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The role of Efr3a in age-related hearing loss
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THE ROLE OF Efr3a IN AGE-RELATED HEARING LOSS
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HAIXIA HU, a,b,cy YAN MA, a,b,cy BIN YE, a,b,c QUAN WANG, a,b,c TAO YANG, a,b,c JINGRONG LV, a,b,c JUN SHI, a,b,c HAO WU b,c AND MINGLIANG XIANG a,b,c*
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a
Department of Otolaryngology & Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China b
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Ear Institute, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
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c
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Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai 200092, China
Abstract—Efr3a has been found to be involved in the functional maintenance and structural degeneration of sensory and motor nervous tissues. Our previous data have suggested that Efr3a may be associated with the initiation of the degeneration of spiral ganglion neurons (SGNs). In this study, we used Efr3a knockdown (Efr3a KD) and Efr3a overexpression (Efr3a OE) mice to determine the role of Efr3a in age-related hearing loss. Measurements of hearing thresholds showed that Efr3a had little or no influence on the hearing threshold at all frequencies in adult mice, whereas in an early stage of senescence, Efr3a reduction resulted in better hearing function, especially at 10 and 12 months of age. No significant differences were observed in hair cell loss among the three groups until 14 months. The number of surviving hair cells in the OE mice was lower than that in the KD mice. As indicated by the density of SGNs in the upper basal turn, the Efr3a OE mice displayed earlier and more severe degeneration than the KD mice. In addition, the p-Akt levels in the cochlear spiral ganglions were higher in adult Efr3a KD mice than in WT and OE mice, although there was no difference in Akt expression among the three groups. Our study suggests that down-regulation of Efr3a might improve hearing function and alleviate the degeneration of SGNs in an early stage of senescence, probably via enhancing the basal expression of activated Akt. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: age-related hearing loss, Efr3a, hair cell, spiral ganglion neuron, degeneration. 14
*Correspondence to: M. Xiang, Department of Otolaryngology & Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China. E-mail address: [email protected] (M. Xiang). y These authors have contributed equally to this work. Abbreviations: ABR, auditory brainstem response; AHL, age-related hearing loss; EDTA, ethylene diamine tetraacetic acid; IHCs, inner hair cells; OHCs, outer hair cells; PCR, polymerase chain reaction; SGN, spiral ganglion neuron. http://dx.doi.org/10.1016/j.neuroscience.2016.11.013 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 1
INTRODUCTION
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Age-related hearing loss (AHL) is a general characteristic of elderly animals and a common sensory disorder in elderly people (Someya and Prolla, 2010). As a progressive neurodegenerative disorder of aging, AHL affects nearly one-third of the population over 65 years of age and approximately 50% of the population over 75 years of age in the US, according to the National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD). AHL is usually not apparent at the onset and progresses slowly, starting at high frequencies. Most people are not conscious of a hearing problem until medium frequency dysfunction appears, which may affect daily communication. AHL is a multi-factorial disorder including cochlear aging, noise exposure and genetic predispositions, and it directly affects patients’ quality of life (Yamasoba et al., 2013). AHL can involve both peripheral and central changes (Someya et al., 2009; Martin del Campo et al., 2012). The detailed mechanisms underlying AHL, however, remain largely unknown. Experimental mouse models also exhibit AHL under normal housing conditions. The onset of AHL and the intrinsic hearing ability differs among different strains (Zheng et al., 1999; Jones et al., 2006). A previous study has indicated an earlier onset of high-frequency hearing loss and an earlier loss of functional outer hair cells (OHCs) at high-frequency areas in C57BL/6 mice than in CBA mice (Park et al., 2010). Therefore, the C57BL/6 mouse strain is an ideal model of early-onset hearing loss and has been widely used for aging research (Keithley et al., 2004; Shiga et al., 2005). The loss of hair cells and spiral ganglion neurons (SGNs) and the atrophy of the stria vascularis are the major features of the cochlea of elderly mice (Kinoshita et al., 2013). AHL progresses from high-frequency sounds to low-frequency sounds during aging and is accompanied by the degeneration of hair cells and SGNs beginning at the basal turn and progressing to the apical turn of the cochlea (Keithley et al., 2004). Efr3a, which is highly homologous to diacylglycerol lipase, has recently been found to be involved in the pathogenesis of degeneration. Its Drosophila homolog, rolling blackout (RBO), is also involved in the degeneration of organic structures and in the preservation of Drosophila sensory nerve function (Huang et al., 2004, 2006; Vijayakrishnan et al., 2009). Efr3a might be associated with an immediate response to hearing deficit and auditory remodeling (Munemoto et al., 2004). In addition, we have previously demonstrated up-regulation of Efr3a in SGNs during the early
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degeneration of the cochleae, thus suggesting that Efr3a may mediate the initiation of cochlear SGN degradation (Nie et al., 2015). To further explore the role of Efr3a in cochlear degeneration and to better understand the subtle morphological changes in the cochlea, we used Efr3a knockdown (Efr3a KD) and overexpression (Efr3a OE) C57BL/6 mice as animal models for AHL. We observed their hearing function and the degeneration of hair cells and SGNs and then explored a possible mechanism for the Efr3a-mediated effects in AHL.
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EXPERIMENTAL PROCEDURES
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Generation of Efr3a-/+ mutant and Efr3a OE mice Efr3a knockdown (Efr3a KD) mice were generated by breeding Efr3afl/fl mice with Ella-Cre mice, which exhibit efficient Cre activity in the one-cell zygote stage of embryonic development (Lakso et al., 1996). The progenies with the second exon of Efr3a deleted (Efr3a-/+) were backcrossed to C57BL/6 mice for over 10 generations to obtain inbred Efr3a-/+ mice. Efr3a overexpression (Efr3a OE, also referred to as Efr3a transgenic) mice were generated by transferring the Efr3a gene into the germ cell of C57BL/6 mice. The progenies of the Efr3a OE mice were also backcrossed to the C57BL/6 mice for over 10 generations. The Efr3a KD mice and the Efr3a OE mice were generously provided by the laboratory of Prof. Huang FD at the Chinese Academy of Sciences. All animals were raised in a specific pathogen-free (SPF) laboratory animal environment and given free access to both water and food. The humidity and temperature were kept at 60 ± 5% and 24 ± 2 °C, respectively. The lights were turned on between 7:00 am and 7:00 pm. The animal care and experimental procedures were approved by the Institutional Authority for Laboratory Animal Care of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China). Genotyping and Efr3a protein analysis
140–160 bp was ctaaaggtTccataggtgag for the WT mice and ctaaaggtGccataggtgag for the OE mice. The Efr3a protein expression in the C57BL/6 WT, Efr3a KD and OE mice was determined by western blotting as previously reported (Nie et al., 2015). The modiolus including the spiral ganglions (but not the intact cochlear organs) of eight cochleae were isolated, digested and centrifuged for further experimentation.
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Auditory brainstem response testing
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The auditory brainstem response (ABR) test was performed at 2 m, 6 m, 8 m, 10 m, 12 m and 14 m of age for each group of mice. The ABR was recorded using an ABR workstation of a MEB-3102 physiological response recorder (Nihon Kohden, Japan) as previously reported (Nie et al., 2015). Briefly, the mice were anaesthetized with ketamine (100 mg/kg) and xylazine (10 mg/ kg) and kept warm with an electric warming pad in a sound-proof room during ABR tests. The active electrode was inserted into the subcutaneous tissue of the vertex with a sterile stainless steel electrode, the reference electrode was inserted into the mastoid process, and the ground electrode was inserted into the contralateral thigh. Sound stimuli, consisting of pure tone bursts at frequencies of 4, 8, 16, 24 and 32 kHz were generated with a Tucker Davis Technologies device (SigGen) at a rate of 21.1 stimuli per second. For each test, 512 responses for each frequency were recorded, and the evoked potentials were filtered with a bandpass of 100–3000 Hz. Acoustic stimulation started at 90 dB for the 2-m and 6m groups and at 110 dB for the 8-m, 10-m, 12-m and 14-m groups, and the stimulation was decreased by 5 dB until the threshold was reached. The ABR threshold was determined as the lowest dB level of a reproducible waveform that could be detected.
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Preparation of cochlear tissue
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The cochleae of the mice were harvested at 6 m, 10 m and 14 m after ABR testing. The animals were euthanized under hyperanesthesia, and the cochleae were obtained from both sides. The cochleae were perfused with 4% phosphate-buffered paraformaldehyde (pH 7.5), immersed in the fixing solution overnight at
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The genotypes of the Efr3a-/+ (Efr3a KD) mice were determined by polymerase chain reaction (PCR) and Southern blotting. The mutant Efr3a-/+ allele was detected using primer F: 50 -TTATT TAGTATGTTGGACGAG-30 and primer R: 50 -ACAAACTTAACCTC CATGTT-30 . A 500-bp band was detected in the Efr3a-/+ mice, whereas no signal was detected in the wild-type mice (WT mice) (data not shown). The genotypes for the Efr3a transgenic (Efr3a OE) mice were determined by PCR and sequencing. For PCR analysis, the Efr3a OE mice were evaluated using primer F: 50 -CACTTGTAAG GAGTGGTGAAGGACCA-30 and Fig. 1. Expression of Efr3a in the spiral ganglions of cochleae from adult WT, Efr3a KD and OE primer R: 50 -ATTGTG mice. (A) Efr3a expression in the spiral ganglions of the cochleae determined by western blotting. CAAGGCCCTGGGCTTAAT-30 . The (B) Relative levels of the Efr3a protein detected by western blotting, with b-actin as the control. The *** sequence of the PCR product at data are presented as the mean ± SEM, n = 8. Represents p < 0.001 versus WT mice. Please cite this article in press as: Hu H et al. The role of Efr3a in age-related hearing loss. Neuroscience (2016), http://dx.doi.org/10.1016/j. neuroscience.2016.11.013
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Table 1. Average thresholds of ABR for C57BL/6 WT, Efr3a KD and Efr3a OE mice. *Indicates a significant (p < 0.05) difference between KD and WT mice. #Indicates a significant (p < 0.05) difference between KD and OE mice 2m (n = 16)
6m (n = 12)
8m (n = 12)
10m (n = 12)
12m (n = 10)
14m (n = 10)
4 kHz
KD WT OE
39.69 ± 1.48 41.56 ± 1.49 44.69 ± 2.01
49.17 ± 2.94 55.42 ± 2.50 53.75 ± 1.86
63.75 ± 2.62 68.75 ± 2.76 68.33 ± 1.28
70.83 ± 3.07 76.25 ± 2.14 79.58 ± 1.89#
83.00 ± 2.13 86.00 ± 3.32 91.50 ± 1.30#
87.50 ± 3.52 88.00 ± 2.91 94.50 ± 2.63
8 kHz
KD WT OE
26.56 ± 1.49 30.31 ± 1.40 29.38 ± 1.70
35.42 ± 2.92 40.83 ± 2.60 46.67 ± 2.71#
50.00 ± 2.04 54.58 ± 3.66 62.08 ± 3.22#
62.50 ± 2.26 64.17 ± 2.45 67.50 ± 1.44
70.00 ± 3.25 72.50 ± 3.35 77.50 ± 1.86
80.50 ± 2.17 81.50 ± 2.99 87.00 ± 2.49
16 kHz
KD WT OE
31.88 ± 2.03 34.69 ± 1.25 36.25 ± 2.30
43.33 ± 3.50 43.33 ± 3.39 52.08 ± 2.08
53.33 ± 2.49 57.92 ± 3.17 64.58 ± 2.57
60.00 ± 3.48 72.08 ± 3.28* 74.58 ± 1.68#
71.50 ± 3.42 83.50 ± 3.80* 85.00 ± 1.97#
81.50 ± 2.48 88.50 ± 2.12* 89.00 ± 2.67#
24 kHz
KD WT OE
42.81 ± 1.29 44.69 ± 1.40 47.81 ± 2.54
57.08 ± 3.96 57.50 ± 4.29 63.33 ± 2.49
84.58 ± 2.85 83.33 ± 5.20 85.00 ± 2.38
87.92 ± 2.64 95.83 ± 3.19* 97.92 ± 1.79#
91.50 ± 1.50 94.50 ± 4.31 100.00 ± 1.67#
94.50 ± 3.37 98.00 ± 1.33 99.00 ± 1.79
32 kHz
KD WT OE
49.06 ± 2.42 51.88 ± 2.58 55.63 ± 2.09
77.92 ± 4.28 77.92 ± 2.71 82.08 ± 1.99
96.67 ± 2.41 99.17 ± 2.74 102.50 ± 1.99
102.50 ± 1.68 103.75 ± 1.96 103.33 ± 1.55
103.00 ± 1.1 103.00 ± 2.13 104.00 ± 2.08
104.50 ± 2.29 104.00 ± 1.25 105.00 ± 1.29
#
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4 °C, and then decalcified in 10% EDTA solution at room temperature for 1 week.
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Hair cell counting
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After decalcification in EDTA, the organ of Corti was carefully isolated and separated into the apical and basal turns. Immunohistochemistry for myosin VIIa was performed to evaluate the cochlear hair cells. The detailed procedure was carried out using a method described previously (Nie et al., 2015), and the samples were mounted on glass slides. Images were acquired under a confocal fluorescent microscope (Zeiss LSM710).
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SGN density counting
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To count the number of SGNs, the cochleae were perfused with 2.5% glutaraldehyde and fixed for at least 2 hours. Afterward, the cochleae were decalcified with EDTA for 1 week and embedded in Eponate 12. Serial sections (1 lm thick) parallel to the modiolus were cut to count cochlear SGNs. Five cochleae were included in each group. In total, Fig. 2. (A) Baseline of the hearing thresholds (at 2 m of age) in WT, Efr3a KD and Efr3a OE mice. 6 cross-sections from Rosenthal’s (B–F) Mean ABR thresholds in C57BL/6 WT, Efr3a KD and OE mice of different ages at 4 kHz, canal were taken from each cochlea 8 kHz, 16 kHz, 24 kHz and 32 kHz. The data are presented as the mean ± SEM, n P 10. at an interval of approximately *Represents p < 0.05, **represents p < 0.01. 30 lm. The SGNs were detected with a 1% toluidine blue stain and Expression of Akt and p-Akt in the spiral ganglions observed under a light microscope. The SGN density For the 2-m-old WT, Efr3a KD and OE mice, the was calculated by dividing the perikaryon number by the modiolus, including the spiral ganglions in the cochleae, cross-sectional area of Rosenthal’s canal, which was were isolated, and the total protein was extracted as measured in NIH ImageJ software.
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mice. Compared with the WT and Efr3a OE mice, the Efr3a KD mice exhibited a slower progression of increasing thresholds at 16 kHz during aging. An early increase in the thresholds at 32 kHz was observed. The elevation was apparent at 8 m and progressed slowly to an almost complete loss of reaction to acoustical signals at older ages, without any significant differences among the three groups (Fig. 2F). At 10 m of age, the KD mice exhibited significant differences in the ABR threshold as compared with those of the WT and Efr3a OE mice at 16 and 24 kHz.
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described previously (Nie et al., 2015). Extracted protein was separated by 10% SDS–PAGE gels for 120 min at 80 V and transferred to a polyvinylidene fluoride membrane (Millipore, USA). After being blocked with 5% skim milk, membranes were incubated with antibodies against GAPDH (Beyotime, 1:1000), Akt (CST, USA; 1:1000), and p-Akt (Ser473) (CST, USA; 1:1000) overnight at 4 °C, and then incubated in HRP-conjugated secondary antibodies for 1 h at room temperature. The immunoreactive bands were detected by enhanced chemiluminescence with a BeyoECL Star kit (Beyotime Biotechnology) according to the manufacturer’s instructions, and the band intensity was semi-quantified using Image Lab software (v 5.2; Bio-Rad, USA).
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Statistical analysis
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The data for the ABR threshold, hair cell counting and SGN density were expressed as the mean ± the standard error, and SPSS statistical software (v 19.0; SPSS INC., USA) was used for all statistical analysis. Data were analyzed using a one-way ANOVA, and p < 0.05 was considered to be statistically significant.
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RESULTS
ABR testing indicated that an Efr3a decrease influenced the ABR threshold in an early stage of senescence, especially at 16 kHz and 24 kHz. To investigate whether the functional defects coincided with the degree of cochlear hair cell loss, we counted the number of hair cells in the basal turn of the basilar membrane. The basilar membranes of the cochleae were subjected to fluorescence staining after the ABR measurements. Degeneration of the hair cell was indicated by irregular arrangement and hair cell loss. As shown in Fig. 3A, at 6 m of age, the hair cells were well-maintained with
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Efr3a KD and OE mice were established successfully
Efr3a reduction did not reduce the loss of hair cells until 14 m of age
To confirm the reduction and overexpression of Efr3a in the Efr3a KD and OE mice, respectively, quantitative analysis of Efr3a protein in the cochleae was performed by western blotting. Consistently with the expected genotypes, the expression of Efr3a in the cochleae was lower and higher in the Efr3a KD and OE mice, respectively (Fig. 1). Efr3a reduction improved hearing function in an early stage of senescence The ABR test, a general method for evaluating hearing function, was performed to assess the progression of AHL in the C57BL/6 WT, Efr3a KD and Efr3a OE mice. The average thresholds at the ages of 2 m, 6 m, 8 m, 10 m, 12 m, and 14 m for pure tones at frequencies of 4, 8, 16, 24 and 32 kHz are shown in Table 1. The result showed that the baseline hearing thresholds at an age of 2 m had no significant difference among the WT, Efr3a KD and Efr3a OE mice at all frequencies (Fig. 2A). The thresholds at 4 kHz (Fig. 2B), 8 kHz (Fig. 2C), 16 kHz (Fig. 2D) and 24 kHz (Fig. 2E) increased gradually during aging in the three groups of
Fig. 3. Surviving cochlear hair cells in C57BL/6 WT, Efr3a KD and OE mice at different ages. (A) Fluorescence immunohistochemistry images of myosin VIIa-labeled IHCs and OHCs in the basal turn at 6 m, 10 m and 14 m of age. Scale bar = 50 lm. (B) Hair cell counts obtained from the basal turns at different ages. Data are presented as the mean ± SEM, n = 6. *Represents p < 0.05, ** represents p < 0.01.
Please cite this article in press as: Hu H et al. The role of Efr3a in age-related hearing loss. Neuroscience (2016), http://dx.doi.org/10.1016/j. neuroscience.2016.11.013
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three rows of OHCs and one row of inner hair cells (IHCs) and arranged in proper order in the three groups of mice. Occasionally, we observed some OHC loss, especially in the Efr3a OE group. The loss and irregular arrangement of the cochlear hair cells increased with age. Animals at 10 m of age began to show a dispersed loss of OHCs and individual IHC loss, particularly the WT and OE mice. By the age of 14 m, a disordered arrangement of OHCs and severe loss of hair cells were observed in the three groups. The surviving OHCs and IHCs progressively decreased during aging in all mice (Fig 3B). There was no statistically significant difference at 6 m and 10 m of age among the three groups. At 14 m of age, the number of surviving IHCs was higher in the KD mice than in the OE mice, and the number of surviving OHCs was higher than in the WT and OE mice. No significant difference in hair cells was observed between the WT and OE mice at all observed time points.
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Efr3a reduction increased the p-Akt levels in the cochlear spiral ganglions The morphological changes of the cochleae in aging mice suggested that the Efr3a deficiency resulted in slower and slighter degeneration of SGNs in Efr3a KD mice than in WT and OE mice. We further sought to determine the molecular mechanisms by which this change occurred by comparing the relative protein levels of Akt and p-Akt in the spiral ganglion among the adult WT, KD and OE mice. There was no significant difference in the Akt expression among the three groups or in the p-Akt levels between the WT and OE mice in the cochlear spiral ganglion (Fig. 5). In contrast, there was a clear increase in p-Akt (Ser473) levels in the Efr3a KD mice compared with those in the WT and OE mice.
Efr3a reduction suppressed SGN degeneration in an early stage of senescence Because Efr3a reduction improved the ABR threshold and did not reduce the loss of hair cells in an early stage of senescence, we investigated the morphology of the SGNs in the cochleae of the aging mice (Fig. 4B). The SGNs in the upper basal turn of the cochleae were quantitatively analyzed via light microscopy and NIH ImageJ software. The result showed that all mice in the WT, KD and OE groups exhibited a progressive loss of SGNs in Rosenthal’s canal. The Efr3a OE mice displayed both earlier and more severe degeneration (Fig. 4A). SGN densities (cells/mm2) decreased with age in all three groups of mice (Fig. 4C). No significant differences were observed between the WT and OE mice at any age. Interestingly, the SGN density in the Efr3a KD mice was greater than that in the Efr3a OE mice at 10 m and 14 m of age. In addition, the SGN densities in the upper basal turn were slightly lower in the WT mice than in the Efr3a KD mice at 10 m and 14 m of age, but the differences were statistically significant only at 10 m of age. Our results indicated that a decrease in Efr3a expression might slow down the degeneration of SGNs in an early stage of senescence.
Fig. 4. Serial light microscopy changes of the cochlear SGNs from C57BL/6 WT, Efr3a KD and OE mice at different ages. (A) Toluidine blue staining of SGNs in the cochleae. Scale bar = 50 lm. (B) Cochlea at low magnification. The red circle represents the upper basal turn where we counted the SGN density. (C) SGN density at the basal turn of the cochlea at different ages. Data are presented as the mean ± SEM, n = 5. *Represents p < 0.05, **represents p < 0.01, ***represents p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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deficiency and irregular arrangement in the older mice. In contrast, the loss of hair cells in the apical turns was lower, and no significant differences were present among the three groups (data not shown). These features were consistent with results of previous reports indicating that hair cell loss mainly occurred in the basal turn, in which the initial and most severe degeneration was present (Mizuta et al., 1993; Simonoska et al., 2009). There were no significant differences in the number of hair cells between the WT and OE mice at all observed time points, but the number of surviving OHCs in the KD mice was higher than that in the WT and OE mice. Similarly to the findings of Shiga et al. (Shiga et al., 2005), the loss of Fig. 5. Protein levels of Akt and p-Akt in the cochlear spiral ganglions of adult C57BL/6 WT, Efr3a IHCs in our study was sparse. HowKD and OE mice. (A) Levels of Akt and p-Akt protein detected by western blotting. (B) The relative ever, the hearing function combined gray intensity of the Akt and p-Akt proteins detected by western blotting, with comparison to with the extent of hair cell loss GAPDH. The data are presented as the mean ± SEM, n = 8. *Represents p < 0.05 (KD versus WT mice), #represents p < 0.05 (KD versus OE mice). between the KD and WT mice was not completely in agreement with the ABR thresholds. Therefore, the functional loss cannot occur because of DISCUSSION the morphological degeneration of cochlear hair cells In AHL, degeneration of the cochlear SGNs is a common alone, although this factor does contribute to auditory dysand slow process of neural degeneration. function. Some previous studies support the hypothesis Morphologically, this degeneration is similar to the dying that SGN loss is the predominant cause of AHL back process, which arises gradually from the distal to (Keithley et al., 1989; Slepecky et al., 2000). the proximal area in neurodegenerative diseases, such As a crucial part of the auditory system, the bipolar as Alzheimer’s disease (Iseki et al., 2001). Efr3a has cochlear SGNs are the primary neurons of the auditory recently been found to be associated with the pathogenepathway that carry auditory information from the hair sis of the dying back phenomenon. Previous studies have cells to the auditory center (Martinez-Monedero et al., shown that Efr3a might play an important role in auditory 2006; Richardson et al., 2006). Moreover, the effect of remodeling or might be involved in initiating the degenercochlear implantation is also thought to be related to the ation of cochlear SGNs after the deprivation of acoustic functional SGN number (Nadol et al., 1989; Gantz et al., signals (Munemoto et al., 2004; Nie et al., 2015). 1993; Xu et al., 2012). Interestingly, in our study, the The hearing function that we observed in the adult and Efr3a OE mice displayed both earlier and more severe aging WT mice was similar to that previously described degeneration than the KD mice at 10 m and 14 m of for C57BL/6 mice (Xiong et al., 2014; Pang et al., age, as indicated by the number of SGNs in the upper 2016). The baseline of ABR thresholds among the adult basal turn. The results indicated that a decrease in Efr3a WT, OE and KD mice was not significantly different at may slow down the degeneration of SGNs in an early all frequencies tested. However, compared with the WT stage of senescence. and Efr3a OE mice, the Efr3a KD mice exhibited a slower We then sought to determine the molecular increase in the hearing threshold during aging. Our results mechanisms underlying the slower degeneration rate of suggested that the decrease in Efr3a expression had little the cochleae in Efr3a KD mice. Rolling blackout (RBO), or no influence on the hearing threshold of the adult mice the Drosophila homolog of the Efr3a protein, forms a but resulted in better hearing function in an early stage of protein complex with PI4KIIIa and modulates the senescence especially at 10 m and 12 m of age. synthesis of PIP2 (Nakatsu et al., 2012). PIP2 can then Hearing loss is one of the most general sensory be converted into PIP3 through PI3K catalysis, thereby disorders, and it is mainly associated with hair cell generating phosphorylated Akt (p-Akt). The resulting pinjury, which causes AHL (Lee, 2013). To investigate Akt can then phosphorylate a large number of substrates, whether the degree of the hair cell loss corresponded with such as Bad, Bcl-2, and Forkhead family members, and hearing functional defects, we counted the hair cells by consequently blocks cell apoptosis (Datta et al., 1999; using immunohistochemistry for myosin VIIa. In the preManning and Cantley, 2007). More importantly, Akt defisent study, the degeneration of cochlear hair cells ciency can significantly elevate the ABR threshold appeared to occur in parallel with aging in all groups of (Brand et al., 2015; Chen et al., 2015), and the Akt and mice. The hair cells in the basal turn exhibited greater p-Akt levels appeared to decrease in different cochlear Please cite this article in press as: Hu H et al. The role of Efr3a in age-related hearing loss. Neuroscience (2016), http://dx.doi.org/10.1016/j. neuroscience.2016.11.013
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regions, including SGNs, during continuous exposure to moderate noise (Selivanova et al., 2007). Thus, we were curious whether the protein levels of Akt and p-Akt were different in the spiral ganglion of the cochlea among the adult WT, KD and OE mice. The results from the present study showed that although the level of total Akt was not different among the three groups, the level of the activated form of Akt (Ser473) was higher in the Efr3a KD mice than in the WT and OE mice. Given that activated Akt might be a critical regulator of cell survival, as well as the higher levels of p-Akt observed in Efr3a KD mice, it is well understood that the degeneration rate of the cochlea during the aging process in Efr3a KD mice is slower than that in WT and Efr3a OE mice.
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CONCLUSION
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Our study suggests that a decrease in Efr3a might improve hearing function and alleviate the degeneration of SGNs in an early stage of senescence, probably by enhancing the basal expression of activated Akt.
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AUTHOR CONTRIBUTION
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Conceived the project: M Xiang; designed the experiments: M Xiang, H Hu and Y Ma; wrote the manuscript: H Hu, M Xiang and T Yang; provided platform support: H Wu; Performed the experiments: H Hu, B Ye, Q Wang, J Lv and J Shi; Analyzed the data: H Hu and Y Ma.
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COMPETING INTERESTS
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The authors declare that they have no competing interest.
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CONSENT FOR PUBLICATION
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All authors consent for the publication of this study.
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Acknowledgments—We thank Prof. Fu De Huang’s lab in Chinese Academy of Sciences for generously providing the Efr3a knockdown and transgenic mice. This study was supported by grant No. 81271088 and No. 81670926 from the National Natural Science Foundation; No.15411950303 and No.14DZ2260300 from the Science and Technology Commission of Shanghai Municipality, China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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(Received 13 September , Accepted 9 November 2016) (Available online xxxx)
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THE ROLE OF Efr3a IN AGE-RELATED HEARING LOSS
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HAIXIA HU, a,b,cy YAN MA, a,b,cy BIN YE, a,b,c QUAN WANG, a,b,c TAO YANG, a,b,c JINGRONG LV, a,b,c JUN SHI, a,b,c HAO WU b,c AND MINGLIANG XIANG a,b,c*
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a
Department of Otolaryngology & Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China b
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Ear Institute, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
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c
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Shanghai Key Laboratory of Translational Medicine on Ear and Nose Diseases, Shanghai 200092, China
Abstract—Efr3a has been found to be involved in the functional maintenance and structural degeneration of sensory and motor nervous tissues. Our previous data have suggested that Efr3a may be associated with the initiation of the degeneration of spiral ganglion neurons (SGNs). In this study, we used Efr3a knockdown (Efr3a KD) and Efr3a overexpression (Efr3a OE) mice to determine the role of Efr3a in age-related hearing loss. Measurements of hearing thresholds showed that Efr3a had little or no influence on the hearing threshold at all frequencies in adult mice, whereas in an early stage of senescence, Efr3a reduction resulted in better hearing function, especially at 10 and 12 months of age. No significant differences were observed in hair cell loss among the three groups until 14 months. The number of surviving hair cells in the OE mice was lower than that in the KD mice. As indicated by the density of SGNs in the upper basal turn, the Efr3a OE mice displayed earlier and more severe degeneration than the KD mice. In addition, the p-Akt levels in the cochlear spiral ganglions were higher in adult Efr3a KD mice than in WT and OE mice, although there was no difference in Akt expression among the three groups. Our study suggests that down-regulation of Efr3a might improve hearing function and alleviate the degeneration of SGNs in an early stage of senescence, probably via enhancing the basal expression of activated Akt. Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved.
Key words: age-related hearing loss, Efr3a, hair cell, spiral ganglion neuron, degeneration. 14
*Correspondence to: M. Xiang, Department of Otolaryngology & Head and Neck Surgery, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China. E-mail address: [email protected] (M. Xiang). y These authors have contributed equally to this work. Abbreviations: ABR, auditory brainstem response; AHL, age-related hearing loss; EDTA, ethylene diamine tetraacetic acid; IHCs, inner hair cells; OHCs, outer hair cells; PCR, polymerase chain reaction; SGN, spiral ganglion neuron. http://dx.doi.org/10.1016/j.neuroscience.2016.11.013 0306-4522/Ó 2016 IBRO. Published by Elsevier Ltd. All rights reserved. 1
INTRODUCTION
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Age-related hearing loss (AHL) is a general characteristic of elderly animals and a common sensory disorder in elderly people (Someya and Prolla, 2010). As a progressive neurodegenerative disorder of aging, AHL affects nearly one-third of the population over 65 years of age and approximately 50% of the population over 75 years of age in the US, according to the National Institutes of Health/National Institute on Deafness and Other Communication Disorders (NIH/NIDCD). AHL is usually not apparent at the onset and progresses slowly, starting at high frequencies. Most people are not conscious of a hearing problem until medium frequency dysfunction appears, which may affect daily communication. AHL is a multi-factorial disorder including cochlear aging, noise exposure and genetic predispositions, and it directly affects patients’ quality of life (Yamasoba et al., 2013). AHL can involve both peripheral and central changes (Someya et al., 2009; Martin del Campo et al., 2012). The detailed mechanisms underlying AHL, however, remain largely unknown. Experimental mouse models also exhibit AHL under normal housing conditions. The onset of AHL and the intrinsic hearing ability differs among different strains (Zheng et al., 1999; Jones et al., 2006). A previous study has indicated an earlier onset of high-frequency hearing loss and an earlier loss of functional outer hair cells (OHCs) at high-frequency areas in C57BL/6 mice than in CBA mice (Park et al., 2010). Therefore, the C57BL/6 mouse strain is an ideal model of early-onset hearing loss and has been widely used for aging research (Keithley et al., 2004; Shiga et al., 2005). The loss of hair cells and spiral ganglion neurons (SGNs) and the atrophy of the stria vascularis are the major features of the cochlea of elderly mice (Kinoshita et al., 2013). AHL progresses from high-frequency sounds to low-frequency sounds during aging and is accompanied by the degeneration of hair cells and SGNs beginning at the basal turn and progressing to the apical turn of the cochlea (Keithley et al., 2004). Efr3a, which is highly homologous to diacylglycerol lipase, has recently been found to be involved in the pathogenesis of degeneration. Its Drosophila homolog, rolling blackout (RBO), is also involved in the degeneration of organic structures and in the preservation of Drosophila sensory nerve function (Huang et al., 2004, 2006; Vijayakrishnan et al., 2009). Efr3a might be associated with an immediate response to hearing deficit and auditory remodeling (Munemoto et al., 2004). In addition, we have previously demonstrated up-regulation of Efr3a in SGNs during the early
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degeneration of the cochleae, thus suggesting that Efr3a may mediate the initiation of cochlear SGN degradation (Nie et al., 2015). To further explore the role of Efr3a in cochlear degeneration and to better understand the subtle morphological changes in the cochlea, we used Efr3a knockdown (Efr3a KD) and overexpression (Efr3a OE) C57BL/6 mice as animal models for AHL. We observed their hearing function and the degeneration of hair cells and SGNs and then explored a possible mechanism for the Efr3a-mediated effects in AHL.
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EXPERIMENTAL PROCEDURES
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Generation of Efr3a-/+ mutant and Efr3a OE mice Efr3a knockdown (Efr3a KD) mice were generated by breeding Efr3afl/fl mice with Ella-Cre mice, which exhibit efficient Cre activity in the one-cell zygote stage of embryonic development (Lakso et al., 1996). The progenies with the second exon of Efr3a deleted (Efr3a-/+) were backcrossed to C57BL/6 mice for over 10 generations to obtain inbred Efr3a-/+ mice. Efr3a overexpression (Efr3a OE, also referred to as Efr3a transgenic) mice were generated by transferring the Efr3a gene into the germ cell of C57BL/6 mice. The progenies of the Efr3a OE mice were also backcrossed to the C57BL/6 mice for over 10 generations. The Efr3a KD mice and the Efr3a OE mice were generously provided by the laboratory of Prof. Huang FD at the Chinese Academy of Sciences. All animals were raised in a specific pathogen-free (SPF) laboratory animal environment and given free access to both water and food. The humidity and temperature were kept at 60 ± 5% and 24 ± 2 °C, respectively. The lights were turned on between 7:00 am and 7:00 pm. The animal care and experimental procedures were approved by the Institutional Authority for Laboratory Animal Care of Xinhua Hospital, Shanghai Jiao Tong University School of Medicine (Shanghai, China). Genotyping and Efr3a protein analysis
140–160 bp was ctaaaggtTccataggtgag for the WT mice and ctaaaggtGccataggtgag for the OE mice. The Efr3a protein expression in the C57BL/6 WT, Efr3a KD and OE mice was determined by western blotting as previously reported (Nie et al., 2015). The modiolus including the spiral ganglions (but not the intact cochlear organs) of eight cochleae were isolated, digested and centrifuged for further experimentation.
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Auditory brainstem response testing
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The auditory brainstem response (ABR) test was performed at 2 m, 6 m, 8 m, 10 m, 12 m and 14 m of age for each group of mice. The ABR was recorded using an ABR workstation of a MEB-3102 physiological response recorder (Nihon Kohden, Japan) as previously reported (Nie et al., 2015). Briefly, the mice were anaesthetized with ketamine (100 mg/kg) and xylazine (10 mg/ kg) and kept warm with an electric warming pad in a sound-proof room during ABR tests. The active electrode was inserted into the subcutaneous tissue of the vertex with a sterile stainless steel electrode, the reference electrode was inserted into the mastoid process, and the ground electrode was inserted into the contralateral thigh. Sound stimuli, consisting of pure tone bursts at frequencies of 4, 8, 16, 24 and 32 kHz were generated with a Tucker Davis Technologies device (SigGen) at a rate of 21.1 stimuli per second. For each test, 512 responses for each frequency were recorded, and the evoked potentials were filtered with a bandpass of 100–3000 Hz. Acoustic stimulation started at 90 dB for the 2-m and 6m groups and at 110 dB for the 8-m, 10-m, 12-m and 14-m groups, and the stimulation was decreased by 5 dB until the threshold was reached. The ABR threshold was determined as the lowest dB level of a reproducible waveform that could be detected.
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Preparation of cochlear tissue
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The cochleae of the mice were harvested at 6 m, 10 m and 14 m after ABR testing. The animals were euthanized under hyperanesthesia, and the cochleae were obtained from both sides. The cochleae were perfused with 4% phosphate-buffered paraformaldehyde (pH 7.5), immersed in the fixing solution overnight at
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The genotypes of the Efr3a-/+ (Efr3a KD) mice were determined by polymerase chain reaction (PCR) and Southern blotting. The mutant Efr3a-/+ allele was detected using primer F: 50 -TTATT TAGTATGTTGGACGAG-30 and primer R: 50 -ACAAACTTAACCTC CATGTT-30 . A 500-bp band was detected in the Efr3a-/+ mice, whereas no signal was detected in the wild-type mice (WT mice) (data not shown). The genotypes for the Efr3a transgenic (Efr3a OE) mice were determined by PCR and sequencing. For PCR analysis, the Efr3a OE mice were evaluated using primer F: 50 -CACTTGTAAG GAGTGGTGAAGGACCA-30 and Fig. 1. Expression of Efr3a in the spiral ganglions of cochleae from adult WT, Efr3a KD and OE primer R: 50 -ATTGTG mice. (A) Efr3a expression in the spiral ganglions of the cochleae determined by western blotting. CAAGGCCCTGGGCTTAAT-30 . The (B) Relative levels of the Efr3a protein detected by western blotting, with b-actin as the control. The *** sequence of the PCR product at data are presented as the mean ± SEM, n = 8. Represents p < 0.001 versus WT mice. Please cite this article in press as: Hu H et al. The role of Efr3a in age-related hearing loss. Neuroscience (2016), http://dx.doi.org/10.1016/j. neuroscience.2016.11.013
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Table 1. Average thresholds of ABR for C57BL/6 WT, Efr3a KD and Efr3a OE mice. *Indicates a significant (p < 0.05) difference between KD and WT mice. #Indicates a significant (p < 0.05) difference between KD and OE mice 2m (n = 16)
6m (n = 12)
8m (n = 12)
10m (n = 12)
12m (n = 10)
14m (n = 10)
4 kHz
KD WT OE
39.69 ± 1.48 41.56 ± 1.49 44.69 ± 2.01
49.17 ± 2.94 55.42 ± 2.50 53.75 ± 1.86
63.75 ± 2.62 68.75 ± 2.76 68.33 ± 1.28
70.83 ± 3.07 76.25 ± 2.14 79.58 ± 1.89#
83.00 ± 2.13 86.00 ± 3.32 91.50 ± 1.30#
87.50 ± 3.52 88.00 ± 2.91 94.50 ± 2.63
8 kHz
KD WT OE
26.56 ± 1.49 30.31 ± 1.40 29.38 ± 1.70
35.42 ± 2.92 40.83 ± 2.60 46.67 ± 2.71#
50.00 ± 2.04 54.58 ± 3.66 62.08 ± 3.22#
62.50 ± 2.26 64.17 ± 2.45 67.50 ± 1.44
70.00 ± 3.25 72.50 ± 3.35 77.50 ± 1.86
80.50 ± 2.17 81.50 ± 2.99 87.00 ± 2.49
16 kHz
KD WT OE
31.88 ± 2.03 34.69 ± 1.25 36.25 ± 2.30
43.33 ± 3.50 43.33 ± 3.39 52.08 ± 2.08
53.33 ± 2.49 57.92 ± 3.17 64.58 ± 2.57
60.00 ± 3.48 72.08 ± 3.28* 74.58 ± 1.68#
71.50 ± 3.42 83.50 ± 3.80* 85.00 ± 1.97#
81.50 ± 2.48 88.50 ± 2.12* 89.00 ± 2.67#
24 kHz
KD WT OE
42.81 ± 1.29 44.69 ± 1.40 47.81 ± 2.54
57.08 ± 3.96 57.50 ± 4.29 63.33 ± 2.49
84.58 ± 2.85 83.33 ± 5.20 85.00 ± 2.38
87.92 ± 2.64 95.83 ± 3.19* 97.92 ± 1.79#
91.50 ± 1.50 94.50 ± 4.31 100.00 ± 1.67#
94.50 ± 3.37 98.00 ± 1.33 99.00 ± 1.79
32 kHz
KD WT OE
49.06 ± 2.42 51.88 ± 2.58 55.63 ± 2.09
77.92 ± 4.28 77.92 ± 2.71 82.08 ± 1.99
96.67 ± 2.41 99.17 ± 2.74 102.50 ± 1.99
102.50 ± 1.68 103.75 ± 1.96 103.33 ± 1.55
103.00 ± 1.1 103.00 ± 2.13 104.00 ± 2.08
104.50 ± 2.29 104.00 ± 1.25 105.00 ± 1.29
#
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4 °C, and then decalcified in 10% EDTA solution at room temperature for 1 week.
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Hair cell counting
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After decalcification in EDTA, the organ of Corti was carefully isolated and separated into the apical and basal turns. Immunohistochemistry for myosin VIIa was performed to evaluate the cochlear hair cells. The detailed procedure was carried out using a method described previously (Nie et al., 2015), and the samples were mounted on glass slides. Images were acquired under a confocal fluorescent microscope (Zeiss LSM710).
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SGN density counting
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To count the number of SGNs, the cochleae were perfused with 2.5% glutaraldehyde and fixed for at least 2 hours. Afterward, the cochleae were decalcified with EDTA for 1 week and embedded in Eponate 12. Serial sections (1 lm thick) parallel to the modiolus were cut to count cochlear SGNs. Five cochleae were included in each group. In total, Fig. 2. (A) Baseline of the hearing thresholds (at 2 m of age) in WT, Efr3a KD and Efr3a OE mice. 6 cross-sections from Rosenthal’s (B–F) Mean ABR thresholds in C57BL/6 WT, Efr3a KD and OE mice of different ages at 4 kHz, canal were taken from each cochlea 8 kHz, 16 kHz, 24 kHz and 32 kHz. The data are presented as the mean ± SEM, n P 10. at an interval of approximately *Represents p < 0.05, **represents p < 0.01. 30 lm. The SGNs were detected with a 1% toluidine blue stain and Expression of Akt and p-Akt in the spiral ganglions observed under a light microscope. The SGN density For the 2-m-old WT, Efr3a KD and OE mice, the was calculated by dividing the perikaryon number by the modiolus, including the spiral ganglions in the cochleae, cross-sectional area of Rosenthal’s canal, which was were isolated, and the total protein was extracted as measured in NIH ImageJ software.
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mice. Compared with the WT and Efr3a OE mice, the Efr3a KD mice exhibited a slower progression of increasing thresholds at 16 kHz during aging. An early increase in the thresholds at 32 kHz was observed. The elevation was apparent at 8 m and progressed slowly to an almost complete loss of reaction to acoustical signals at older ages, without any significant differences among the three groups (Fig. 2F). At 10 m of age, the KD mice exhibited significant differences in the ABR threshold as compared with those of the WT and Efr3a OE mice at 16 and 24 kHz.
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described previously (Nie et al., 2015). Extracted protein was separated by 10% SDS–PAGE gels for 120 min at 80 V and transferred to a polyvinylidene fluoride membrane (Millipore, USA). After being blocked with 5% skim milk, membranes were incubated with antibodies against GAPDH (Beyotime, 1:1000), Akt (CST, USA; 1:1000), and p-Akt (Ser473) (CST, USA; 1:1000) overnight at 4 °C, and then incubated in HRP-conjugated secondary antibodies for 1 h at room temperature. The immunoreactive bands were detected by enhanced chemiluminescence with a BeyoECL Star kit (Beyotime Biotechnology) according to the manufacturer’s instructions, and the band intensity was semi-quantified using Image Lab software (v 5.2; Bio-Rad, USA).
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Statistical analysis
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The data for the ABR threshold, hair cell counting and SGN density were expressed as the mean ± the standard error, and SPSS statistical software (v 19.0; SPSS INC., USA) was used for all statistical analysis. Data were analyzed using a one-way ANOVA, and p < 0.05 was considered to be statistically significant.
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RESULTS
ABR testing indicated that an Efr3a decrease influenced the ABR threshold in an early stage of senescence, especially at 16 kHz and 24 kHz. To investigate whether the functional defects coincided with the degree of cochlear hair cell loss, we counted the number of hair cells in the basal turn of the basilar membrane. The basilar membranes of the cochleae were subjected to fluorescence staining after the ABR measurements. Degeneration of the hair cell was indicated by irregular arrangement and hair cell loss. As shown in Fig. 3A, at 6 m of age, the hair cells were well-maintained with
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Efr3a KD and OE mice were established successfully
Efr3a reduction did not reduce the loss of hair cells until 14 m of age
To confirm the reduction and overexpression of Efr3a in the Efr3a KD and OE mice, respectively, quantitative analysis of Efr3a protein in the cochleae was performed by western blotting. Consistently with the expected genotypes, the expression of Efr3a in the cochleae was lower and higher in the Efr3a KD and OE mice, respectively (Fig. 1). Efr3a reduction improved hearing function in an early stage of senescence The ABR test, a general method for evaluating hearing function, was performed to assess the progression of AHL in the C57BL/6 WT, Efr3a KD and Efr3a OE mice. The average thresholds at the ages of 2 m, 6 m, 8 m, 10 m, 12 m, and 14 m for pure tones at frequencies of 4, 8, 16, 24 and 32 kHz are shown in Table 1. The result showed that the baseline hearing thresholds at an age of 2 m had no significant difference among the WT, Efr3a KD and Efr3a OE mice at all frequencies (Fig. 2A). The thresholds at 4 kHz (Fig. 2B), 8 kHz (Fig. 2C), 16 kHz (Fig. 2D) and 24 kHz (Fig. 2E) increased gradually during aging in the three groups of
Fig. 3. Surviving cochlear hair cells in C57BL/6 WT, Efr3a KD and OE mice at different ages. (A) Fluorescence immunohistochemistry images of myosin VIIa-labeled IHCs and OHCs in the basal turn at 6 m, 10 m and 14 m of age. Scale bar = 50 lm. (B) Hair cell counts obtained from the basal turns at different ages. Data are presented as the mean ± SEM, n = 6. *Represents p < 0.05, ** represents p < 0.01.
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three rows of OHCs and one row of inner hair cells (IHCs) and arranged in proper order in the three groups of mice. Occasionally, we observed some OHC loss, especially in the Efr3a OE group. The loss and irregular arrangement of the cochlear hair cells increased with age. Animals at 10 m of age began to show a dispersed loss of OHCs and individual IHC loss, particularly the WT and OE mice. By the age of 14 m, a disordered arrangement of OHCs and severe loss of hair cells were observed in the three groups. The surviving OHCs and IHCs progressively decreased during aging in all mice (Fig 3B). There was no statistically significant difference at 6 m and 10 m of age among the three groups. At 14 m of age, the number of surviving IHCs was higher in the KD mice than in the OE mice, and the number of surviving OHCs was higher than in the WT and OE mice. No significant difference in hair cells was observed between the WT and OE mice at all observed time points.
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Efr3a reduction increased the p-Akt levels in the cochlear spiral ganglions The morphological changes of the cochleae in aging mice suggested that the Efr3a deficiency resulted in slower and slighter degeneration of SGNs in Efr3a KD mice than in WT and OE mice. We further sought to determine the molecular mechanisms by which this change occurred by comparing the relative protein levels of Akt and p-Akt in the spiral ganglion among the adult WT, KD and OE mice. There was no significant difference in the Akt expression among the three groups or in the p-Akt levels between the WT and OE mice in the cochlear spiral ganglion (Fig. 5). In contrast, there was a clear increase in p-Akt (Ser473) levels in the Efr3a KD mice compared with those in the WT and OE mice.
Efr3a reduction suppressed SGN degeneration in an early stage of senescence Because Efr3a reduction improved the ABR threshold and did not reduce the loss of hair cells in an early stage of senescence, we investigated the morphology of the SGNs in the cochleae of the aging mice (Fig. 4B). The SGNs in the upper basal turn of the cochleae were quantitatively analyzed via light microscopy and NIH ImageJ software. The result showed that all mice in the WT, KD and OE groups exhibited a progressive loss of SGNs in Rosenthal’s canal. The Efr3a OE mice displayed both earlier and more severe degeneration (Fig. 4A). SGN densities (cells/mm2) decreased with age in all three groups of mice (Fig. 4C). No significant differences were observed between the WT and OE mice at any age. Interestingly, the SGN density in the Efr3a KD mice was greater than that in the Efr3a OE mice at 10 m and 14 m of age. In addition, the SGN densities in the upper basal turn were slightly lower in the WT mice than in the Efr3a KD mice at 10 m and 14 m of age, but the differences were statistically significant only at 10 m of age. Our results indicated that a decrease in Efr3a expression might slow down the degeneration of SGNs in an early stage of senescence.
Fig. 4. Serial light microscopy changes of the cochlear SGNs from C57BL/6 WT, Efr3a KD and OE mice at different ages. (A) Toluidine blue staining of SGNs in the cochleae. Scale bar = 50 lm. (B) Cochlea at low magnification. The red circle represents the upper basal turn where we counted the SGN density. (C) SGN density at the basal turn of the cochlea at different ages. Data are presented as the mean ± SEM, n = 5. *Represents p < 0.05, **represents p < 0.01, ***represents p < 0.001. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
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deficiency and irregular arrangement in the older mice. In contrast, the loss of hair cells in the apical turns was lower, and no significant differences were present among the three groups (data not shown). These features were consistent with results of previous reports indicating that hair cell loss mainly occurred in the basal turn, in which the initial and most severe degeneration was present (Mizuta et al., 1993; Simonoska et al., 2009). There were no significant differences in the number of hair cells between the WT and OE mice at all observed time points, but the number of surviving OHCs in the KD mice was higher than that in the WT and OE mice. Similarly to the findings of Shiga et al. (Shiga et al., 2005), the loss of Fig. 5. Protein levels of Akt and p-Akt in the cochlear spiral ganglions of adult C57BL/6 WT, Efr3a IHCs in our study was sparse. HowKD and OE mice. (A) Levels of Akt and p-Akt protein detected by western blotting. (B) The relative ever, the hearing function combined gray intensity of the Akt and p-Akt proteins detected by western blotting, with comparison to with the extent of hair cell loss GAPDH. The data are presented as the mean ± SEM, n = 8. *Represents p < 0.05 (KD versus WT mice), #represents p < 0.05 (KD versus OE mice). between the KD and WT mice was not completely in agreement with the ABR thresholds. Therefore, the functional loss cannot occur because of DISCUSSION the morphological degeneration of cochlear hair cells In AHL, degeneration of the cochlear SGNs is a common alone, although this factor does contribute to auditory dysand slow process of neural degeneration. function. Some previous studies support the hypothesis Morphologically, this degeneration is similar to the dying that SGN loss is the predominant cause of AHL back process, which arises gradually from the distal to (Keithley et al., 1989; Slepecky et al., 2000). the proximal area in neurodegenerative diseases, such As a crucial part of the auditory system, the bipolar as Alzheimer’s disease (Iseki et al., 2001). Efr3a has cochlear SGNs are the primary neurons of the auditory recently been found to be associated with the pathogenepathway that carry auditory information from the hair sis of the dying back phenomenon. Previous studies have cells to the auditory center (Martinez-Monedero et al., shown that Efr3a might play an important role in auditory 2006; Richardson et al., 2006). Moreover, the effect of remodeling or might be involved in initiating the degenercochlear implantation is also thought to be related to the ation of cochlear SGNs after the deprivation of acoustic functional SGN number (Nadol et al., 1989; Gantz et al., signals (Munemoto et al., 2004; Nie et al., 2015). 1993; Xu et al., 2012). Interestingly, in our study, the The hearing function that we observed in the adult and Efr3a OE mice displayed both earlier and more severe aging WT mice was similar to that previously described degeneration than the KD mice at 10 m and 14 m of for C57BL/6 mice (Xiong et al., 2014; Pang et al., age, as indicated by the number of SGNs in the upper 2016). The baseline of ABR thresholds among the adult basal turn. The results indicated that a decrease in Efr3a WT, OE and KD mice was not significantly different at may slow down the degeneration of SGNs in an early all frequencies tested. However, compared with the WT stage of senescence. and Efr3a OE mice, the Efr3a KD mice exhibited a slower We then sought to determine the molecular increase in the hearing threshold during aging. Our results mechanisms underlying the slower degeneration rate of suggested that the decrease in Efr3a expression had little the cochleae in Efr3a KD mice. Rolling blackout (RBO), or no influence on the hearing threshold of the adult mice the Drosophila homolog of the Efr3a protein, forms a but resulted in better hearing function in an early stage of protein complex with PI4KIIIa and modulates the senescence especially at 10 m and 12 m of age. synthesis of PIP2 (Nakatsu et al., 2012). PIP2 can then Hearing loss is one of the most general sensory be converted into PIP3 through PI3K catalysis, thereby disorders, and it is mainly associated with hair cell generating phosphorylated Akt (p-Akt). The resulting pinjury, which causes AHL (Lee, 2013). To investigate Akt can then phosphorylate a large number of substrates, whether the degree of the hair cell loss corresponded with such as Bad, Bcl-2, and Forkhead family members, and hearing functional defects, we counted the hair cells by consequently blocks cell apoptosis (Datta et al., 1999; using immunohistochemistry for myosin VIIa. In the preManning and Cantley, 2007). More importantly, Akt defisent study, the degeneration of cochlear hair cells ciency can significantly elevate the ABR threshold appeared to occur in parallel with aging in all groups of (Brand et al., 2015; Chen et al., 2015), and the Akt and mice. The hair cells in the basal turn exhibited greater p-Akt levels appeared to decrease in different cochlear Please cite this article in press as: Hu H et al. The role of Efr3a in age-related hearing loss. Neuroscience (2016), http://dx.doi.org/10.1016/j. neuroscience.2016.11.013
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regions, including SGNs, during continuous exposure to moderate noise (Selivanova et al., 2007). Thus, we were curious whether the protein levels of Akt and p-Akt were different in the spiral ganglion of the cochlea among the adult WT, KD and OE mice. The results from the present study showed that although the level of total Akt was not different among the three groups, the level of the activated form of Akt (Ser473) was higher in the Efr3a KD mice than in the WT and OE mice. Given that activated Akt might be a critical regulator of cell survival, as well as the higher levels of p-Akt observed in Efr3a KD mice, it is well understood that the degeneration rate of the cochlea during the aging process in Efr3a KD mice is slower than that in WT and Efr3a OE mice.
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CONCLUSION
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Our study suggests that a decrease in Efr3a might improve hearing function and alleviate the degeneration of SGNs in an early stage of senescence, probably by enhancing the basal expression of activated Akt.
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AUTHOR CONTRIBUTION
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Conceived the project: M Xiang; designed the experiments: M Xiang, H Hu and Y Ma; wrote the manuscript: H Hu, M Xiang and T Yang; provided platform support: H Wu; Performed the experiments: H Hu, B Ye, Q Wang, J Lv and J Shi; Analyzed the data: H Hu and Y Ma.
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COMPETING INTERESTS
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The authors declare that they have no competing interest.
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CONSENT FOR PUBLICATION
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All authors consent for the publication of this study.
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Acknowledgments—We thank Prof. Fu De Huang’s lab in Chinese Academy of Sciences for generously providing the Efr3a knockdown and transgenic mice. This study was supported by grant No. 81271088 and No. 81670926 from the National Natural Science Foundation; No.15411950303 and No.14DZ2260300 from the Science and Technology Commission of Shanghai Municipality, China. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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(Received 13 September , Accepted 9 November 2016) (Available online xxxx)
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