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.

Characterization of the Expression of Basigin Gene Products Within the Pineal Gland of Mice.

The expression of Basigin gene products and monocarboxylate transporter-1 (MCT1) has been investigated within the mammalian neural retina and suggests a role for these proteins in cellular metabolism within that tissue. The purpose of the present study was to investigate the expression of these same proteins in the pineal gland of the mouse brain. Mouse pineal gland and neural retina RNA and protein were subjected to quantitative reverse transcription-polymerase chain reaction and immunoblotting analyses. In addition, paraffin-embedded sections of each tissue were analyzed for expression of Basigin gene products and MCT1 via immunohistochemistry. The results indicate that MCT1 and Basigin variant-2, but not Basigin variant-1, are expressed within the mouse pineal gland. The expression of Basigin variant-2 and MCT1 was localized to the capsule surrounding the gland. The position and relative amounts of the gene products suggest that they play a much less prominent role within the pineal gland than in the neural retina.

Dose and developmental responses of Anopheles merus larvae to salinity.

Saltwater tolerance is a trait that carries both ecological and epidemiological significance for Anopheles mosquitoes that transmit human malaria, as it plays a key role in determining their habitat use and ecological distribution, and thus their local contribution to malaria transmission. Here, we lay the groundwork for genetic dissection of this trait by quantifying saltwater tolerance in three closely related cryptic species and malaria vectors from the Afrotropical Anopheles gambiae complex that are known to differ starkly in their tolerance to salinity: the obligate freshwater species A. gambiae and A. coluzzii, and the saltwater-tolerant species A. merus. We performed detailed comparisons of survivorship under varying salinities, using multiple strains of A. gambiae, A. coluzzii and A. merus, as well as F1 progeny from reciprocal crosses of A. merus and A. coluzzii. Additionally, using immunohistochemistry, we compared the location of three ion regulatory proteins (Na(+)/K(+)-ATPase, carbonic anhydrase and Na(+)/H(+)-antiporter) in the recta of A. coluzzii and A. merus reared in freshwater or saline water. As expected, we found that A. merus survives exposure to high salinities better than A. gambiae and A. coluzzii. Further, we found that exposure to a salinity level of 15.85 g NaCl l(-1) is a discriminating dose that kills all A. gambiae, A. coluzzii and A. coluzzii-A. merus F1 larvae, but does not negatively impact the survival of A. merus. Importantly, phenotypic expression of saltwater tolerance by A. merus is highly dependent upon the developmental time of exposure, and based on immunohistochemistry, salt tolerance appears to involve a major shift in Na(+)/K+-ATPase localization in the rectum, as observed previously for the distantly related saline-tolerant species A. albimanus.

Countering a bioterrorist introduction of pathogen-infected mosquitoes through mosquito control.

The release of infected mosquitoes or other arthropods by bioterrorists, i.e., arboterrorism, to cause disease and terror is a threat to the USA. A workshop to assess mosquito control response capabilities to mount rapid and effective responses to eliminate an arboterrorism attack provided recommendations to improve capabilities in the USA. It is essential that mosquito control professionals receive training in possible responses, and it is recommended that a Council for Emergency Mosquito Control be established in each state to coordinate training, state resources, and actions for use throughout the state.

Environmental DNA monitoring of oncogenic viral shedding and genomic profiling of sea turtle fibropapillomatosis reveals unusual viral dynamics.

Pathogen-induced cancers account for 15% of human tumors and are a growing concern for endangered wildlife. Fibropapillomatosis is an expanding virally and environmentally co-induced sea turtle tumor epizootic. Chelonid herpesvirus 5 (ChHV5) is implicated as a causative virus, but its transmission method and specific role in oncogenesis and progression is unclear. We applied environmental (e)DNA-based viral monitoring to assess viral shedding as a direct means of transmission, and the relationship between tumor burden, surgical resection and ChHV5 shedding. To elucidate the abundance and transcriptional status of ChHV5 across early, established, regrowth and internal tumors we conducted genomics and transcriptomics. We determined that ChHV5 is shed into the water column, representing a likely transmission route, and revealed novel temporal shedding dynamics and tumor burden correlations. ChHV5 was more abundant in the water column than in marine leeches. We also revealed that ChHV5 is latent in fibropapillomatosis, including early stage, regrowth and internal tumors; higher viral transcription is not indicative of poor patient outcome, and high ChHV5 loads predominantly arise from latent virus. These results expand our knowledge of the cellular and shedding dynamics of ChHV5 and can provide insights into temporal transmission dynamics and viral oncogenesis not readily investigable in tumors of terrestrial species.

Molecular characterization of a marine turtle tumor epizootic, profiling external, internal and postsurgical regrowth tumors.

Sea turtle populations are under threat from an epizootic tumor disease (animal epidemic) known as fibropapillomatosis. Fibropapillomatosis continues to spread geographically, with prevalence of the disease also growing at many longer-affected sites globally. However, we do not yet understand the precise environmental, mutational and viral events driving fibropapillomatosis tumor formation and progression.Here we perform transcriptomic and immunohistochemical profiling of five fibropapillomatosis tumor types: external new, established and postsurgical regrowth tumors, and internal lung and kidney tumors. We reveal that internal tumors are molecularly distinct from the more common external tumors. However, they have a small number of conserved potentially therapeutically targetable molecular vulnerabilities in common, such as the MAPK, Wnt, TGFß and TNF oncogenic signaling pathways. These conserved oncogenic drivers recapitulate remarkably well the core pan-cancer drivers responsible for human cancers. Fibropapillomatosis has been considered benign, but metastatic-related transcriptional signatures are strongly activated in kidney and established external tumors. Tumors in turtles with poor outcomes (died/euthanized) have genes associated with apoptosis and immune function suppressed, with these genes providing putative predictive biomarkers.Together, these results offer an improved understanding of fibropapillomatosis tumorigenesis and provide insights into the origins, inter-tumor relationships, and therapeutic treatment for this wildlife epizootic.

The 2M6 antigen is a Müller cell-specific intracellular membrane-associated protein of the sarcolemmal-membrane-associated protein family and is also TopAP.

PURPOSE: The differentiation marker 2M6 has been used to identify Müller cells within the developing chick retina for several years, although the molecular identity of 2M6 was not known. This study was aimed at determining the identity of the protein antigen recognized by the 2M6 monoclonal antibody. METHODS: Affinity chromatography and subsequent mass spectrometry were used to determine the molecular identity of the 2M6 antigen. Immunohistochemistry of monolayer preparations and paraffin-embedded sections of chick retina were performed to localize expression of the 2M6 antigen within cells of the chick retina. RESULTS: Mass spectrometry analyses revealed that the 2M6 antigen is identical (with 95% probability) to the protein known as Top(AP), which is a member of the sarcolemmal membrane-associated protein family of proteins. The 2M6 polypeptide is expressed by Müller glial cells as well as boundary cells within the chick retina. Expression localizes to intracellular membrane structures within those cells. CONCLUSIONS: Members of the sarcolemmal membrane-associated protein family of proteins have been implicated in structural and functional roles related to the cytoskeleton and Ca(+2) release from internal stores. It is thought that 2M6 plays a similar role in Müller cells of the vertebrate retina.

Organ-associated muscles in Aedes albopictus (Diptera: Culicidae) respond differentially to Sindbis virus.

Differential host cell responses to the alphavirus Sindbis were observed in visceral muscles of the adult female mosquito Aedes albopictus. Following intrathoracic inoculation with SIN, muscles associated with the midgut, hindgut, and ovary resulted in clearance, persistence, and refractoriness to virus, respectively. Prominent sarcomeres characteristic of myofilaments were identified in muscles associated with these three organs by phalloidin labeling of actin, confirming these cells as muscle. The location of virus antigen mimicked the distribution of actin in both mid- and hindgut-associated muscles. Furthermore, these myofilaments remained intact following virus clearance from midgut muscles and during virus persistence in hindgut muscles. Changes in the temporal onset of virus antigen following high titer inoculum compared with standard titer inoculum was observed in anterior midgut muscles, but not in muscles associated with the posterior midgut or hindgut. Muscle bundles closely approximated the gut surface, while a wispy association was displayed at the ovary surface. Prominent ultrastructural differences were observed in the basal lamina attached to the gut compared with the ovary. Additionally, ultrastructural evidence for virus-associated pathology was observed in gut-associated muscles and gut epithelium. Visceral muscles, all composed of the same tissue type, but associated to three different organs in the insect abdomen, responded differentially to Sindbis. We speculate that variations in structure, function or physiology and ultrastructure inherent to insect host cells or organs interactions reflect the complicated milieu of the organism and contribute to differential virus phenotypic expression in muscle cells.

Physiological and pharmacological characterizations of the larval Anopheles albimanus rectum support a change in protein distribution and/or function in varying salinities.

Ion regulation is a biological process crucial to the survival of mosquito larvae and a major organ responsible for this regulation is the rectum. The recta of anopheline larvae are distinct from other subfamilies of mosquitoes in several ways, yet have not yet been characterized extensively. Here we characterize the two major cell types of the anopheline rectum, DAR and non-DAR cells, using histological, physiological, and pharmacological analyses. Proton flux was measured at the basal membrane of 2%- and 50%-artificial sea water-reared An. albimanus larvae using self-referencing ion-selective microelectrodes, and the two cell types were found to differ in basal membrane proton flux. Additionally, differences in the response of that flux to pharmacological inhibitors in larvae reared in 2% versus 50% ASW indicate changes in protein function between the two rearing conditions. Finally, histological analyses suggest that the non-DAR cells are structurally suited for mediating ion transport. These data support a model of rectal ion regulation in which the non-DAR cells have a resorptive function in freshwater-reared larvae and a secretive function in saline water-reared larvae. In this way, anopheline larvae may adapt to varying salinities.

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.

Ectopic expression of L1CAM ectodomain alters differentiation and motility, but not proliferation, of human neural progenitor cells.

Adult human neural progenitor and stem cells have been implicated as a potential source of brain cancer causing cells, but specific events that might cause cells to progress towards a transformed phenotype remain unclear. The L1CAM (L1) cell adhesion/recognition molecule is expressed abnormally by human glioma cancer cells and is released as a large extracellular ectodomain fragment, which stimulates cell motility and proliferation. This study investigates the effects of ectopic overexpression of the L1 long ectodomain (L1LE; ˜180 kDa) on the motility, proliferation, and differentiation of human neural progenitor cells (HNPs). L1LE was ectopically expressed in HNPs using a lentiviral vector. Surprisingly, overexpression of L1LE resulted in reduced HNP motility in vitro, in stark contrast to the effects on glioma and other cancer cell types. L1LE overexpression resulted in a variable degree of maintenance of HNP proliferation in media without added growth factors but did not increase proliferation. In monolayer culture, HNPs expressed a variety of differentiation markers. L1LE overexpression resulted in loss of glutamine synthetase (GS) and ß3-tubulin expression in normal HNP media, and reduced vimentin and increased GS expression in the absence of added growth factors. When co-cultured with chick embryonic brain cell aggregates, HNPs show increased differentiation potential. Some HNPs expressed p-neurofilaments and oligodendrocytic O4, indicating differentiation beyond that in monolayer culture. Most HNP-L1LE cells lost their vimentin and GFAP (glial fibrillary acidic protein) staining, and many cells were positive for astrocytic GS. However, these cells rarely were positive for neuronal markers ß3-tubulin or p-neurofilaments, and few HNP oligodendrocyte progenitors were found. These results suggest that unlike for glioma cells, L1LE does not increase HNP cell motility, but rather decreases motility and influences the differentiation of normal brain progenitor cells. Therefore, the effect of L1LE on increasing motility and proliferation appears to be limited to already transformed cells.

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.

Expression, purification, kinetic, and structural characterization of an alpha-class carbonic anhydrase from Aedes aegypti (AaCA1).

Carbonic anhydrases (CAs) are zinc-containing metalloenzymes that catalyze the interconversion of carbon dioxide and bicarbonate. The alpha-class CAs are found predominantly in vertebrates, but they are also expressed in insects like mosquitoes. Recently, an alpha-CA from the midgut of Aedes aegypti larvae (AaCA1) was identified, cloned, and subsequently shown to share high sequence homologous to human CA I (HCA I). This paper presents the bacterial expression, purification, and kinetic characterization of the soluble CA domain of AaCA1. The data show AaCA1 is a highly active CA that displays inhibition by methazolamide and ethoxzolamide with nM affinity. Additionally, a homology model of AaCA1, based on the crystal structure of HCA I, is presented and the overall structure, active site, and surface charge properties are compared to those of HCA I and II. Measurements of catalysis show that AaCA1 is more like HCA II in terms of proton transfer, but more similar to HCA I in terms of conversion of carbon dioxide to bicarbonate, and these differences are rationalized in terms of structure. These results also indicate that amino acid differences in the active site of AaCA1 compared to human CAs could be used to design specific CA inhibitors for the management of mosquito populations.

Characterization of Carbonic Anhydrase 9 in the Alimentary Canal of Aedes aegypti and Its Relationship to Homologous Mosquito Carbonic Anhydrases.

In the mosquito midgut, luminal pH regulation and cellular ion transport processes are important for the digestion of food and maintenance of cellular homeostasis. pH regulation in the mosquito gut is affected by the vectorial movement of the principal ions including bicarbonate/carbonate and protons. As in all metazoans, mosquitoes employ the product of aerobic metabolism carbon dioxide in its bicarbonate/carbonate form as one of the major buffers of cellular and extracellular pH. The conversion of metabolic carbon dioxide to bicarbonate/carbonate is accomplished by a family of enzymes encoded by the carbonic anhydrase gene family. This study characterizes Aedes aegypti carbonic anhydrases using bioinformatic, molecular, and immunohistochemical methods. Our analyses show that there are fourteen Aedes aegypti carbonic anhydrase genes, two of which are expressed as splice variants. The carbonic anhydrases were classified as either integral membrane, peripheral membrane, mitochondrial, secreted, or soluble cytoplasmic proteins. Using polymerase chain reaction and Western blotting, one of the carbonic anhydrases, Aedes aegypti carbonic anhydrase 9, was analyzed and found in each life stage, male/female pupae, male/female adults, and in the female posterior midgut. Next, carbonic anhydrase 9 was analyzed in larvae and adults using confocal microscopy and was detected in the midgut regions. According to our analyses, carbonic anhydrase 9 is a soluble cytoplasmic enzyme found in the alimentary canal of larvae and adults and is expressed throughout the life cycle of the mosquito. Based on previous physiological analyses of adults and larvae, it appears AeCA9 is one of the major carbonic anhydrases involved in producing bicarbonate/carbonate which is involved in pH regulation and ion transport processes in the alimentary canal. Detailed understanding of the molecular bases of ion homeostasis in mosquitoes will provide targets for novel mosquito control strategies into the new millennium.

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.

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.

Loss of MCT1, MCT3, and MCT4 expression in the retinal pigment epithelium and neural retina of the 5A11/basigin-null mouse.

PURPOSE: The neural retina expresses multiple monocarboxylate transporters (MCTs) that are likely to play a key role in the metabolism of the outer retina. Recently, it was reported that targeting of MCT1 and -4 to the plasma membrane requires association with 5A11/basigin (CD147). In the present study, the hypothesis that reduced amplitudes in the electroretinograms in the 5A11/basigin null mouse (Bsg(-/-)) may be linked to altered expression of MCTs was studied. METHODS: The expression and subcellular distribution of MCTs in Bsg(-/-) mice was analyzed by immunofluorescence microscopy with isoform-specific antibodies. Protein expression was analyzed by Western blot analysis, and mRNA expression was examined with RT-PCR. RESULTS: Immunofluorescence labeling of tissue sections from the Bsg(-/-) mice revealed a dramatic reduction in labeling with MCT antibodies. There was a loss of MCT1 labeling in the apical membrane of the RPE and in the neural retina. MCT3, which is expressed in the basolateral membrane of the RPE wild-type mouse, was expressed at very low levels in both the apical and basolateral membranes of the Bsg(-/-) mouse. There was no change in expression or distribution of the glucose transporter (GLUT)-1 in the RPE and retina of the Bsg(-/-) mouse. Western blot analysis of detergent-soluble lysates prepared from wild-type and Bsg(-/-) eyes confirmed that the levels of MCT1, MCT3, and MCT4 protein were severely reduced in Bsg(-/-) mice. RT-PCR analyses of mRNA levels from wild-type and Bsg(-/-) mice demonstrated that the MCT1 transcript was expressed at normal levels in Bsg(-/-) mice. CONCLUSIONS: In Bsg(-/-) mice, there is a severe reduction in accumulation of the MCT1 and -3 proteins in the RPE and a concomitant reduction in MCT1 and -4 in the neural retina supporting a role for 5A11/basigin in the targeting of these transporters to the plasma membrane. Decreased expression of MCT1 and -4 on the surfaces of Müller and photoreceptor cells may compromise energy metabolism in the outer retina, leading to abnormal photoreceptor cell function and degeneration.