Wnt activation protects against neomycin-induced hair cell damage in the mouse cochlea

Recent studies have reported the role of Wnt/β-catenin signaling in hair cell (HC) development, regeneration, and differentiation in the mouse cochlea; however, the role of Wnt/β-catenin signaling in HC protection remains unknown. In this study, we took advantage of transgenic mice to specifically knockout or overactivate the canonical Wnt signaling mediator β-catenin in HCs, which allowed us to investigate the role of Wnt/β-catenin signaling in protecting HCs against neomycin-induced damage. We first showed that loss of β-catenin in HCs made them more vulnerable to neomycin-induced injury, while constitutive activation of β-catenin in HCs reduced HC loss both in vivo and in vitro. We then showed that loss of β-catenin in HCs increased caspase-mediated apoptosis induced by neomycin injury, while β-catenin overexpression inhibited caspase-mediated apoptosis. Finally, we demonstrated that loss of β-catenin in HCs led to increased expression of forkhead box O3 transcription factor (Foxo3) and Bim along with decreased expression of antioxidant enzymes; thus, there were increased levels of reactive oxygen species (ROS) after neomycin treatment that might be responsible for the increased aminoglycoside sensitivity of HCs. In contrast, β-catenin overexpression reduced Foxo3 and Bim expression and ROS levels, suggesting that β-catenin is protective against neomycin-induced HC loss. Our findings demonstrate that Wnt/β-catenin signaling has an important role in protecting HCs against neomycin-induced HC loss and thus might be a new therapeutic target for the prevention of HC death.

Inner ear hair cells (HCs) are responsible for hearing. Aminoglycosides can be ototoxic and induce caspasemediated apoptosis in HCs. During mammalian inner ear development, canonical Wnt signaling is critical for otocyst induction and directs the formation of the vestibular organs. 1,2 Wnt signaling also has an important role in the cochlear HC development, and knockout of β-catenin inhibits HC differentiation from sensory progenitors thus reducing HC generation. 3,4 Recently, the Wnt signaling downstream target genes Lgr5 and Axin2 have been reported to mark inner ear HC progenitors. Lgr5-positive HC progenitors can self-renew to regenerate HCs after isolation in vitro and can spontaneously regenerate HCs after HC damage in the neonatal mouse cochlea in vivo. [5][6][7][8][9][10] Recent studies have also shown that Wnt signaling has dual roles in controlling the proliferation and differentiation of HC progenitors; 3,4 however, the role of Wnt/β-catenin signaling in HC survival and damage protection in the mouse cochlea remains unclear.
In other organs, the Wnt/β-catenin signaling pathway has been shown to function in various cell processes, including cellular protection. [11][12][13][14][15] The pro-survival activity of the Wnt pathway has been reported in many tissues, and is believed to be mediated by the induction of specific anti-apoptotic genes. 16,17 For example, in retinal ganglion cells Wnt activation reduces apoptosis by increasing the expression of protective growth factors including NT3, BDNF, and NGF. 11 In the intestine, overexpression of Wnt2a glycoprotein ligand of the Wnt proteins decreases bacterial-induced intestinal epithelial cell death. In the liver, Wnt/β-catenin signaling acts as a transcriptional co-activator of hypoxia inducible factor-1α signaling and has a protective role against hypoxia-induced liver injury. Forkhead box O3 transcription factor (Foxo3) and 1 Bim, which belong to the BCL-2 family members and are the downstream target gene of Foxo3, 18 have been reported to regulate the expression of stress-response proteins and to be involved in apoptosis in multiple organs. [19][20][21] Overactivation of Wnt signaling inhibits Foxo3-induced apoptosis through upregulation of serum and glucocorticoid-inducible kinase 1 (SGK1), 22 and overexpression of Wnt/β-catenin signaling inhibits Foxo3 signaling in 3,5-diethoxycarbonyl-1,4-dihydrocollidine (DDC)-induced liver injury. 23 However, the protective role of Wnt/β-catenin signaling against neomycin-induced HC loss in the mouse inner ear has been unclear.
In this study, we used loss-of-function and gain-of-function mouse models to investigate the role of Wnt/β-catenin signaling in protecting HCs against aminoglycoside-induced ototoxicity in the mouse cochlea both in vivo and in vitro. We found that β-catenin regulates Foxo3 and Bim expression and controls reactive oxygen species (ROS) levels, thus protecting HCs against caspase-mediated apoptosis after neomycin injury.
Western blot results revealed that the protein expression of β-catenin in HCs was significantly decreased in Gfi1-Cre/β-catenin flox(exon2-6) mice and increased in Gfi1-Cre/β-catenin flox(exon3) mice ( Figure 1e). The activation level of Wnt signaling in HCs was further confirmed by the mRNA expression of the Wnt downstream target genes Axin2 and Lgr5. Quantitative real-time PCR (qPCR) results showed that the mRNA expression of Axin2 and Lgr5 in HCs were both significantly decreased in Gfi1-Cre/β-catenin flox (exon2-6) mice and increased in Gfi1-Cre/β-catenin flox(exon3) mice ( Figure 1f). Finally, FM1-43, a marker of functional Figure 2 Knockout of β-catenin makes HCs more vulnerable to neomycin-induced ototoxicity in vivo. (a) The diagram of the assay for (b and c). Cochlear sensory epithelium samples from P2 wild-type mice were dissected out and allowed to recover for 12 h. The samples were treated with 1 mM neomycin for 6 h, allowed to recover for 3 h, and then used for immunostaining and western blot experiments. Samples for qPCR were collected at 0, 3, 6, 9, and 12 h after the beginning of neomycin treatment. (b and c) Immunofluorescence and western blot revealed the increased β-catenin expression in the HCs after neomycin injury. (d) qPCR results showed that the expression of β-catenin and the Wnt target genes Axin2 and Lgr5 were significantly upregulated after neomycin injury. ***Po0.001, **Po0.01, n = 5, versus no neomycin group. (e) The diagram of the assay for (f and g). Cochlear sensory epithelium samples from P2 Gfi1-Cre/β-catenin flox(exon2-6) mice were dissected out and allowed to recover for 12 h. The samples were treated with 1 mM neomycin for 6 h, allowed to recover for 24 h, and then stained with Myosin antibody. Littermates lacking the Gfi1-Cre allele were used as controls. (f and g) β-Catenin knockout mice had significantly greater HC loss than control mice in the apical and middle turns of the cochlea after neomycin treatment in newborn mice. Scale bar = 20 μm. ***Po0.001, **Po0.01, n = 5 mechanotransduction channels in HCs, was used to detect the mechanotransduction function of HCs in transgenic mice. FM1-43 staining revealed normal function of mechanotransduction channels in HCs in both Gfi1-Cre/β-catenin flox(exon2- 6) and Gfi1-Cre/β-catenin flox(exon3) mice ( Figure 1g).
The Wnt/β-catenin pathway was activated in the cochlear HCs after neomycin injury. We next explored the expression of β-catenin and Wnt target genes in the cochlear HCs after neomycin treatment. Both immunofluorescence and western blot results revealed increased β-catenin expression in the HCs after neomycin injury (Figures 2b and c). qPCR results showed that the expression of β-catenin and the Wnt target genes Axin2 and Lgr5 were significantly upregulated after neomycin treatment (Figure 2d). These results demonstrated that the Wnt/β-catenin pathway was activated in the cochlear HCs after neomycin injury, indicating that Wnt/β-catenin might be a protective physiological mechanism against neomycin injury.
Knockout of β-catenin makes the HCs more vulnerable to neomycin-induced ototoxicity in vitro. In this experiment, cochlear sensory epithelium samples from postnatal day (P) 2 Gfi1-Cre/β-catenin flox(exon2-6) and control mice were cultured in vitro and treated with neomycin. Without neomycin, there were no reductions in HCs either in control cochleae or in Gfi1-Cre/β-catenin flox(exon2-6) cochleae (Figures 2f and g). With neomycin treatment, Gfi1-Cre/β-catenin flox(exon2-6) cochleae had significantly greater HC loss than control cochleae in the apical and middle turns (Figures 2f and g and Supplementary Table 1). This result suggested that β-catenin has an important role in regulating the sensitivity of cochlear HCs to neomycin-induced injury.
Overactivation of the Wnt/β-catenin signaling pathway protects against neomycin-induced HC damage in vitro. To investigate the protective role of β-catenin against neomycin-induced HC damage, we used the Wnt agonist Bio and β-catenin overexpressing transgenic mice in two independent in vitro experiments. First, cochlear sensory epitheliums from P2 wild-type (WT) mice were cultured with Bio (5 μM) for 48 h (Figure 3a). Immunofluorescence results demonstrated upregulated expression of β-catenin in Biotreated HCs (Figure 3b). qPCR data showed that Bio-treated cochleae had significantly higher expression of the Wnt downstream target genes Lgr5 and Axin2 than the control group ( Figure 3c). When treated with neomycin in the presence of Bio, Myosin7a staining showed that Bio-treated cochleae had significantly less HC loss than the control cochleae in all three turns (Figures 3d and e and   Supplementary Table 1), which suggested that Wnt/β-catenin signaling could protect HCs against neomycin-induced damage in vitro.
In a separate experiment, we investigated the protective role of β-catenin using Gfi1-Cre/β-catenin flox(exon3) transgenic mice. Compared with control cochleae, Gfi1-Cre/β-catenin flox(exon3) cochleae had significantly reduced HC loss in all three turns after neomycin treatment (Figures 3g and h and Supplementary  Table 1), which was consistent with the Wnt agonist treatment and demonstrated that overexpression of Wnt/β-catenin protects against neomycin-induced HC damage in vitro.
Knockout of β-catenin in HCs leads to partial hearing loss and scattered HC loss in vivo. To investigate the role of β-catenin in HC survival in vivo, we measured hearing function of Gfi1-Cre/β-catenin flox(exon2-6) mice using pure-tone auditory brainstem response (ABR) and then dissected out the cochlear sensory epithelium for immunohistochemistry staining at P30 and P60 ( Figure 4a). The same litter control mice have normal hearing, and no HC loss was observed at P30 or P60 (Figures 4b and d). In Gfi1-Cre/β-catenin flox(exon2-6) mice, we observed a 5-10 dB threshold shift at P30 and a 5-15 dB threshold shift at P60 compared with controls (Figures 4c and d). We also found scattered HC loss in middle and basal turns, but the total HC number showed no significant difference compared with controls at P30 and P60 (Figures 4e and f). These results demonstrated that deletion of β-catenin in HCs leads to partial hearing loss and scattered HC loss in vivo.
Overexpression of Wnt/β-catenin protects against neomycin-induced hearing loss and HC loss in vivo. Here, Gfi1-Cre/β-catenin flox(exon3) transgenic mice were given daily subcutaneous injections of neomycin from P7 to P14, which is the ototoxic-sensitive period in the cochlea. 25 At P30 and P60, we measured hearing function and then dissected out the cochlear sensory epithelium for immunohistochemistry staining (Figure 5a). Control mice had significant hearing loss, and the ABR thresholds were significantly increased at all frequencies at both P30 and P60 ( Figure 5c). In Gfi1-Cre/β-catenin flox(exon3) mice, the ABR threshold shifts were significantly lower at all frequencies compared with the control littermates at both P30 and P60 (Figure 5c), suggesting that overexpression of Wnt/β-catenin protects against neomycin-induced hearing loss in vivo. Immunohistochemistry results showed that Gfi1-Cre/β-catenin flox(exon3) mice had significantly reduced outer hair cell (OHC) loss compared with the control littermates at both P30 and P60 (Figures 5b, d and e and Supplementary Table 2). This demonstrated that overexpression of Wnt/β-catenin (e and f) Statistical data showing that the total HC number was not significantly different between Gfi1-Cre/β-catenin flox(exon2-6) and control mice. Scale bar = 20 μm. n = 5 protects against neomycin-induced OHC loss in vivo. There was almost no inner hair cell (IHC) loss in either Gfi1-Cre/βcatenin flox(exon3) mice or control littermates at P30 or P60 (Figures 5b and f).
Foxo3 expression is regulated by Wnt/β-catenin signaling in HCs after neomycin injury. Previous studies have reported that Wnt/β-catenin inhibits the pro-apoptotic transcription factor Foxo3 and protects against oxidative stress-induced apoptosis though downregulation of Foxo3. 14,15,28 We investigated the Foxo3 expression in neomycin-treated cochleae. At 6 h after neomycin treatment, intense nuclear Foxo3 staining was observed in control HCs, which is indicative of active Foxo3 signaling in response to neomycin-induced HC damage (Figure 8b). In Gfi1-Cre/βcatenin flox(exon3) cochleae, HCs had significantly reduced Foxo3 staining intensity (Figure 8b). In Gfi1-Cre/β-catenin flox(exon2-6) cochleae, HCs had significantly greater Foxo3 staining intensity (Figure 8b). qPCR and western blot data showed that Foxo3 expression was significantly reduced in Gfi1-Cre/β-catenin flox(exon3) cochleae and significantly increased in Gfi1-Cre/β-catenin flox(exon2-6) cochleae compared with the controls (Figures 8d and e). Bim is one of the BCL-2 family members participating in the progress of apoptosis, and is the downstream target gene of Foxo3. 18 qPCR data revealed that Bim expression was also significantly decreased in Gfi1-Cre/β-catenin flox(exon3) cochleae and increased in Gfi1-Cre/β-catenin flox(exon2-6) cochleae (Figure 8d). These results suggested that after neomycin injury Foxo3 and Bim expression was inhibited when β-catenin was overexpressed in HCs and increased when β-catenin was knocked out in HCs. We also noticed that the mRNA expression of Foxo1, which is another member of the Foxo protein superfamily, was not significantly changed (Figure 8d), suggesting that Foxo1 might not be regulated by Wnt signaling in the cochlea.
Antioxidant treatment rescues β-catenin deficiencyinduced HC loss after neomycin injury. To further investigate whether the increase in ROS levels contributes to the increased injury sensitivity to aminoglycosides of β-catenin-deficient HCs, the antioxidant N-acetylcysteine (NAC), which is a reduced glutathione provider and a direct scavenger of reactive oxygen intermediates, 34 was used to treat the explant cultured cochlea with neomycin injury. After NAC treatment, HC loss dramatically decreased in both Gfi1-Cre/β-catenin flox(exon2-6) and control mice, and the number of surviving HCs was not significantly different between Gfi1-Cre/β-catenin flox(exon2-6) and control mice (Figures 10b and c). Moreover, MitoSOX Red immunofluorescence showed that ROS levels significantly decreased in HCs of Gfi1-Cre/β-catenin flox(exon2-6) mice after NAC treatment (Figures 10e and f), suggesting that the rescue of the HCs was associated with a decrease in oxidative stress. Together, these results showed that antioxidant treatment successfully rescued the β-catenin deficiency-induced HC loss in Gfi1-Cre/β-catenin flox(exon2-6) mice after neomycin injury, and demonstrated that ROS accumulation was the  major cause of the high injury sensitivity to aminoglycosides in β-catenin-deficient HCs.

Discussion
The role of Wnt/β-catenin signaling in cochlear development and HC regeneration has been extensively studied in the mouse inner ear. Recently, Wnt/β-catenin has been reported to be required for HC differentiation in the mouse cochlea. Knockout of β-catenin inhibits prosensory cells from differentiating into HCs, but β-catenin is not required to maintain HC fate once it is specified. 4 However, whether Wnt/β-catenin signaling is required for HC survival has not been investigated. In this study, we observed increased susceptibility of HCs to Previous studies reported that several pathways are involved in β-catenin deficiency-induced cell death, including increased apoptosis of hepatic progenitor cells due to enhanced expression of cleaved caspase-9 and caspase-3 when β-catenin expression is blocked, 16 increased apoptosis in cisplatin-resistant lung adenocarcinoma cells when DKK3 is used to inhibit the Wnt/β-catenin pathway, 35 and increased si-LGR5-induced apoptosis when the mitochondrial membrane potential is disrupted in colorectal cancer cells. 36 In our study, significantly greater HC loss was observed in β-catenin knockout cochleae compared with controls after neomycin treatment (Figure 2), which was accompanied by upregulation of the pro-apoptotic transcription factor Foxo3 and its downstream target gene Bim (Figure 8). Foxo3 is a pro-apoptotic transcription factor that regulates the expression of stressresponse proteins and leads to apoptosis in many tissues. [19][20][21] In neuronal cells, activation of Foxo3 induces two sequential ROS waves by induction of its transcriptional target Bim. 18 ROS can oxidize cell constituents, such as DNA, and can lead to DNA damage that activates multiple apoptotic pathways, including caspase-mediated apoptosis and p53-dependent apoptosis. [37][38][39] Foxo3 also activates an ROS rescue pathway by inducing Sestrin3, which is responsible for the biphasic ROS accumulation. 18 Previous studies have reported that Wnt/β-catenin protects against oxidative Figure 10 The NAC rescue assay. (a) The scheme of the assay for (b and c). P2 Gfi1-Cre/β-catenin flox(exon2-6) cochlear epithelium samples were dissected out and cultured with 1 mM neomycin for 6 h with NAC (20 μM), then allowed to recover for 24 h in the presence of NAC before analysis. (b) Myosin7a immunofluorescence showed that NAC treatment rescued β-catenin-deficient HCs from neomycin injury. (c) Statistical data showing that the number of surviving HCs was not significantly different between Gfi1-Cre/βcatenin flox(exon2-6) and control mice after NAC treatment. (d) The scheme of the assay for (e and f). P2 Gfi1-Cre/β-catenin flox(exon2-6) cochlear epithelium samples were dissected out and cultured with 1 mM neomycin for 6 h with NAC (20 μM), then allowed to recover for 6 h in the presence of NAC before analysis. (e) MitoSOX Red immunofluorescence showed that ROS levels significantly decreased in the HCs of Gfi1-Cre/β-catenin flox(exon2-6) mice after NAC treatment. (f) The number of Myosin7a/MitoSOX Red double-positive cells. Scale bar = 20 μm. ***Po0.001. n.s., no significant difference. n = 5 stress-induced apoptosis through downregulation of Foxo3. 14,15,28 In β-catenin-deficient cochleae, we found that upregulation of Foxo3 expression was accompanied by decreased expression of antioxidant enzymes (Figure 9), increased mitochondrial ROS accumulation (Figure 9), and significantly higher expression levels of Casp3, Casp9, Bax, Apaf1, and p53 (Figures 6 and 7), suggesting that the increased susceptibility of β-catenin-deficient HCs to neomycin treatment is attributed to Foxo3 activation and ROS accumulation. The precise role of Foxo3 in the oxidative stress in cochlear HCs needs to be investigated in the future.
Previous studies reported that several genes have protective functions against aminoglycoside-induced HC loss. Overexpression of XIAP inhibits caspase expression and prevents neomycin-induced HC death and subsequent hearing loss. 25 Insulin-like growth factor 1 (IGF-1) protects HCs from aminoglycosides by upregulating growth-associated protein 43 and netrin 1. 40 In many organs and cell lines, Wnt/β-catenin has been reported to have a protective function against apoptosis. In human HCT116 colon cancer cells, Wnt/β-catenin negatively regulates the pro-apoptotic transcription factor Foxo3 and inhibits Foxo3-induced apoptosis. 28 In the liver, Wnt/β-catenin protects against hepatotoxin DDC-induced liver injury and inhibits Foxo3 expression thus inhibiting oxidative stress-induced apoptosis. 14 In the rat sensory epithelium OC1 cell line, Wnt/β-catenin protects the OC1 cells against cisplatin-induced cell death. 41 Here, we found that after neomycin treatment overexpression of β-catenin in mouse HCs significantly inhibits the expression of Foxo3 and Bim (Figure 8), enhances the expression of antioxidant enzymes (Figure 9), reduces ROS accumulation (Figure 9), and inhibits caspase-induced apoptosis (Figures 6  and 7), and thus protects HCs against neomycin-induced damage (Figures 3 and 5). These results indicate that Wnt/β-catenin has an important role in protecting against neomycin-induced HC damage.
Aminoglycosides are widely used in clinics to treat bacterial infections, but all aminoglycosides have ototoxic side effects, which limit their clinical use. Mammalian sensory HCs have many mitochondria and high oxygen consumption, which makes them very sensitive to oxidative stress, especially when challenged by external stimulation such as noise or aminoglycosides. 42 Here, we found that knockout of β-catenin in HCs increases caspase-mediated HC apoptosis after neomycin treatment and that overexpression of β-catenin in HCs inhibits caspase-mediated HC apoptosis after neomycin treatment (Figures 6 and 7). Besides the canonical Wnt/ β-catenin pathway, two β-catenin-independent pathways have been described, including the Wnt/Ca 2+ and Wnt/PCP (planar cell polarity) pathways. 43,44 In the zebrafish lateral line, after exposure to aminoglycosides, dying HCs undergo a transient increase in intracellular Ca 2+ that occurs shortly after mitochondrial membrane potential collapse. Inhibition of intracellular Ca 2+ elevation mitigates toxic effects of aminoglycoside exposure. 45 Under physiological conditions, calcium and ROS act as signaling molecules inside the cell and their pathways can interact. However, under pathological conditions dysfunction in either of the systems might affect the other system thus potentiating harmful effects that might contribute to cell death. 46 The role of non-canonical Wnt signaling in cochlear HC survival and the role of calcium overload in β-catenin deficiency-induced increased HC susceptibility to neomycin need to be investigated in the future.
Previous studies reported that Wnt/β-catenin is protective against oxidative stress-induced apoptosis through inhibition of Foxo3 in the liver, bones, and SH-SY5Y cells. 14,15,47 There are also other papers that suggest that Wnt has the opposite effect on mitochondria. Yoon et al. 48 reports that increased Wnt signals are a potent activator of mitochondrial biogenesis and ROS generation, leading to DNA damage and acceleration of cellular senescence in primary cells. 48 This might be because Wnt signaling has diverse functions in different environments and stages, and sometimes these effects are in opposition to each other. For example, promoting proliferation and promoting differentiation are usually two opposing effects, and during embryonic stages Wnt/β-catenin signaling promotes proliferation during early mitotic phases of development and also promotes HC differentiation in the differentiating organ of Corti. 3 Here, we found that neomycin-induced HC damage was accompanied by Foxo3 upregulation and mitochondrial ROS accumulation. Knockout of β-catenin in HCs upregulated Foxo3 expression and increased the accumulation of ROS even more, while overexpression of β-catenin in HCs inhibited Foxo3 expression and decreased the accumulation of ROS after neomycin injury. This finding indicates that Wnt/β-catenin protects HCs against neomycin injury by regulating Foxo3 expression and controlling ROS levels.
In summary, we showed that deletion of β-catenin in HCs increases neomycin-induced HC loss. Next, we reported that overexpression of β-catenin in HCs protects against neomycin-induced HC loss. Last, we demonstrated that Wnt/β-catenin signaling in HCs regulates Foxo3 expression, antioxidant enzymes, and ROS levels, thus protecting HCs against caspase-mediated apoptosis after neomycin injury. Our data suggest that Wnt/β-catenin signaling is essential for HC protection against neomycin-induced HC loss, and thus might be a new therapeutic target for the prevention of aminoglycoside-induced HC death.
Mice were housed with open access to food and water at the Experimental Animal Center, Shanghai Medical College of Fudan University, China. Postnatal day (P) 0 was defined as the day of birth. Mice received a daily subcutaneous injection of neomycin (200 mg/kg) or sterile saline from P7 to P14. This study was carried out in strict accordance with the 'Guiding Directive for Humane treatment of Laboratory Animals' issued by the Chinese National Ministry of Science and Technology in September 2006. All experiments were approved by the Shanghai Medical Experimental Animal Administrative Committee (Permit Number: 2009-0082). All efforts were made to minimize suffering and reduce the number of animals used.
Organotypic culture of neonatal mice cochlea. The mice were killed at P2, then the cochlear sensory epithelium was isolated and seeded intact on a glass coverslip coated with Cell-Tak (BD Biosciences, Franklin Lakes, NJ, USA). 52 The explanted cochleae were treated with 1 mM neomycin (Sigma-Aldrich, St. Louis, MO, USA) and/or 5 μM Bio (Sigma-Aldrich) or 20 mM NAC (Sigma-Aldrich). PBS was used as the vehicle control.
ABR test. The hearing thresholds of the mice were examined with the ABR test. In this test, changes in the electrical activity of the brain in response to sound were recorded via electrodes that were placed on the scalp of the mice. Animals were anesthetized with ketamine (100 mg/kg) and xylazine (25 mg/kg) and placed on a thermostatic heating pad in a sound-attenuating chamber to maintain their body temperatures at 38°C. Frequency-specific auditory responses were measured using the Tucker-Davis Technology system III (Tucker-Davies Technologies, Gainesville, FL, USA) as previously described. 53 All ABR tests were performed on mice older than P21.
Tissue preparation for quantitative RT-PCR and western blot. After killing the mice, the otic capsule was immediately isolated, rapidly frozen in liquid nitrogen, and stored at − 70°C until further processing. To obtain the total RNA, 10 cochleae were pooled in TRIzol (Invitrogen, Carlsbad, CA, USA) following the manufacturer's instructions. The RNA concentration was measured with a Bio-Rad spectrophotometer (Applied Biosystems, Foster City, CA, USA). cDNA was synthesized from 1 μg total RNA by reverse transcription using the GoScript Reverse Transcription System (Promega, Madison, WI, USA) following the manufacturer's protocols. qPCR was performed using GoTaq qPCR Master Mix (Promega) on a Bio-Rad 7500 detection system (Applied Biosystems, Foster City, CA, USA). GAPDH was used as a housekeeping gene for control purposes. Primer sequences are listed in Supplementary Table 6. For protein extraction, 10 cochleae were pooled in 100 μl RIPA lysis buffer with 1% PMSF, sonicated, incubated on ice for 30 min, and stored at − 80°C. Extracts were boiled with 5 × loading buffer, subjected to PAGE (Mini-Protean TGX Systems; Bio-Rad, Hercules, CA, USA), transferred onto an Immobilon-P membrane (Millipore, Bedford, MA, USA), probed with anti-Foxo3 antibody (Cell Signaling Technology, Danvers, MA, USA), anti-βcatenin (BD Biosciences), and anti-GAPDH (Kangchen Biotech, Shanghai, China) and finally incubated with HRP-conjugated secondary antibodies. Signal was detected with the Supersignal West Femto Trial Kit (Thermo Fisher Scientific, Rockford, IL, USA) on a FluorChem M system (ProteinSimple, San Jose, CA, USA).
Cell counts. For HC quantification in the neomycin-treated samples, we imaged the entire cochlea using a 40 × 3 objective and counted the Myosin7a+ HCs that remained. The same procedure was used to quantify cleaved caspase-3+/ Myosin7a+, TUNEL+/Myosin7a+ cells, and myosin7a+/MitoSOX Red+ cells. For all experiments, only one cochlea from each mouse was used for immunofluorescence and quantification. Thus, n represents the number of mice examined.
Statistical analyses. Statistical analyses were conducted using Microsoft Excel and GraphPad Prism software (GraphPad Software, La Jolla, CA ,USA). Data were expressed as mean ± S.E.M. ABR thresholds were analyzed by two-way ANOVA followed by a Newman-Keuls post hoc test. Immunofluorescence analysis was performed with a two-tailed, unpaired Student's t-test when comparing two groups or with a one-way ANOVA followed by a Dunnett's multiple comparisons test when comparing more than two groups. Po0.05 was considered as statistically significant.