Oxidative stress and ROS metabolism via down-regulation of sirtuin 3 expression in Cmah-null mice affect hearing loss.

CMP-Neu5Ac hydroxylase (Cmah) disruption caused several abnormalities and diseases including hearing loss in old age. However, underling molecular mechanisms that give rise to age-related hearing loss (AHL) in Cmah-null mouse are still obscure. In this study, Cmah-null mice showed age-related decline of hearing associated with loss of sensory hair cells, spiral ganglion neurons, and/or stria vascularis degeneration in the cochlea. To identify differential gene expression profiles and pathway associated with AHL, we performed microarray analysis using Illumina MouseRef-8 v2 Expression BeadChip and pathway-focused PCR array in the cochlear tissues of Cmah-null mouse. Pathway and molecular mechanism analysis using differentially expressed genes provided evidences that altered biological pathway due to oxidative damage by low expressed antioxidants and dysregulated reactive oxygen species (ROS) metabolism. Especially, low sirtuin 3 (Sirt3) gene expressions in Cmah-null mice decreased both of downstream regulator (Foxo1 and MnSod) and regulatory transcription factor (Hif1α and Foxo3a) gene expression. Taken together, we suggest that down-regulation of Sirt3 expression leads to oxidative stress and mitochondrial dysfunction by regulation of ROS and that it could alter various signaling pathways in Cmah-null mice with AHL.


INTRODUCTION
activity of some complexes which as a result leads to chronic inflammation and prompting of apoptotic cell death pathways [8]. In another studies, rat F344/NHsd strain showed an increased mitochondrial DNA deletions in the cochlea at 6-and 9-months of age [9,10]. As a result, mitochondrial DNA deletions or mutations causes impairment in mitochondrial function and eventually leads to buildup of cochlear oxidative stress and consequent cochlear degeneration and AHL [11]. Even though there is growing recognition that oxidative stress and reactive oxygen species (ROS) may be an important factor for underlying mechanism of hearing loss, the studies of molecular mechanisms of AHL is still infancy in Cmah-null mouse.
Recent study provided a possible mechanism of sirtuin 3 (Sirt3) in ROS and oxidative stress induced metabolism in mammals [12,13]. Sirtuins are NAD +dependent protein deacetylases [14]. They possess the ability to regulate life span in lower organisms and have turned up as immense cellular fate regulators and mammalian biology [15]. There are seven sirtuins in mammals; they demonstrate diverse cellular localization [16,17]. Among them, Sirt3 localized in mitochondria stimulation results in activation of oxidative metabolism through deacetylation of numerous mitochondrial enzymes [18,19]. Sirt3 has been found to predominantly regulate the production of ROS at the electron transport chain (ETC), further the detoxification of ROS via activation of antioxidant enzymes [20][21][22][23][24][25]. A current study implied that mitochondrial Sirt3 arbitrates the anti-aging effects of calorie restriction (CR) on AHL by promoting the glutathione-mediated mitochondrial antioxidant defense system in mice [23]. As CR is recognized to provoke Sirt3 protein levels in wild-type (WT) mice, there is a possibility that Sirt3 expression levels can mediate ROS management, mitochondrial integrity, and sensory function, at least in those neurons that constrain hearing [26][27][28]. CR-mediated aging retardation regulated by Sirt3 in mammal. However, CR failed and impaired to reduce oxidative damage in multiple tissues of Sirt3 null mouse compare to control and it leads to the progression of AHL [29].
Despite growing interest in the relationship between loss of Cmah activity and age-related hearing loss, how genome-wide altered gene expression in the cochlear tissues of Cmah-null mouse affects hearing loss with aging remains unclear. In this study, we first explored differential gene expression profile in the cochlear tissues of Cmah-null mouse. We performed the functional enrichment analyses using annotations from both Gene Ontology (GO) and pathways. More importantly, we identified the pathway that related to the oxidative damage and ROS metabolism, supporting the pathogenesis hypothesis of age-related dysfunction. Finally, we analyzed the expression of their genes that involved Sirtuins, oxidative stress regulation, Wnt signaling, mitochondrial dysfunction identified by KEGG (Kyoto Encyclopedia of Genes and Genome) pathway analysis on microarray and a pathway-focused PCR array to address the mechanisms underlying AHL in Cmah-null mouse. In conclusion, we suggest a hypothetical model that down-regulated Sirt3 lead to oxidative damage and mitochondrial dysfunction by ROS and could alter various signaling pathways in Cmah-null mice with AHL. In this study, we propose a possible hypothetical model that down-regulated Sirt3 lead to oxidative damage and mitochondrial dysfunction by ROS and it could alter various signaling pathways involved in Cmah-null mice with AHL.

Cmah disruption result in histological abnormalities in vestibular otoconial epithelia and the cochlear sensory epithelium, and neuronal cell loss of inner ears
Cmah-null mouse, like the previous observations, showed age-related hearing loss [5,30]. To determine the lack of CMAH protein expression, we performed immunohistochemistry (IHC) in inner ear from WTand Cmah-null mice. As shown in Fig. 1A, the Neu5Gc, which is a final product by hydroxylation of CMP-Neu5Ac to CMP-Neu5Gc by CMP-Neu5Ac hydroxylase, was highly expressed in the WT, whereas the Cmah-null mice were completely deficient in inner ears. In histological analysis, 12 month-but not 5 week-old ( Supplementary Fig. 1). Cmah-null mice had histological abnormalities in the vestibular otoconial epithelia and the cochlear sensory epithelium of inner ears (Fig. 1B). The apical surface of the vestibular otoconial epithelia contains unusual deposits of apparently a cellular material among the stereociliary bundles (Fig. 1B upper). Further, we observed degeneration of outer hair cell throughout the cochlea, with collapse of the outer organ of corti in the basal (high-frequency) turn of 12 month-old mice ( Fig. 1B; bottom), whereas both of the cochlear and vestibular sensory epithelia of WT-mice appeared normal, even in older mice. Next, we performed the TUNEL assay to investigate the apoptotic cell death. The result shows significantly increased numbers of apoptotic cells in supporting cells of cochlear tissues from Cmah-null mice, compared to control (Fig. 1C).
In order to examine neuronal cell loss in Cmah-null mouse, we investigate the several neuronal cell-specific www.impactaging.com gene expressions in cochlear tissues. In this study, we examined glial fibrillary acidic protein (GFAP) expression for a neural injury marker or astrocyte marker, neurofilament (NF) expression for an axonal injury marker, vimentin (VIM) expression for a neural precursor cells marker, and enolase (NSE) expression for neuronal specific marker. In IHC, cochlear tissues of Cmah-null mice showed significant reductions in expres-sions of several neuronal cell markers such as GFAP, NSE, NF, and VIM in with respect to control mice (Fig.  1D). Reverse transcriptase quantitative real-time polymerase chain reaction (RT-qPCR) analysis also showed significantly down-regulated gene expression for same neuronal cell markers (Gfap, Nse, Nf, and Vim) compared to control (Fig. 1E), indicate that RT-qPCR results mimic IHC results. www.impactaging.com

Cmah-null mice
Expression profiles in cochlear tissues of WT-and Cmah-null mice (n=3, respectively) were compared using an Illumina MouseRef-8 v2 Expression BeadChip, which is composed of 25,697 probe sets for mouse mRNA. To analyze differential gene expression in WT-and Cmah-null mice, we focused on the gene expressions, which showed ≥1.5 folds changes with a significant p-value (p < 0.05). In this study, we performed scatter plot analysis in order to estimate global gene expression change. As shown in Fig. 2A, mRNA expression was normalized by the scatter plots: of 25,697 genes, 631 genes were up-regulated, whereas 729 genes were down-regulated in Cmah-null micederived cochlear tissues compared to control micederived cochlear tissues (Fig. 2B). Additional File 1 and 2 showed complete lists of these genes.

Alteration of genes involved in oxidative damage and ROS metabolism
To clarify differential mRNAs expression of oxidative stress and antioxidant defense-related genes, we examined the expression of genes related to oxidative damage and ROS metabolism pathway in the cochlear tissues from WT-and Cmah-null mice, using a pathway-focused PCR array. RT-qPCR analysis revealed a significant down-regulation in most of gene expressions in Cmah-null mice with AHL, except one gene, Apoe (Table 3). Down-regulated genes consisted of 3 specific subsets for oxidative damage and dysregulated ROS metabolism as follows: 1) glutathion peroxidases (Gpxs), peroxiredoxins (Tpxs), peroxidases, and antioxidant-related genes, 2) superoxide dismutases (Sod), genes involved in superoxide metabolism, genes involved in ROS metabolism, and oxidative damage responsive genes for ROS metabolism, and 3) oxygen transporters. Therefore, these results suggested specific subsets of oxidative stress genes that appear to play an important role in AHL in the Cmah-null mice.

Cmah-null mice with hearing loss
To address the underlying mechanisms of AHL in Cmah-null mouse, we analyzed the expression of their genes that involved sirtuins, oxidative stress regulation, Wnt signaling, mitochondrial dysfunction identified by pathway analysis on microarray and a pathway-focused PCR array. As shown in Fig. 4A, Sirt3 protein expression was significantly reduced in cochlear tissues of Cmah-null mouse, compared to their control. Also, RT-qPCR analysis showed that Sirt3, 4, and 5 gene expressions is significantly reduced in Cmah-null mice than in controls (Fig. 4B). To determine the expression of AHL related genes in the cochlear tissues of Cmahnull mouse, we checked the expression profiles of these www.impactaging.com genes on the microarray analysis (Fig. 4C). The result shows that the data sets obtained from the microarray analysis accurately matched with RT-qPCR analysis in Cmah-null mouse ( Fig. 4C and D). Like that, expressions of Hif1α and Foxo3a mRNAs as transcription factor regulated by Sirt3 as well as Foxo1 and MnSod mRNAs expressions as downstream regulator of Sirt3 was decreased in Cmah-null mice (Fig. 4D).
To observe the regulatory factors in Wnt signaling, which might control by ROS in Cmah-null mouse, we analyzed the expression of Wnt signaling-related genes (Lrp5, Lrp6, Wnt3a, Wnt5a, Gsk3β, and Catenin). The result shows that Wnt signaling-related gene expressions in Cmah-null mouse were significantly decreased, whereas the Wnt receptor gene expression was not altered (Fig. 4D). Next, we examined mitochond-rial dysfunction-related gene expression. The expression of these genes (Crb1, Mtfp1, RhoT2, Soc2, and Atp5f1) with a role as regulators and mediators of mitochondrial molecular transport was significantly down-regulated (Fig. 4D). We therefore investigated whether altered genes affect the mitochondria expression in sensory neurons of Cmah-null mouse by immunofluorescence. As shown in Fig. 4E, mitochondrial positive signals were significantly decreased in Cmah-null mice, compared to control mice. Taken together, we proposed a key underlying mechanism for explaining AHL in Cmah-null mice (Fig. 4F), suggested that AHL in Cmah-null mice was caused by down-regulation of Sirt3 mRNA expression, which resulted in histological abnormalities in vestibular otoconial epithelia and the cochlear sensory epithelium, and neuronal cell loss in inner ears due to mitochondrial dysfunction by excessive oxidative stress.  Alzheimer's disease, Parkinson's disease, and Huntington's disease [31]. Also, we have identified stress response and Wnt signaling abnormality, and mitochondrial dysfunction, which are up-and downregulated by the loss of Cmah activity. Especially, we identified a molecular mechanism of oxidative stress regulated by Sirt3 expression, which lead to mitochondrial dysfunction by ROS, in Cmah-null mice. Therefore, we concluded that Sir3 dysfunction in Cmahnull mice altered various signaling pathways, which are responsible for the most common sensory disorder in the elderly.

Antioxidants
Generally, AHL is closely associated with an impaired inner ear, which generally shows loss of sensory hair cells and/or stria vascularis degeneration in the cochlea and neuronal loss (spiral ganglion neurons) in brain [32]. It is generally well-known that GFAP is a neural injury marker or astrocyte marker; NF for an axonal injury marker, VIM for a neural precursor cells marker in CNS, and NSE for neuronal marker. To this end, we studied the alteration of GFAP, NSE, NF, and VIM in cochlear tissues during different stages of AHL used Cmah-null mice mimicking AHL. Immunohistochemically there is combined loss of staining for GFAP, NF, and VIM in cochlear tissues of Cmah-null mice, whereas NSE staining patterns was optically not changed (Fig. 1). However, all of Gfap, Nse, Nf, and Vim mRNA expressions in Cmah-null mice are significantly down-regulated, compared to control. Of note, cochlear tissues of Cmah-null mice showed extensive apoptosis, whereas control mouse showed a moderate apoptosis. Our results suggested that there was a potential astrocyte regenerative response to neuron degeneration in the adult cochlear tissues during the onset and progression stages of AHL-like disease. Therefore, the regenerative responses in the adult cochlear tissues of AHL-like mice may be a potential pathway in attempting to repair the degenerated neurons and restore the dysfunctional neural circuitry.
Having identified differentially expressed genes between Cmah-null and control mice, we searched for differential gene expression profiles contributing to hearing loss with aging. In this study, we determined the 11 most significantly enriched pathways with differentially expressed genes identified on microarray analysis by KEGG pathway analysis to explore their functional role. Remarkably, most of them are associated with the oxidative stress, metabolism and degenerative nerve diseases, supporting the pathogenesis hypothesis of age-related dysfunction ( Table 1). Previous studies suggest that in case of AHL, oxidative stress is playing significant role. ROS generated in cochleae increase high-intensity noise [33]. Mice lacking antioxidant enzymes demonstrate enhanced age-related cochlear hair cell loss and susceptibility to noise-induced hearing loss [34,35]. Moreover, oxidative stress increases protein damage, which directly related to age in the cochleae of CBA mice [36]. To address the possibility that in Cmah-null mice the cochlear tissues generates excessive ROS, we examined gene expressions that involved in oxidative stress and antioxidant defense using a pathway-focused PCR array. We found that expression of antioxidant and ROS metabolism related genes was significantly downregulated and/or dysregulated in the cochlear tissues of Cmah-null mouse. These observations suggested that oxidative stress in the cochlear tissues of Cmah-null mouse played an important role in the pathogenesis of AHL.
In this study, we found that Wnt signaling (Lrp5, Lrp6, Wnt3a, Wnt5a, Gsk3β, and Catenin)-and mitochondrial molecular transport (Crb1, Mtfp1, RhoT2, Soc2, and Atp5f1)-regulator gene expressions in the cochlear tissues of Cmah-null mice with AHL were significantly down-regulated. Previous studies suggest that Wnt/βcatenin signaling play an important role in critical hepatic development, regeneration, and carcinogenesis [37][38][39]. The data from these studies suggested that βcatenin regulate hepatic energy metabolism and mitochondrial function [40]. Further, studies from mammalian cells and Caenorhabditis elegans showed that β-catenin and Foxos expression in response to oxidative stress have a cross-talk mechanism [41,42]. Taken our data with previous observations, ROS accumulations in the cochlear tissues of Cmah-null mouse might be caused by reduction of both antioxidant-and Wnt/β-catenin signaling-related gene expressions, which ultimately induced mitochondrial dysfunction (Table 3 and Fig. 4). In conclusion, these findings suggest that Cmah disruption could affect a variety of signaling pathways accompanying orchestrated gene expression changes. To the best of our knowledge, our results for the first time showed that down-regulated β-catenin expression in Wnt signaling give rise to ROS in the cochlear tissues of Cmah-null mice with AHL (Table 3).
Finally, we found that Hif1α and Foxo3a mRNAs as well as Foxo1 and MnSod mRNAs expressions, which located in downstream of Sirt3 gene expression, is significantly decreased. Also, we found that Sirt3 mRNA expression in the cochlear tissues of Cmah-null mouse is significantly down-regulated (Fig. 4). It is well known that Sirt3 are localized in mitochondria and that Sirt3 is the primary deacetylase, which plays an important role in energy production, metabolism, www.impactaging.com apoptosis, and cell signaling during aging [43][44][45]. Further, recent several studies also showed that Sirt3 is as an essential player to enhance glutathione antioxidant defense system in mitochondria under CR conditions and demonstrated that Sirt3-dependent mitochondrial adaptations may be a central mechanism of aging retardation in mammals [29,46,47]. These observation suggested that ROS accumulations in the cochlear tissues of Cmah-null mice with AHL down-regulated Sirt3 expression. As a result, Sirt3 dysregulation leads to oxidative damage and mitochondrial dysfunction (Fig. 4). Therefore, this study could provide a possible mechanism of Sirt3 in ROS and oxidative stress induced metabolism in Cmah-null mouse. Based on our results and previous reports, we suggested a hypothetical model that down-regulated Sirt3 lead to oxidative damage and mitochondrial dysfunction and could be alter various signaling pathways in Cmah-null mice with AHL. In conclusion, the present study suggests that mice with Cmah deficiency serve an important model for studying hearing loss with aging caused by evolutionary loss of Neu5Gc synthesis in human.
Of course, mice studies are not complementary to human. Furthermore, we cannot expect that mice with a human-like deficiency of Neu5Gc will mimic all consequences of Neu5Gc loss in a hominid ancestor. In this study, we selected Cmah-null mice as a model system to represents a state of at least partial characterization of biochemistry, physiology and metabolism and it could serve as a model system for studying hearing loss with aging caused by evolutionary loss of Neu5Gc synthesis in human. However many additional studies are required to prove hearing loss with aging caused by evolutionary loss of Neu5Gc synthesis in human. overnight. After the incubation, the samples were washed and incubated with horseradish peroxidaseconjugated secondary antibody. Samples were then stained with ImmPACT TM DAB peroxidase substrate (Vector Laboratories; CA, USA) to visualize the signal. Samples were also stained with Hematoxylin QS to provide background information for reference. The samples were mounted using VECTORSHIELD HardSet mounting medium (Vector Laboratories; CA, USA) and observed using fluorescence microscopy (Olympus; Japan). For immunofluorescence, the samples were incubated with primary antibodies for mitochondrial marker (1:1000 dilution, Abcam, Cambridge, UK) at 4°C overnight. After incubate with Alexa Fluor 488-labeled goat anti-mouse (1:500), the samples were mounted using VECTORSHIELD HardSet mounting medium with DAPI (Vector Laboratories) and observed using fluorescence microscopy (Olympus; Japan).

METHODS
Apoptotic cell staining. The terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) method was employed for detection of apoptosis using the in situ cell detection kit, tetramethylrhodamine red (TMR, red fluorescence) from Roche Applied Science (Lewes, East Sussex, UK) according to the manufacturer's instructions. For detection of the characteristic chromatin condensation and nuclear fragmentation associated with apoptosis, the samples were mounted using VECTORSHIELD HardSet mounting medium with DAPI (Vector Laboratories) and observed using fluorescence microscopy (Olympus; Japan).

Microarray analysis of gene expression. WT-and
Cmah-null mice with same age (12 months) and genetic background (C57BL/6J) were used (n=3 per each group) for microarray analysis. Total RNA was extracted and purified from the cochlear tissues of WTand Cmah-null mice using Trizol and RNeasy columns In silico data analysis. Differentially expressed genes (DEG) were subjected to intensive investigation to identify their biological functions. GO analysis was performed as described using DAVID (http://david.abcc.ncifcrf.gov) to study the biological function of the regulated genes [48]. The differentially expressed genes were compared to all of the genes on the microarrays and a P value computed using a Fisher's exact test. KEGG is a collection of online databases dealing with genomes, enzymatic pathways, and biological chemicals [49]. The KEGG PATHWAY Database records networks of molecular interactions in the cell that includes organism-specific network maps (http://www.genome.jp/kegg/). In addition, canonical pathways and functions analyses were created by IPA (Ingenuity Systems, Mountain View, CA, USA), which assists with microarray data interpretation via grouping DEG into known functions, pathways and gene interaction networks, based primarily on human and rodent studies. Significance was set at a P value of 0.05.
Pathway-focused PCR array. The Mouse Oxidative Stress and Antioxidant Defense RT² Profiler™ PCR array (SABiosciences Corp., Valencia, CA) was used to measure the alteration of gene expression in the cochlear tissues from WT-and Cmah-null mice. The RT 2 Profiler™ PCR Array (SABiosciences Corp.) is a commercially available set of optimized RT-qPCR primer assays on a 96-well plate. Total RNA was then treated with DNase I, reverse-transcribed using an RT 2 First Strand Kit (Qiagen), and brought to a final volume of 120 μL. cDNA from individual samples was used as a template for the PCR array, according to the array instructions, using SYBR green on an ABI ViiA TM 7 system (Applied Biosystems). Data were analyzed using SABiosciences RT2 Profiler PCR Data Analysis software, available at http://pcrdataanalysis. sabiosciences.com/pcr/arrayanalysis.php, and were considered significant at >4-fold change. Relative quantitation for each gene was determined by normalization to 5 housekeeping genes (Actb, B2m, Gapdh, Gusb, and Hsp90ab1), comparing the WT and Cmah-null groups by using the 2-ΔΔCt method.
Quantitative real-time polymerase chain reaction (RT-qPCR). The total RNA obtained from the cochlea of WT-and Cmah-null mice was reverse-transcribed with the QuantiTect Reverse Transcription Kit (Qiagen; Valencia, CA, USA) as recommended by the manufacturer. To evaluate gene expression, RT-qPCR was performed on an ABI ViiA TM 7 system (Applied Biosystems; Foster City, CA, USA) with SYBR Green as the fluorescence detection method (Bio-Rad; Hercules, CA, USA). The mouse Gadph gene was used as an internal control to normalize the RT-qPCR efficiency and for quantification of gene expression (Supplementary Table 1 and 2). The RT-qPCR was performed in triplicate for each sample.
Statistical analysis. All experimental data was presented as means ± standard deviation (SD). Each experiment was performed at least three times. For statistical analysis ( Fig. 1 and 4), one-way analysis of variance (ANOVA) was performed to determine whether there were differences among the groups, and Fisher's posttest was performed to determine significance between pairs of groups. In all experiments, *P < 0.05, **P < 0.01, and ***P < 0.001 were considered significant.