no treatment for these side effects.

Aging is the most significant risk factor for a range of diseases, including many cancers, neurodegeneration, cardiovascular disease, and diabetes. Caloric restriction (CR) without malnutrition delays aging in diverse species, and therefore offers unique insights into age-related disease vulnerability. Previous studies suggest that there are shared mechanisms of disease resistance associated with delayed aging, however quantitative support is lacking. We therefore sought to identify a common response to CR in diverse tissues and species and determine whether this signature would reflect health status independent of aging. We analyzed gene expression datasets from eight tissues of mice subjected to CR and identified a common transcriptional signature that includes functional categories of mitochondrial energy metabolism, inflammation and ribosomal structure. This signature is detected in flies, rats, and rhesus monkeys on CR, indicating aspects of CR that are evolutionarily conserved. Detection of the signature in mouse genetic models of slowed aging indicates that it is not unique to CR but rather a common aspect of extended longevity. Mice lacking the NAD-dependent deacetylase SIRT3 fail to induce mitochondrial and anti-inflammatory elements of the signature in response to CR, suggesting a potential mechanism involving SIRT3. The inverse of this transcriptional signature is detected with consumption of a high fat diet, obesity and metabolic disease, and is reversed in response to interventions that decrease disease risk. We propose that this evolutionarily conserved, tissue-independent, transcriptional signature of delayed aging and reduced disease vulnerability is a promising target for developing therapies for age-related diseases.

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.

Thioredoxin 2 (TXN2) is a small redox protein found in nearly all organisms. As a mitochondrial member of the thioredoxin antioxidant defense system, TXN2 interacts with peroxiredoxin 3 (PRDX3) to remove hydrogen peroxide. Accordingly, TXN2 is thought to play an important role in maintaining the appropriate mitochondrial redox environment and protecting the mitochondrial components against oxidative stress. In the current study, we investigated the effects of Txn2 haplodeficiency on cochlear antioxidant defenses, auditory function, and cochlear cell loss across the lifespan in wild-type (WT) and Txn2 heterozygous knockout (Txn2+/-) mice backcrossed onto CBA/CaJ mice, a well-established model of age-related hearing loss. Txn2+/- mice displayed a 58% decrease in TXN2 protein levels in the mitochondria of the inner ears compared to WT mice. However, Txn2 haplodeficiency did not affect the thioredoxin or glutathione antioxidant defense in both the mitochondria and cytosol of the inner ears of young mice. There were no differences in the levels of mitochondrial biogenesis markers, mitochondrial DNA content, or oxidative DNA and protein damage markers in the inner ears between young WT and Txn2+/- mice. In a mouse inner ear cell line, knockdown of Txn2 did not affect cell viability under hydrogen peroxide treatment. Consistent with the tissue and cell line results, there were no differences in hair cell loss or spiral ganglion neuron density between WT and Txn2+/- mice at 3-5 or 23-25 months of age. Furthermore, Txn2 haplodeficiency did not affect auditory brainstem response threshold, wave I latency, or wave I amplitude at 3-5, 15-16, or 23-25 months of age. Therefore, Txn2 haplodeficiency does not affect cochlear antioxidant defenses, accelerate degeneration of cochlear cells, or affect auditory function in mice across the lifespan.

Mitochondrial DNA (mtDNA) mutations are thought to have a causative role in age-related pathologies. We have shown previously that mitochondrial mutator mice (PolgD257A/D257A), harboring a proofreading-deficient version of the mtDNA polymerase gamma (POLG), accumulate mtDNA mutations in multiple tissues and display several features of accelerated aging. Calorie restriction (CR) is known to delay the onset of age-related diseases and to extend the lifespan of a variety of species, including rodents. In the current study we investigated the effects of CR on the lifespan and healthspan of mitochondrial mutator mice. Long-term CR did not increase the median or maximum lifespan of PolgD257A/D257A mice. Furthermore, CR did not reduce mtDNA deletions in the heart and muscle, accelerated sarcopenia, testicular atrophy, nor improve the alterations in cardiac parameters that are present in aged mitochondrial mutator mice. Therefore, our findings suggest that accumulation of mtDNA mutations may interfere with the beneficial action of CR in aging retardation.

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.

The glutathione transferase (GST) detoxification system converts exogenous and endogenous toxins into a less toxic form by conjugating the toxic compound to reduced glutathione (GSH) by a variety of GST enzymes. Of the ~20 GST isoforms, GSTA4 exhibits high catalytic efficiency toward 4-hydroxynonenal (4-HNE), one of the most abundant end products of lipid peroxidation that contributes to neurodegenerative diseases and age-related disorders. Conjugation to GSH by GSTA4 is thought to be a major route of 4-HNE elimination. In the current study, we investigated the effects of Gsta4 deficiency on age-related cochlear pathology and hearing loss using young (3-5 months old) and old (24-25 months old) Gsta4+/+ and Gsta4-/- mice that were backcrossed onto the CBA/CaJ mouse strain, a well-established model of age-related hearing loss (AHL). At 3-5 months of age, loss of Gsta4 resulted in decreased total GSTA activity toward 4-HNE in the inner ears of young mice. However, there were no differences in the levels of 4-HNE in the inner ears between Gsta4+/+ and Gsta4-/- mice at 3-5 or 24-25 months of age. No histological abnormalities were observed in the cochlea and no hearing impairments were observed in young Gsta4-/- mice. At 24-25 months of age, both Gsta4+/+ and Gsta4-/- mice showed elevated ABR thresholds compared to 3-month-old mice, but there were no differences in ABR thresholds, cochlear spiral ganglion neuron densities, or stria vascularis thickness between Gsta4+/+ and Gsta4-/- mice. Together, these results suggest that under normal physiological conditions or during normal aging, GSTA4 is not essential for removal of 4-HNE in mouse inner ears.

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.

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.

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.

Presbycusis is characterized by an age-related progressive decline of auditory function, and arises mainly from the degeneration of hair cells or spiral ganglion (SG) cells in the cochlea. Here we show that caloric restriction suppresses apoptotic cell death in the mouse cochlea and prevents late onset of presbycusis. Calorie restricted (CR) mice, which maintained body weight at the same level as that of young control (YC) mice, retained normal hearing and showed no cochlear degeneration. CR mice also showed a significant reduction in the number of TUNEL-positive cells and cleaved caspase-3-positive cells relative to middle-age control (MC) mice. Microarray analysis revealed that CR down-regulated the expression of 24 apoptotic genes, including Bak and Bim. Taken together, our findings suggest that loss of critical cells through apoptosis is an important mechanism of presbycusis in mammals, and that CR can retard this process by suppressing apoptosis in the inner ear tissue.

Mitochondrial DNA (mtDNA) mutations may contribute to aging and age-related diseases. Previously, we reported that accumulation of mtDNA mutations is associated with age-related hearing loss in mice carrying a mutator allele of the mitochondrial Polg DNA polymerase. To elucidate the role of mtDNA mutations in the pathogenesis of age-related hearing loss or presbycusis, we performed large scale gene expression analysis to identify mtDNA mutation-responsive genes and biological process categories associated with mtDNA mutations by comparing the gene expression patterns of cochlear tissues from 9-month-old mitochondrial mutator and control mice. mtDNA mutations were associated with transcriptional alterations consistent with impairment of energy metabolism, induction of apoptosis, cytoskeletal dysfunction, and hearing dysfunction in the cochlea of aged mitochondrial mutator mice. TUNEL staining and caspase-3 immunostaining analysis demonstrated that the levels of apoptotic markers were significantly increased in the cochleae of mitochondrial mutator mice compared to age-matched controls. These observations support a new model of how mtDNA mutations impact cochlear function whereby accumulation of mtDNA mutations lead to mitochondrial dysfunction, an associated impairment of energy metabolism, and the induction of an apoptotic program. The data presented here provide the first global assessment at the molecular level of the pathogenesis of age-related disease in mitochondrial mutator mice and reveal previously unrecognized biological pathways associated with mtDNA mutations.

Age-related hearing loss (AHL) is the progressive loss of auditory function with aging. Mutations in the Cdh23 gene of DBA/2J mice result in AHL by 3 months of age. Hearing function was analyzed by auditory brainstem response (ABR) which confirmed that severe age-related hearing loss occurred in 8-month-old mice, whereas mild hearing loss occurred in 2-month-old mice. Cochlear gene expression of 2-month-old and 8-month-old DBA/2J mice was measured using Affymetrix microarrays. Comprehensive gene expression analysis identified significant expression changes correlated with AHL in over 4000 cochlear genes. AHL-correlated genes in the cochlea of 8-month-old DBA/2J mice were statistically associated with 15 mitochondrial process categories, including "mitochondrial electron transport chain", "oxidative phosphorylation", "respiratory chain complex I", "respiratory chain complex IV", and "respiratory chain complex V". Furthermore, 31 genes encoding components of the mitochondrial respiratory chain complexes I, II, III, IV, and V were significantly down-regulated in the cochlea. Quantitative RT-PCR (QRT-PCR) validated the microarray data in a selected set of genes. Thus, these observations provide evidence that AHL is associated with profound down-regulation of genes involved in the mitochondrial respiratory chain complexes in the cochlea of aged DBA/2J mice.

Despite recent advances in genome engineering technologies, traditional transgenic mice generated on a mixed genetic background of C57BL/6 and 129/Sv mice remain widely used in age-related hearing loss (AHL) research, since C57BL/6 mice exhibit early onset and progression of AHL due to a mutation in cadherin 23-encoding gene (Cdh23753G>A). In these transgenic mice, backcrossing for more than 10 generations results in replacement of the donor background (129/Sv) with that of the recipient (C57BL/6), so that approximately 99.9% of genes are C57BL/6-derived and are considered congenic. However, the regions flanking the target gene may still be of 129/Sv origin, creating a so-called "passenger gene problem" where the normal 129/Sv-derived Cdh23753G allele can travel with the target gene. In this study, we investigated the role of fatty acid-binding protein 7 (Fabp7), which is important for cellular uptake and intracellular trafficking of fatty acids in the cochlea, using traditional Fabp7 knockout (KO) mice on the C57BL/6 background. We found that Fabp7 KO mice showed delayed AHL progression and milder cochlear degeneration. However, the genotype of the Cdh23 region flanking Fabp7 was still that of 129/Sv origin (Cdh23753GG). Our findings reveal the potential risk of contamination for traditional transgenic mice generated on the C57BL/6 background.

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.