Over-expression of X-linked inhibitor of apoptosis protein slows ...

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Neurobiology of Aging xxx (2008) xxx–xxx

Over-expression of X-linked inhibitor of apoptosis protein slows presbycusis in C57BL/6J mice Jian Wang a,b,∗,1 , Trevor Menchenton b,1 , Shankai Yin a,∗∗,1 , Zhiping Yu b , Manohar Bance b,c,g , David P. Morris c , Craig S. Moore d , Robert G. Korneluk e , George S. Robertson d,f a

The Affiliated Sixth People’s Hospital, Otolaryngology Institute of Shanghai Jiao Tong University, Shanghai, China b School of Human Communication Disorders, Dalhousie University, Halifax, Canada c Division of Otolaryngology, Department of Surgery, Dalhousie University, Halifax, Canada d Department of Pharmacology, Dalhousie University, Halifax, Canada e Apoptosis Research Centre, Children’s Hospital of Eastern Ontario Research Institute, Ottawa, Canada f Department of Psychiatry and Pharmacology, Dalhousie University, Halifax, Canada g Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Canada Received 8 April 2008; received in revised form 5 July 2008; accepted 23 July 2008

Abstract Apoptosis of cochlear cells plays a significant role in age-related hearing loss or presbycusis. In this study, we evaluated whether overexpression of the anti-apoptotic protein known as X-linked Inhibitor of Apoptosis Protein (XIAP) slows the development of presbycusis. We compared the age-related hearing loss between transgenic (TG) mice that over-express human XIAP tagged with 6-Myc (Myc-XIAP) on a pure C57BL/6J genetic background with wild-type (WT) littermates by measuring auditory brainstem responses. The result showed that TG mice developed hearing loss considerably more slowly than WT littermates, primarily within the high-frequency range. The average total hair cell loss was significantly less in TG mice than WT littermates. Although levels of Myc-XIAP in the ear remained constant at 2 and 14 months, there was a marked increase in the amount of endogenous XIAP from 2 to 14 months in the cochlea, but not in the brain, in both genotypes. These results suggest that XIAP over-expression reduces age-related hearing loss and hair cell death in the cochlea. © 2008 Elsevier Inc. All rights reserved. Keywords: X-linked inhibitor of apoptosis protein; Apoptosis; Presbycusis; Mouse

1. Introduction Age-related hearing loss (AHL), or presbycusis, is a common neurodegenerative disorder which affects approximately 40% of the population by 65 years of age (Seidman et al., 2002). Many factors such as noise exposure and miscellaneous ototoxic insults can injure receptor hair cells (HC) and ∗ Corresponding author at: The Affiliated Sixth People’s Hospital, Otolaryngology Institute of Shanghai Jiao Tong University, Shanghai 200233, China. Tel.: +1 902 4945149; fax: +1 902 4945151. ∗∗ Corresponding author. E-mail address: [email protected] (J. Wang). 1 These authors made equal contribution to the study.

spiral ganglion neurons (SGNs) in the cochlea, and collectively these are thought to contribute to AHL. At present, it is difficult to distinguish between the effects of aging per se from the cumulative action of these environmental factors. The HCs and SGNs are terminally differentiated cells and cannot be replaced by mitosis. Despite intense efforts to promote regeneration of HCs and SGNs in mammals, it is not yet possible to prevent the loss of these cells and the ensuing impairment of hearing. A large body of evidence implicates apoptosis in agerelated cochlear cell death (Alam et al., 2001; Iwai et al., 2001; Pickles, 2004; Spicer and Schulte, 2002; Zheng et al., 1998). During the process of aging, caspase-mediated apoptosis can be triggered by a variety of factors (Spicer and

0197-4580/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.neurobiolaging.2008.07.016

Please cite this article in press as: Wang, J., et al., Over-expression of X-linked inhibitor of apoptosis protein slows presbycusis in C57BL/6J mice. Neurobiol Aging (2008), doi:10.1016/j.neurobiolaging.2008.07.016

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ARTICLE IN PRESS J. Wang et al. / Neurobiology of Aging xxx (2008) xxx–xxx

Schulte, 2002). Activation of these cysteinyl proteases in the cochlea causes the death of HCs and SGNs (Zheng et al., 1998). Hence, caspase inhibition as a method of preventing cochlear cell death may be a novel treatment strategy. In support of this approach, caspase inhibitors such as z-DEVD-fmk (caspase-3) and z-LEHD-fmk (caspase-9) have been shown to protect cochlear hair cells from cisplatin-induced death (Liu et al., 1998; Wang et al., 2004; Wu et al., 2005; Zhang et al., 2003). In addition, direct caspase inhibitor application to the inner ear protects vestibular hair cells against aminoglycoside toxicity (Matsui et al., 2003). Other interventions that partially prevent ototoxin-induced hair cell loss include the use of minocycline (Wei et al., 2005), neurotrophins (Ding et al., 1999a; Ernfors et al., 1996; Zheng et al., 1995), calpain inhibitors (Wang et al., 1999) and anti-oxidant therapy (Garetz et al., 1994; Lautermann et al., 1995; Ohinata et al., 2003). These treatments all block apoptosis. Unfortunately, the short duration of action of these chemical inhibitors or anti-oxidants limits their clinical utility in the treatment of presbycusis. Members of the Inhibitor of Apoptosis Proteins (IAPs) such as X-linked IAP (XIAP), human-IAP1 (HIAP1/cIAP2) and human-IAP2 (HIAP2/cIAP1) inhibit apoptosis by blocking both the intrinsic (caspase-9; XIAP) and extrinsic (caspase-8; HIAP1/2) pathways that converge on the executioner caspases-3 and -7, which, in turn, are both inactivated directly by XIAP (Deveraux et al., 1998; Deveraux et al., 1997; Roy et al., 1997; Suzuki et al., 2001). As a result, elevating IAP expression increases the survival of many cells types when challenged with a variety of apoptotic triggers (Liston et al., 1996; Robertson et al., 2000). In the central nervous system (CNS), virally mediated over-expression of XIAP reduces the loss of Cornu Ammonis (CA)1 hippocampal neurons and preserves spatial navigation memory after transient forebrain ischemia (Xu et al., 1999). Virally mediated IAP expression also delays the death of cultured cerebellar granule neurons following potassium withdrawal (Simons et al., 1999). In hepatocytes, over-expression of HIAP2 inhibits apoptosis induced by various cytokines (Schoemaker et al., 2002). In the inner ear, blocking caspase activity by XIAP overexpression exhibits at least two advantages over the use of exogenous inhibitors. Firstly, virally mediated XIAP expression in the inner ear produces more prolonged caspase inhibition than chemical inhibitors (Chan et al., 2007; Cooper et al., 2006). Secondly, XIAP also blocks non-caspasemediated cell death, such as that produced by activation of the c-Jun terminal kinase pathway, which is also implicated in cochlear hair cell loss (Kaur et al., 2005; Suckfuell et al., 2007). Thirdly, XIAP by inhibiting caspase-3 and -7, XIAP blocks both extrinsic and intrinsic cell death pathways. These latter two features make XIAP the most potent of all known inhibitors of apoptosis (Deveraux and Reed, 1999; Deveraux et al., 1999; Kaur et al., 2005). Yet another advantage over small molecule caspase inhibitors such as z-VAD-fmk or DEVD-fmk, is that these small inhibitors are not selec-

tive caspase inhibitors (Schotte et al., 1999) but may block cell death by inhibiting a variety of cysteinyl proteases. The non-selective and irreversible cysteinyl protease inhibition produced by z-VAD-fmk or -DEVD-fmk increases the potential for toxicity. Here we report the protective effects of XIAP overexpression in the inner ear in slowing the development of hearing loss that are potentially related to aging, using a transgenic mouse in which expression of the human XIAP gene is under control of the ubiquitin promoter (ubXIAP). The ubXIAP transgene was engineered to produce XIAP that contains a 6-Myc tag (Myc-XIAP), which is expressed in most cells types in the cochlea. Consistent with slow presbycusis, XIAP over-expression also reduced hair cell loss in the cochlea.

2. Materials and methods 2.1. Subjects and procedures Transgenic founders were generated by microinjection of a linearized plasmid construct consisting of the Ubiquitin C promoter, 6 repeats of the 9E10 Myc epitope tag fused to the amino terminus of the human XIAP coding region, and a polyadenylation signal from SV40. The construct was microinjected into the male pronucleus of C57BL/6J X C3H F1 zygotes. C57BL/6J X C3H F1 offspring were backcrossed over 15 generations against wild-type (WT) C57BL/6J mice to obtain ubXIAP transgenic animals on a pure C57BL/6J genetic background. To obtain wild-type littermates for experimentation, ubXIAP animals were crossed with WT C57BL/6J mice. Transgenic status within the colony was determined by PCR targeting the 6-Myc tag. All transgenic mice used in this experiment were heterozygous. The Myc-XIAP C57 transgenic (TG) mice and wild-type (WT) littermates were bred in the animal facility at Dalhousie University. In total, 48 mice were used in this study and longitudinally observed for development of hearing loss with time; 24 animals comprised each of the WT and TG groups with a matched number of mice of each gender in the two groups. Over the 14-month duration of the experiment, a few mice died, so that by the end of the experiment, 17 TG and 15 WT mice had survived. Hearing status was evaluated using frequency-specific auditory brainstem responses (ABR) that were performed every 2 months from the ages of 2–14 months. After the final ABR testing, the animals were sacrificed and both cochleas were harvested; one was used for evaluation of hair cell loss and the other for the quantification of both endogenous and transgenic XIAP. In total, 19 cochleas were taken from each group for constructing cytocochleograms (two mice from TG and 4 from WT groups contributed two cochleas). Western blotting was used for the quantification of both Myc-XIAP (65 kDa) and endogenous XIAP (endo-XIAP; 55 kDa). A piece of brain tissue was also taken from the temporal lobe of each animal for measurement of endo-XIAP and Myc-XIAP. Western blotting was success-




ful in 14 cochleae in the TG group and 11 in the WT group. To further probe the interactions of age and XIAP expression, an additional 34 young mice (2 months old, 17 in each genotype group) were examined to evaluate XIAP expression with Western blotting.

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with 5% EDTA solution for 72 h. The organ of Corti was dissected and surface preparations were made on slides. Cytococochleograms were established using normative data for C57BL/6J mice using custom-written software. 2.4. Western blotting

2.2. Auditory brainstem response measurement Mice were anesthetized with a ketamine and xylazine mixture [60–80 mg/kg (i.p.) + 10 mg/kg (i.p.), respectively] and put on a thermostatic heating pad to keep the body temperature at 38.5 ◦ C. Signal generation and ABR acquisition employed Tucker-Davis hardware and BioSig software (Tucker-Davis Technology system III). The stimuli consisted of tone bursts at 2, 4, 8, 16, 32, 48 and 64 kHz, with a duration of 10 ms and rise/fall of 1 ms (Blackman window). The signals were generated digitally using the TDT system III and delivered to the subjects through a electrostatic speaker (ES1, TDT) which presented a flat frequency response from 2 to 100 kHz. The speaker was placed 10 cm above the head of the subject. The sound level was calibrated using a 1/4-in. B&K condense microphone (Mode 4349) which was placed at the position that would be occupied by the head of the animal. The output of the microphone was examined using SigCal software from TDT. The stimulation rate was of 21.1/s, and 1000 evoked responses were averaged for each trial. At each frequency, the ABR was tested by starting with 90 dB sound pressure level (SPL) and then decreasing stimulation SPL in 5–10 dB steps until the threshold for detecting a repeatable response was reached. The evoked responses were recorded by sub-dermal electrodes, band-pass filtered between 100 and 3000 Hz, before amplification. If the evoked response was not detected at the highest sound presentation level (90 dB SPL) at any given frequency, the threshold at this frequency was labeled as 100 dB SPL. 2.3. Cytocochleogram The methods for determining cochlear morphology are similar to those reported by others (Ding et al., 2001, 1999b). The cytocochleogram was determined by the spatialpercentage count of missing hair cells along the cochlear duct. The mice were deeply anesthetized with an over-dose of ketamine, and the cochleas rapidly harvested after the final ABR test. Surrounding soft tissues were removed, and the round window and oval window were both opened. A small hole was made with a needle at the apex of the cochlea for perfusion and staining. The staining solution for succinate dehydrogenase (SDH) histochemistry was freshly prepared by mixing 0.2 M sodium succinate (2.5 ml), phosphatebuffered saline (2.5 ml) and nitro-tetranitro blue tetrazolium (nitro-BT, 5 ml). The cochlea was gently perfused through the hole at the cochlear apex and the opened round and oval windows. Following this, the cochlea was immersed in the SDH solution for 45 min at 37 ◦ C, and then fixed with 10% formalin for 4 h. After fixation, the cochlea was decalcified

Western blotting was employed to quantify endogenous XIAP and Myc-XIAP in both cochlear and brain tissues. As much soft tissue as possible was harvested from each cochlea in addition to a 2-mm3 piece of brain tissue dissected from the left temporal lobe of each mouse. Each cochlea was used as one independent sample for Western blots. Tissues were homogenized in RIPA buffer (1% Triton X-100, 1% SDS, 8.77% NaCl, 2.42 Tris–HCl base and 5% deoxycholic acid, pH 8) and then centrifuged at 14,000 × g for 10 min at 4 ◦ C. Supernatants were transferred to a new 1.5-ml tube. Protein concentrations were estimated using Bio-Rad reagent and a microplate reader (ELx 800 UV, Bio-tek Instrument Inc.). Following this step, 20 ␮g of protein from each sample was transferred into a tube containing RIPA, 2× SDS sample buffer (7.5 ␮l each) and DTT (15 mg/ml). The samples were stored at −80 ◦ C for later use. The samples were then separated by 10–15% SDS-polyacrylamide gel electrophoresis in running buffer and then transferred to PDVF membrane. The membrane was blocked with a solution containing 1 M Tris–HCl 25 ml, 1 M NaCl 150 ml and Tween-20 500 ␮l, 5% non-fat milk powder in 1 l overnight at 4 ◦ C. The blots were then probed with a primary antibody directed against an epitope common to both endogenous the XIAP and the MycXIAP (1:1500, XIAP Ab mouse, BD Biosciences 610762), equivalent protein loading was confirmed using an antibody against ␤-actin (1:20,000; Sigma A5441). XIAP was visualized using an anti-mouse IgG horseradish peroxidase-linked antibody (1:10,000; Vector Laboratories, PI-2000) and an ECL Plus Kit (GE Health Care). The volume–density values of the immunoreactive bands were read with a Storm 840 gel analysis system. ␤-actin band in the same blotting film was used as internal reference to obtain the fold ratios as relative levels of both endo-XIAP and Myc-XIAP. 2.5. Data analysis and determination of cochlear hair loss ABR thresholds were displayed as a function of frequency, and these were compared between the two groups over time. Two-way ANOVAs were performed against the factors of genotype and sound frequency at each time point at which ABR hearing sensitivity was evaluated. If a significant effect of genotype was found, Tukey’s post hoc tests were performed to determine at which frequencies differences between the two groups occurred. Hair cell loss was measured using the cochleograms of the surviving 17 mice in the TG group and the 15 mice in the WT group. Hair cell loss was determined separately for IHCs and OHCs, within continuous 0.24 mm segments that encompassed the apex to base


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of each cochlea. These values were then converted into percentage hair cell loss by using the hair cell densities revealed from young mice (1–2 months of age). The densities of IHCs and OHCs along the cochlear were roughly 93/0.24 mm and 300/0.24 mm, respectively. The number varies slightly, but not significantly, along the cochlea from apex to the base. The percentage loss is plotted as a function of distance from the apex in Fig. 5. The number of inner hair cells (IHCs) and outer hair cells (OHCs) were counted and the percentage loss determined by dividing the number of IHCs and OHCs calculated in the controls to generate percentage loss of hair cells as a function of distance along the cochlea (Fig. 5). The total loss was compared between the two groups. The levels of endo-XIAP and Myc-XIAP were calculated as a volume ratio between either endo-XIAP or Myc-XIAP and ␤-actin by using ImageQuant software. The expression of endogenous XIAP was evaluated against three factors (age: 2 versus 14 months, genotype: TG versus WT, and tissue: cochlea versus the temporal region of the brain) using a threeway ANOVA with significance set at α = 0.05. Unpaired post hoc tests were performed within the age and tissue factors if significance was achieved by ANOVA testing. The expression of Myc-XIAP was also evaluated against age and tissue type using a two-way ANOVA (p < 0.05).

3. Results 3.1. Hearing loss progress with age is slowed by XIAP over-expression In general, C57BL/6J mice develop hearing loss rapidly starting at a very early age. In comparison to WT littermates, ubXIAP TG mice appeared to have a little better ABR thresholds at the two highest frequencies (48 and 64 kHz) tested at 2 months of age (Fig. 1A). The thresholds were 76.19 and 83.04 dB SPL at 48 and 64 kHz for the WT mice, compared to 60.41 and 75.93 dB SPL for the TG mice. A two-way ANOVA indeed identified a significant effect of genotype. The difference between the groups was found to be significant at 48 kHz (Mann–Whitney Rank Sum Test, T = 649, P < 0.05, as indicated by the asterisk in Fig. 1A) but not at 64 kHz. The development of hearing loss was found to be slower in the TG group compared to WT littermates, and this is demonstrated in two different ways in Fig. 1. Firstly, Fig. 1B compares the averaged ABR audiograms between the two groups at the age of 6 months. A significant difference was found in favor of the TG group at frequencies of 4 kHz and above, suggesting a slower development of high-frequency hearing loss in this TG group. Secondly, Fig. 1C and D shows the changes of ABR thresholds from 2 to 6 months in the two groups. In the TG group, the ABR thresholds remained generally unchanged at frequencies below 16 kHz from the values at 2 months of age (Fig. 1D). In the WT group, however, the threshold elevation was found to be larger than 5 dB at all fre-

quencies and was statistically significant at the frequencies indicated by asterisks (Fig. 1C). In the later stages of the experiment, the threshold differences between the two groups were further exacerbated in the high-frequency region, but the opposite trend was seen in the low-frequency region. Fig. 2 shows the ABR-threshold changes observed at 10, 12 and 14 months. At 10 months, TG mice show superior thresholds across all the frequencies tested relative to WT littermates. Comparing the data shown in panels A–C demonstrates that TG animals retained superior ABR thresholds in the high-frequency region (16, 32, 48 and 64 kHz) relative to WT mice, whereas within the lowfrequency regions (2, 4 and 8 kHz) the differences became smaller with aging and disappeared by 14 months. The difference in hearing loss between the TG and WT mice illustrated in Fig. 3 shows the data in two different ways. In the first manner (Fig. 3A), the ABR thresholds were averaged across two separate frequency segments: 2, 4 and 8 kHz as the low-frequency (LF) region (solid lines), and 16, 32, 48 and 64 kHz as the high-frequency (HF) region (dashed lines) The difference between the two groups in the high-frequency region starts to be significant by 2 months of age, showing better hearing in the TG group. In the TG group, the averaged HF threshold slowly increased with aging, roughly in parallel with the WT group up to 6–8 months of age, and then stabilized. The HF thresholds in the WT group continued to deteriorate, albeit at a slightly slower rate than that seen from 2 to 8 months, resulting in an increasing HF differential between the two groups. The averaged thresholds in the LF region are very close to each other between the two groups early in life (2 and 4 months). The development of LF hearing loss is slower before 8 months age period in TG mice compared to WT littermates resulting in a larger difference between the groups during this period. In contrast to the HF region, LF hearing loss did not seem to stabilize in the TG group after 8 months, but rather continued to progress at a higher rate than in WT littermates. Therefore, LF hearing loss in the TG group was similar to WT mice at 8 months of age and the same as the WT group by 14 months. To further illustrate the protective effects of Myc-XIAP on age-related hearing loss, we analyzed the data in a second fashion (Fig. 3B). The averaged ABR-threshold audiogram in the WT group obtained at 6 months was compared with that from the TG group at 14 months The thresholds at the three high-frequencies (16, 32 and 48 kHz) obtained from the 14-month TG mice were superior to those from 6-month WT mice, suggesting that the HF hearing loss was slowed by more than 8 months. 3.2. XIAP over-expression reduces cochlear hair cell loss associated with aging Hair cell loss was evaluated from 19 cochleas in each group. Fig. 4 shows representative cochlear surface preparation images from two mice (the two in the left panel from a TG mouse, and the two in the right from a WT mouse).




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Fig. 1. ABR-threshold audiograms in young (2–6-month-old) WT and TG mice. (A) ABR audiograms for TG and WT mice at 2 months of age; (B) ABR audiograms for TG and WT mice at 6 months of age; (C) aging-related hearing loss at 2 and 6 months of age in WT littermates; (D) aging-related hearing loss at 2 and 6 months of age in TG mice. Each circle represents mean ± S.E.M. of 15–17 animals. Asterisks in A–C indicate the frequencies at which the differences were statistically significant, p < 0.05.

A smaller degree of OHC loss is seen in the TG cochlea at the basal location (left panel, basal turn image in two different magnifications). Only scattered IHC loss was observed at the very basal end of the cochlea. Images taken from the WT cochlea at comparable locations show that OHC loss is much more severe. In addition, significant IHC loss was also noted in these two images of the WT cochlea (Fig. 4, right panel). Fig. 5 compares the average loss of both IHCs (4A) and OHCs (4B) in TG mice and WT littermates. Generally, the loss of IHC was much less than that of OHC in both groups and was only seen at the high-frequency end of the cochleas. In the WT group (right panel), the OHC loss was over 70% for the basal (HF) end of the cochlea, with spread to the middle of the cochlear duct. In the TG group, the OHC loss was less than 30% for the basal cochlear duct, while the loss was mostly restricted to the high-frequency region. Combining the IHC and OHC losses, the average HC loss in the WT group was 665.5 ± 95.9 cells per cochlea (mean ± S.E.M.), and in the TG group was 220.0 ± 57.6 (mean ± S.E.M.) cells per cochlea. This difference is highly significant (t-test, t = 3.982, p < 0.001).

3.3. XIAP changes with age Western blot analysis was performed on 14 TG and 11 WT cochleas obtained from the mice that had been tested for 14 months and had completed hearing assessment by ABR at 14 months. To further explore the effects of age on the expression of both endogenous XIAP (endo-XIAP) and Myc-XIAP, equivalent numbers of young mice (2 months old) for each genotype were tested. Samples used for Western blot analysis were divided into four groups according to age and genotype (young-TG, young-WT, old-TG, and old-WT). From each animal, the tissue from one cochlea and a piece of temporal lobe were used to sample both Myc-XIAP and endo-XIAP. Relative levels of endo-XIAP and Myc-XIAP were calculated in comparison to levels of ␤-actin to generate a volume ratio. To avoid confounding factors from potential technical variations across different gels, the samples were arranged, as indicated in Fig. 6, so that each gel contained 8 samples from both (2) brain tissue and ear tissue from all 4 groups (2 × 4). Interestingly, it was found that while Myc-XIAP expression appeared not to change with age, levels of endo-


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XIAP increased in the ear, but not the brain of older animals (14 months). As shown in Fig. 7, endo-XIAP levels appear to be higher in the cochleae than in the brains for both TG and WT mice at 14 months of age. A three-way ANOVA was performed to identify the impact of age (2 versus 14 months), genotype (WT versus TG) and tissue (brain versus cochlea) on the endo-XIAP levels. A significant effect was found for both the age and tissue factors (p < 0.001), but not for genotype (p = 0.1). The lack of effect of genotype suggested that the ubXIAP transgene did not interfere with expression of endo-XIAP. For this reason, we analyzed the age effect by grouping the samples from both genotypes according to age and tissue type alone. Within the age factor, endo-XIAP levels were compared between the 25 old cochleas (tissue from 14-month-old mice after final ABR testing) and cochleas from 2-month-old mice of the same sample size. The endoXIAP levels were much higher in older cochleas (1.4 ± 0.08 for 14-month cochleas versus 0.73 ± 0.06 for 2-month-old cochleas (mean ± S.E.M.), t = 6.437, p < 0.001). By contrast, an unpaired t-test failed to show a significant difference in levels of endo-XIAP in the brain between the two ages (0.53 ± 0.03 (mean ± S.E.M.) for 2 months and 0.71 ± 0.05 (mean ± S.E.M.) for 14 months (n = 24 for each age group). Therefore, the age effect was largely due to increased levels of endo-XIAP in the cochlea of 14-month-old animals. Lastly, a significant tissue effect was indicated by the fact that levels of endo-XIAP were found to be higher in the cochlea than in the brain at both ages (t = 2.921 and p = 0.007 for the age of 2 months; t = 4.354 and p < 0.001 for the age of 14 months). The levels of Myc-XIAP were independent of age but differed in cochlear and brain tissues. Fig. 8 shows the expression of Myc-XIAP in both ears and brains for 2-and 14-month-old TG mice. A two-way ANOVA was performed for the two factors (age and tissue) to assess their impact on levels of Myc-XIAP. A significant effect of tissue was found (p < 0.001). Unlike the endo-XIAP, levels of MycXIAP were found to be at a higher level in the brain than in the ear. For example, at 14 months, the Myc-XIAP was 0.601 ± 0.06 (mean ± S.E.M.) in the ear and 1.3 ± 0.11 in the brain (t = 6.621, p < 0.001). However, the effect of age was not significant, suggesting that ubXIAP was expressed in a stable manner that did not change with age.

4. Discussion Fig. 2. ABR-threshold audiograms at 10–14 months in WT and TG mice. (A) At 10 months hearing thresholds across all frequencies were superior for TG relative to WT mice. (B) At 12 months, hearing thresholds for WT and TG mice are similar at low frequencies (2, 4 and 8 kHz). (C) At 14 months of age, the averaged ABR thresholds were similar for WT and TG mice at the three low frequencies (2, 4 and 8 kHz) tested while TG mice still displayed superior ABR sensitivities at the higher frequencies. In (B) and (C), asterisks indicate the frequencies at which TG mice displayed superior ABRs (p < 0.05). Each circle represents the mean ± S.E.M. of 15–17 animals.

The major finding of our study is that age-related hearing loss (AHL) in C57BL/6J mice can be significantly slowed by over-expression of XIAP in the cochlea. The development of presbycusis is faster in WT than in TG group, especially in the high-frequency region. This is shown in Fig. 3B: the HF HL for 6-month WT mice was larger than that for 14-month TG mice. Nevertheless, superior hearing sensitivity was also observed at lower frequencies (8–16 kHz) in 6–10-month-old TG mice compared to WT littermates (Figs. 1A; 2B, C). The increased survival of hair cells in TG mice compared to WT




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Fig. 3. Comparison of aging-related hearing loss in WT littermates and TG mice. (A) The ABR thresholds were averaged into two distinct frequency segments: 2–8 kHz as the low-frequency (LF) region (solid symbols) and 16–64 kHz as the high-frequency (HF) region (open symbols). (B) The ABR-threshold audiogram from WT group at 6 months (dashed line, open circle) is compared with that from TG group at 14 months (solid line, solid circle). In (A), each point represents the mean of 15–17 animals. In (B), each circle represents the mean ± S.E.M. of 15–17 animals.

mice at 14 months of age indicates a strong cytoprotective effect of XIAP. This result suggests that cochlear hair cells die during aging, at least in part, by a form of apoptosis that can be attenuated by XIAP over-expression. It is worth noting that the transgenic mice used in the present study still showed a more rapid development and more severe hearing loss compared to other strains of mice such as CBA/J or CBA/CaJ (Ohlemiller, 2004). Inhibitors of apoptosis proteins (IAPs) are one of two major families of cellular apoptotic inhibitors. Unlike members of the Bcl-2 family that can block only the intrinsic (mitochondrial) pathway by preventing the release of cytochrome c, IAPs can block both the intrinsic and extrinsic

cell death pathways. XIAP exerts its anti-apoptotic functions by directly binding to and inhibiting effector caspases (3 and 7) and sterically hindering the action of caspase-9 (which is the initiator caspase in the intrinsic apoptotic pathway) in the apoptosome (Eckelman et al., 2006; Holcik, 2003; Holcik et al., 2001; Salvesen and Duckett, 2002). XIAP is a prototypical IAP characterized by three baculoviral IAP repeats (BIRs) and a Ring Zinc2+ finger motif at the C terminus. Based upon the results of in vitro kinetic studies, XIAP is thought to be the most potent caspase inhibitor in the IAP family (Deveraux and Reed, 1999). XIAP contains three BIR domains. The BIR2 and BIR3 domains are located towards the N-terminus of XIAP and are sufficient to protect cells

Fig. 4. Representative hair cell loss images from one TG cochlea (left) and one WT cochlea. The samples were treated with SDH staining. The images were taken from matched spots in the basal turns of the two cochleae. Upper row: high magnification images; bottom row: overall view of basal turns.


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Fig. 5. Hair cell loss as a percentage for both TG and WT mice (n = 19 per group). Filled and open circles represent the mean ± S.E.M. for hair cell loss in TG mice and WT littermates, respectively.

from Fas-induced apoptosis by inhibiting the executioner caspases-3 and -7 upon which the intrinsic and extrinsic pathways converge. XIAP can also promote the ubiquitination of caspase-3 and -7, thus targeting these proteases for degradation by the proteosome (Deveraux and Reed, 1999; Suzuki et al., 2001). XIAP-based gene therapy has been evaluated in many different settings. For example, over-expression of XIAP through genetic manipulation has been demonstrated to prevent neuronal death in models of stroke and Parkinson’s disease (Crocker et al., 2003; Trapp et al., 2003; Xu et al., 1999). The survival advantage offered by XIAP overexpression in the brain is conferred by inhibition of at least two cell death pathways: inhibition of caspase-3 (Deveraux and Reed, 1999) and c-Jun N-terminal kinase (JNK) (Igaki et al., 2002). C57BL/6J mice are well known to display early onset (2–3 months of age) and progressive sensorineural hearing loss with aging (Henry and Chole, 1980; Hunter and Willott, 1987; Li and Borg, 1991; Mikaelian, 1979; Spongr et al., 1997; Willott, 1986). Moreover, the aging process of this mouse strain appears to be more rapid in the inner ear than in the CNS, thus resulting in “old ears” connected to a young brain at the early-to-middle life span of these animals (Parham and Willott, 1988; Willott, 1986). The reason(s) for this faster aging specifically in the auditory system remain unclear. Up to three major genes have been identified as major contributors to AHL, and mice strains examined to date may contain one to three of these genes (Erway et al., 1993). For example, a major AHL gene (an allele of Cdh23 for a stereocilia protein) has been mapped in C57BL/6J mice to chromosome 10 that is thought to be a major contributor to AHL in nine other inbred mouse strains (Erway et al., 1993; Johnson et al., 1997, 2000). C57BL/6J mice also carry Ahl3 on chromosome 17. The protein product from this gene is still unknown (Johnson et al., 2000). It is not known, however,

precisely how many different AHL genes are present in each mouse strain (Johnson et al., 2000). Furthermore, differences in AHL onset and development across different strains cannot be attributed to the allelic heterogeneity of the AHL genes (Johnson et al., 2000). Lastly, it is not known whether these genes are related to the apoptotic cell death associated with AHL. For example, we do not know so far whether or why the AHL allele of Cdh23 causes hair cell death. However, the results of the present study suggest that the XIAP overexpression in the transgenic mice not only prevents hair cell from dying, but also preserves their function. This may be an indication that the stereocilia abnormality with aging related to Cdh23 mutation may be prevented or diminished through some intracellular mechanisms by the transgene. Consistent with the results reported here, previous studies have suggested that apoptosis is involved in degenerative cell death in brain as well as in aged cochleas (Alam et al., 2001; Iwai et al., 2001; Pickles, 2004; Spicer and Schulte, 2002; Zheng et al., 1998). A significant increase of caspase-3 associated with aging was reported in the organ of Corti, in SGNs, and in the lateral wall of the cochlea in gerbils (Alam et al., 2001; Zheng et al., 1998). The apoptotic pathways can be triggered by various mechanisms in the cochlea of aging gerbils, including accumulated damage from free-radicals and deteriorating mitochondrial structure and function (Zheng et al., 1998). Very recently, induction of apoptotic markers have been correlated with mutations in mitochondrial DNA (mtDNA) that accumulate during aging, and with increased markers of oxidative stress (Kujoth et al., 2005). The deleterious effects of progressive mtDNA mutations have long been associated with presbycusis (Ohlemiller, 2004; Seidman et al., 2002). In aging, the correlation between the accumulation of mtDNA mutations and apoptotic markers suggests that compromised mitochondrial function may promote apoptosis resulting in cell death (Kujoth et al., 2005). Although AHL is defined as hearing loss due to aging in the absence of obvious




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Fig. 6. Representative Western blot showing Myc-XIAP and endo-XIAP levels in the ear and brain (temporal lobe) of 2- and 14-month-old WT and TG animals. Endo-XIAP levels were higher in the ear than brain, particularly in older age mice at 14 months (14 mo).

environmental risk factors, it is possible that cochlear presbycusis occurs as the consequence of an interplay between environmental factors and genes that regulate the survival and repair of cochlear cells (Johnson et al., 2000). For example, several studies have showed that the AHL gene renders C57BL/6 mice more susceptible to noise-induced hearing loss (NIHL) (Davis et al., 2001; Erway et al., 1996; Harding et al., 2005; Jimenez et al., 2001; Ohlemiller et al., 2000). Another major finding in this study is the increase in endoXIAP in the cochlea alone of old mice. This result suggests that endo-XIAP accumulates in the cochlea in response to the cumulative effects of cellular stressors associated with apoptosis with aging. Interestingly, aging-related increases in endo-XIAP observed in the cochlea were not observed in brain tissue from the same mice, suggesting that apoptosis is more predominant in the aged cochlea than in the brain. This finding is consistent with the fact that aging in the cochlea is more rapid than in the brain in many strains of mice and provides additional support for the role of apoptosis in AHL. The present study also shows that levels of Myc-XIAP derived from the ubXIAP transgene do not change with age. This indicates that expression of the transgene is not associated with AHL. We hypothesize that the stable expression of Myc-XIAP provides continuous protection against apop-

Fig. 7. Histogram showing the impact of all three factors (genotype, tissue and age) on the levels of endo-XIAP. Bars represent mean ± S.E.M. Endogenous XIAP levels were found to be higher in ears than in brains in both genotypes at 14 months compared to 2 months of age. The difference was statistically significant at 14 months of age. *p < 0.05 relative to brain.

Fig. 8. Histogram showing quantification of Myc-XIAP levels in brain and ears at 2 and 14 months of age. Each bar represents the mean ± S.E.M. At both ages, Myc-XIAP was significantly higher in brain than ear (p < 0.05).

tosis in the TG mice and that expression of the transgene is not influenced by factors which regulate the expression of endogenous XIAP. These hypotheses are supported by the observation that endo-XIAP levels in the cochlea were similar in TG mice and WT littermates. Thus endo-XIAP, despite its increase with aging, is not adequate to protect the cochlea from apoptotic cell death. It is possible that the Myc-XIAP expression conferred sufficient additional protection to reduce the deleterious effects of aging on the cochlea. Although intense effort has been made to understand the biological control of XIAP expression, it is still not entirely clear how XIAP expression is regulated in responses to various insults which induce apoptosis. XIAP is under transcriptional control by the transcriptional activator NF-␬B (Stehlik et al., 1998) and protein levels regulated by ubiquitination (post-translation regulating). However, it is also possible that transcription of the gene is promiscuous (Holcik, 2003), and that regulation is mostly post-transcriptional to allow for differential expression in tissues that require more or less XIAP protein. This regulation is mediated by the extensive 5- and 3-untranslated regions (UTRs) in the messenger RNA of XIAP and the internal ribosome entry site (IRES) in the 5-UTR (Holcik et al., 1999). Several binding proteins have been described that regulate XIAP expression at the RNA level (for review see (Holcik, 2003). Because only the coding domains of the human XIAP gene are located in Myc-XIAP mice, it is possible that the finding of stable Myc-XIAP levels with aging is due to the lack of sites that are present in endogenous XIAP mRNA that mediate more dynamic expression of this anti-apoptotic gene. Over-expression of the Myc-XIAP transgene resulted in better protection at the high-frequency region than in the lowfrequency region. Consistent with previous reports (Li and Borg, 1991; McFadden et al., 2001; Mikaelian, 1979), our ABR results showed that age-related hearing loss starts at the high-frequency end of the hearing range of mice, and spreads out towards middle and low-frequency regions with


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ageing. A significant hearing loss in the low-to-middle frequency regions has also been reported in the previous studies and was confirmed by the present study. We also did not see a clear downward spreading of hearing loss with age from HF to LF region. Rather, the LF hearing loss was evident even when comparing the hearing of 2- and 4-month-old mice, especially in the WT group (Fig. 1). The hearing loss in this region developed slightly later and progressed much slower in the TG group than in the WT group up to 8 months of age (Fig. 3A). LF hearing loss was, however, comparable in both genotype groups by the end of the experiment. The LF hearing loss appears to accelerate after 8 months of age in the TG group so that at the end of the experiment there is no statistical difference between the two groups (Figs. 2 and 3). More importantly, the amount of hair cell loss appears to match the degree of hearing loss in the highfrequency region in the two groups. In the low-frequency region, however, we generally observed no hair cell loss in the TG group and only slight hair cell loss in the WT group. Therefore, the LF hearing loss is not due to hair cell loss. A similar discrepancy between the hair cell loss and the elevation of the thresholds has also been demonstrated in previous studies (McFadden et al., 2001; Spongr et al., 1997). Since we did not specifically examine for pathology in other parts or tissues of the cochleae, we cannot be certain which degenerative changes are responsible for the LF hearing loss shown in this strains during aging. Since the LF hearing loss is not protected by XIAP over-expression, the pathology or degenerative changes involved are not likely to be due to apoptosis. In the predominant conceptual framework for AHL or presbycusis proposed by Schuknecht (Schuknecht, 1964; Schuknecht and Gacek, 1993), the three major cochlear elements (organ of Corti, spiral ganglion neurons (SGNs), and stria vascularis (SV)) can degenerate separately, thereby contributing to AHL independently. The apical turn of the cochlea has been found to be prone to primary SGN loss in humans and many animal strain including C57BL/6J mice (Covell and Rogers, 1957; Dazert et al., 1996; Felder and Schrott-Fischer, 1995; Keithley et al., 1992; Ohlemiller and Gagnon, 2004b; Willott et al., 1998). In some recent reports, SGN loss has shown an apical-to-basal gradient during the development of presbycusis (Ohlemiller, 2004; Ohlemiller and Gagnon, 2004a,b). This is opposite to the HC loss that develops from the basal turn to the apex. Other abnormalities include regions in the spiral limbus, pillar cells and Reissner’s membrane. However, it is not clear how these changes are related to the death of SGNs and if they are mediated by apoptosis. Even though we did not examine the histopathology of the SV and SGNs, we postulate that a non-apoptotic lesion is more likely to occur in the strial vascularis (SV) than the SGNs. This is supported by the previous studies that suggest apoptosis is often involved in aging-related SGN death, while the degenerative changes in the SV can be atrophic in nature. In one report, for example, DNA fragmentation (an

indicator of an endonuclease activation associated with apoptosis) was predominantly found in the OHCs and SGNs, but not in the stria cells, which showed marked atrophy (Zheng et al., 1998). A later study reported that caspase-3 expression increased with aging in the organ of Corti, SGNs, as well as lateral wall of the cochlea in gerbils. This increase in active caspases has been claimed to be responsible for functional hearing deterioration (Alam et al., 2001). At the present time, however, we do not have definitive anatomical evidence to support this claim. It is noted that a recent study fail to find significant SV degeneration in association with a normal endocochlear potential up to 26 mouths of age in the C57BL/6J strain (Ohlemiller et al., 2006). Further studies are clearly needed to clarify this matter. Another possible explanation for the LF hearing loss that does not involve the HC is that conductive hearing loss occurs as a result of middle ear pathology, which is often low-frequency biased. Although we did not see any obvious middle ear abnormalities or fluid by visual inspection when cochleae were harvested at the end of the experiments, this simple inspection cannot rule out entirely the possibility of ageing related changes in the middle ear structures. In conclusion, our study demonstrated that overexpression of XIAP by genetic manipulation provides protection in C57BL/6J mice against age-related hearing loss, and this loss is appears to be a result of the accumulation of apoptotic processes in the cochlea with aging. Expression of the transgene product (Myc-XIAP) was not altered in response to apoptosis rather it remained steady throughout the life span of TG mice. The transgene gene also did not interfere with the expression of the endo-XIAP gene. By contrast, endogenous XIAP gene expression increased with age in the cochlea but not in the brain, suggesting that the cochlea is a predominant site for apoptosis in this strain. Over-expression of XIAP protected against AHL in the high-frequency region but not in the low-frequency region where the degenerative pathology may not be apoptotic, or inner ear related. These finds suggest that therapeutic approaches designed to elevate XIAP in the cochlea may have utility in the treatment of presbycusis.

Conﬂicts of interest None.

Acknowledgements This study is supported by grant of Canadian Institute of Health Research (MOP-79452) and the grant of National Nature Science Foundation of China (30672294/C030310). We thank Dr. Peter Liston, who had generated the ubXIAP transgenic mice and generously provided this valuable resource for our study.




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