The roles of NADPH and isocitrate dehydrogenase in cochlear mitochondrial antioxidant defense and aging.

Hearing loss is the third most prevalent chronic health condition affecting older adults. Age-related hearing loss affects one in three adults over 65 years of age and is caused by both extrinsic and intrinsic factors, including genetics, aging, and exposure to noise and toxins. All cells possess antioxidant defense systems that play an important role in protecting cells against these factors. Reduced nicotinamide adenine dinucleotide phosphate (NADPH) serves as a co-factor for antioxidant enzymes such as glutathione reductase and thioredoxin reductase and is produced by glucose-6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, isocitrate dehydrogenase 1 (IDH1) or malic enzyme 1 in the cytosol, while in the mitochondria, NADPH is generated from mitochondrial transhydrogenase, glutamate dehydrogenase, malic enzyme 3 or IDH2. There are three isoforms of IDH: cytosolic IDH1, and mitochondrial IDH2 and IDH3. Of these, IDH2 is thought to be the major supplier of NADPH to the mitochondrial antioxidant defense system. The NADP+/NADPH and NAD+/NADH couples are essential for maintaining a large array of biological processes, including cellular redox state, and energy metabolism, mitochondrial function. A growing body of evidence indicates that mitochondrial dysfunction contributes to age-related structural or functional changes of cochlear sensory hair cells and neurons, leading to hearing impairments. In this review, we describe the current understanding of the roles of NADPH and IDHs in cochlear mitochondrial antioxidant defense and aging.

Txn2 haplodeficiency does not affect cochlear antioxidant defenses or accelerate the progression of cochlear cell loss or hearing loss across the lifespan.

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.

Effects of Gsta4 deficiency on age-related cochlear pathology and hearing loss in mice.

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.

GSTA4 mediates reduction of cisplatin ototoxicity in female 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.

Increased burden of mitochondrial DNA deletions and point mutations in early-onset age-related hearing loss in mitochondrial mutator mice.

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.

Loss of IDH2 Accelerates Age-related Hearing Loss in Male Mice.

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.

GSR is not essential for the maintenance of antioxidant defenses in mouse cochlea: Possible role of the thioredoxin system as a functional backup for GSR.

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.

G6pd Deficiency Does Not Affect the Cytosolic Glutathione or Thioredoxin Antioxidant Defense in Mouse Cochlea.

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.

Effects of Long-Term Exercise on Age-Related Hearing Loss in Mice.

Regular physical exercise reduces the risk for obesity, cardiovascular diseases, and disability and is associated with longer lifespan expectancy (Taylor et al., 2004; Pahor et al., 2014; Anton et al., 2015; Arem et al., 2015). In contrast, decreased physical function is associated with hearing loss among older adults (Li et al., 2013; Chen et al., 2015). Here, we investigated the effects of long-term voluntary wheel running (WR) on age-related hearing loss (AHL) in CBA/CaJ mice, a well established model of AHL (Zheng et al., 1999). WR activity peaked at 6 months of age (12,280 m/d) and gradually decreased over time. At 24 months of age, the average WR distance was 3987 m/d. Twenty-four-month-old runners had less cochlear hair cell and spiral ganglion neuron loss and better auditory brainstem response thresholds at the low and middle frequencies compared with age-matched, non-WR controls. Gene ontology (GO) enrichment analysis of inner ear tissues from 6-month-old controls and runners revealed that WR resulted in a marked enrichment for GO gene sets associated with immune response, inflammatory response, vascular function, and apoptosis. In agreement with these results, there was reduced stria vascularis (SV) atrophy and reduced loss of capillaries in the SV of old runners versus old controls. Given that SV holds numerous capillaries that are essential for transporting oxygen and nutrients into the cochlea, our findings suggest that long-term exercise delays the progression of AHL by reducing age-related loss of strial capillaries associated with inflammation. SIGNIFICANCE STATEMENT: Nearly two-thirds of adults aged 70 years or older develop significant age-related hearing loss (AHL), a condition that can lead to social isolation and major communication difficulties. AHL is also associated with decreased physical function among older adults. In the current study, we show that regular exercise slowed AHL and cochlear degeneration significantly in a well established murine model. Our data suggest that regular exercise delays the progression of AHL by reducing age-related loss of strial capillaries associated with inflammation.

Sirt1 deficiency protects cochlear cells and delays the early onset of age-related hearing loss in C57BL/6 mice.

Hearing gradually declines with age in both animals and humans, and this condition is known as age-related hearing loss (AHL). Here, we investigated the effects of deficiency of Sirt1, a member of the mammalian sirtuin family, on age-related cochlear pathology and associated hearing loss in C57BL/6 mice, a mouse model of early-onset AHL. Sirt1 deficiency reduced age-related oxidative damage of cochlear hair cells and spiral ganglion neurons and delayed the early onset of AHL. In cultured mouse inner ear cell lines, Sirt1 knockdown increased cell viability under oxidative stress conditions, induced nuclear translocation of Foxo3a, and increased acetylation status of Foxo3a. This resulted in increased activity of the antioxidant enzyme catalase. In young wild-type mice, both Sirt1 and Foxo3a proteins resided in the cytoplasm of the supporting cells within the organ of Corti of the cochlea. Therefore, our findings suggest that SIRT1 promotes early-onset AHL through suppressing FOXO3a-mediated oxidative stress resistance in the cochlea of C57BL/6 mice.