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| ORIGINAL ARTICLE |
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| Year : 2021 | Volume
: 64
| Issue : 2 | Page : 61-71 |
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Tau phosphorylation and cochlear apoptosis cause hearing loss in 3×Tg-AD Mouse Model of Alzheimer's Disease
Sheue-Er Wang1, Chung-Hsin Wu2
1 School of Life Sciences, National Taiwan Normal University, Taipei; Department of Pathological, Saint Paul's Hospital, Taoyuan City, Taiwan
2 School of Life Sciences, National Taiwan Normal University, Taipei, Taiwan
| Date of Submission | 12-Oct-2020 |
| Date of Decision | 21-Feb-2021 |
| Date of Acceptance | 05-Mar-2021 |
| Date of Web Publication | 21-Apr-2021 |
Correspondence Address:
Dr. Chung-Hsin Wu
School of Life Sciences, National Taiwan Normal University, Taipei
Taiwan
 Source of Support: None, Conflict of Interest: None  | 7 |
DOI: 10.4103/CJP.CJP_79_20
Clinically typical dementia Alzheimer's disease (AD) is associated with abnormal auditory processing. However, possible molecular mechanisms responsible for the auditory pathology of AD patients are not known. According to our past research findings that the thresholds of auditory brainstem response, but not distortion product otoacoustic emissions, were significantly increased in AD mice from 9 months of age and thereafter. Thus, we further explored the possible mechanism of auditory degradation of 3×Tg-AD mice in this study. Our histochemical staining evidence showed the cochlear spiral ganglion neurons (SGN), but not the cochlear hair cells, were lost significantly in the cochlea of 3×Tg-AD mice from 9 months of age and thereafter. Our immunostaining and western blotting evidence showed that phosphorylated tau protein (p-Tau), p-glycogen synthase kinase 3, neurofilament, and apoptosis-related p53, Bcl2-associated X protein, cytochrome c, caspase-9, and caspase-3 were gradually increased, but B-cell lymphoma 2 was gradually decreased with age growth in the cochlea of 3×Tg-AD mice. We suggested that tau hyperphosphorylation and p-Tau 181 aggregation, and mitochondria- and endoplasmic reticulum stress-mediated apoptosis may play a role in the degeneration of SGN in the cochlea. Progressive SGN degeneration in the cochlea may contribute to hearing loss of aging 3×Tg-AD mice.
Keywords: Alzheimer's disease, apoptosis, auditory brainstem response, cochlear pathology, hearing loss, neurofilament, spiral ganglion neurons, tau hyperphosphorylation, transgenic mice
How to cite this article:
Wang SE, Wu CH. Tau phosphorylation and cochlear apoptosis cause hearing loss in 3×Tg-AD Mouse Model of Alzheimer's Disease. Chin J Physiol 2021;64:61-71 |
How to cite this URL:
Wang SE, Wu CH. Tau phosphorylation and cochlear apoptosis cause hearing loss in 3×Tg-AD Mouse Model of Alzheimer's Disease. Chin J Physiol [serial online] 2021 [cited 2023 Nov 30];64:61-71. Available from: https://www.cjphysiology.org/text.asp?2021/64/2/61/314189 |
| Introduction | |  |
Alzheimer's disease (AD) is the most common dementia of mental decline in the elderly and reflects a progressive neurodegenerative disorder characterized by cognitive, motor, and behavioral dysfunction.[1] The histopathological landmarks of AD are represented by neurofibrillary tangles (NFTs) and neuritic β-amyloid (Aβ) plaques found mostly in the association cortex and subcortical nuclei.[2],[3],[4] The main component of NFTs is the cytoskeleton protein known as the tau protein, in hyperphosphorylated form.[5] The presence of phosphorylated tau protein (p-Tau) also appears to be involved in this toxic process.[6] It has been reported that tau protein may be hyperphosphorylated by activation of glycogen synthase kinase 3 (GSK-3).[7] Furthermore, GSK3 is a key mediator of apoptosis and thereby might directly contribute to neuronal loss in AD.[8]
Clinically typical dementia AD is associated with deficient perceptual and semantic processing of environmental sounds and melodies.[3],[9],[10],[11],[12] Early studies have found a correlation between hearing loss and dementia in elderly populations.[13],[14],[15] Several studies have suggested that auditory pathology in AD is associated with the auditory cortex. Within the auditory cortex, only occasional NFTs and few neuritic plaques were found in the primary auditory cortex (Brodmann areas 41 and 42), while substantial increases were reported in the number of NFTs in the auditory association cortex (Brodmann area 22) in AD patients.[16],[17],[18] It has been suggested that central auditory dysfunction may precede the onset of dementia.[19]
The relationship between peripheral hearing loss and AD has been studied previously and the interaction of these two medical conditions has been found to interact with each other to cause greater disability than either one alone. It is accepted that the strong interaction between these conditions can be devastating to communication and somewhat attributed to age-related decline of the cochlea and/or auditory nerve. It appears that individuals with AD have a greater probability of peripheral hearing loss. However, peripheral auditory dysfunction in individuals with AD has not been investigated extensively. According to our past research findings that the thresholds of auditory brainstem response (ABR), but not distortion product otoacoustic emissions (DPOAE), were significantly increased in AD mice from 9 months of age and thereafter.[20] By H and E staining evidence, we have observed that 3×Tg-AD preserved quite intact their outer and inner hair cells, but lost their cochlear spiral ganglion neurons (SGN) compared to those of their wild-type controls from 9 months of age and thereafter.[20] Thus, we further explored the possible mechanism of auditory degradation of 3×Tg-AD mice in this study. By immunostaining and western blotting evidence, our result suggested that tau hyperphosphorylation and p-Tau aggregation, and mitochondria- and endoplasmic reticulum (ER) stress-mediated apoptosis may be involved in the degeneration of SGN in the cochlea that may contribute to peripheral hearing loss of late-aged 3×Tg-AD mice.
| Materials and Methods | | |
Animal preparation
According to our past research, we have compared cochlear structure between 3×Tg-AD mice and their wild-type by using H and E staining.[20] In this study, we further explored the possible mechanism of auditory degradation of 3×Tg-AD mice in this study. Thus, only male 3×Tg-AD mice (Jackson Laboratory, Bar Harbor, ME) were used in this study. The 3×Tg-AD mice harbor human transgenes APPSwe, PS1M146V, and tauP301L (co-express mutant human APP, PS1, and tau protein, respectively).[21],[22],[23] The 3×Tg-AD mice were housed in air-conditioned colony rooms (temperature 21°C ± 1°C, humidity 60%) on a natural light/dark cycle, with food in pellets and tap water available ad libitum. Body weight was recorded throughout the observation period, and rectal temperature was maintained close to 37°C by means of heating lamps. Animal sacrifice at the end of the study was performed under general anesthesia with sodium pentobarbital (50 mg/kg) intraperitoneally (i.p.). In accordance with the Institutional Guidelines of the Animal Care and Use Committee of National Taiwan Normal University (NTNU), the 3×Tg-AD mice were maintained in the animal facility of NTNU under specific pathogen-free conditions. All 3×Tg-AD mice experiments were approved by the Institutional Animal Care and Use Committee of NTNU (Protocol number: NTNU Animal Experiments No. 106030).
Functional hearing assessment in 3×Tg-AD mice
We assessed thresholds of ABR and DPOAE of 3×Tg-AD mice at 3, 6, 9, and 12 months of age. As suggested in a previous study of Zheng et al,[24] the ABR and DPOAE recordings were performed in a sound-attenuating, electrically shielded booth located in a sound-attenuating room. 3×Tg-AD mice were first treated with atropine (0.5 mg/kg im) and then anesthetized with urethane (1.2 mg/kg i.p.; Sigma, St. Louis, MO, USA). Subdermal needles were used as electrodes (Rochester Electro-Medical, Tampa, FL, USA). The active electrode was inserted at the vertex, the reference electrode ventrolateral to the left ear, and the ground electrode ventrolateral to the right ear. During recording, the body temperature of mice was monitored by a rectal probe and maintained at 37°C ± 1°C with a nonelectrical heating pad.
The ABR of 3×Tg-AD mice were recorded using Tucker Davis Technologies (TDT, Alachua, FL) System and BioSig System III (TDT, Alachua, FL, USA). Acoustic stimulation and simultaneous recordings were performed with a Tucker-Davis (TDT). Acoustic stimuli were generated digitally using SigGenRP software (TDT) and RX6 Piranha Multifunction Processor hardware (TDT). Before the ABR recording, stimuli were calibrated using SigCal software (TDTs) and an ER-10B+ low noise microphone system (Etymotic Research Inc., Elk, Groove, IL, USA). Stimuli were delivered monaurally (right ear) into the external auditory meatus of the mice using an EDC1 electrostatic speaker driver (TDTs) through an EC-1 electrostatic speaker (TDTs). The evoked potentials were filtered (0.3–3.0 kHz), averaged (500 waveforms), and stored in computer hardware for offline analysis later. To determine the threshold of ABR, the evoked responses were recorded in a 10 dB-step descending from a maximum stimulus intensity of 100 dB sound pressure level (SPL). The background activity was measured before the stimulus onset. The ABR threshold was defined as the stimulus intensity that evoked waveforms with a peak-to-peak voltage greater than 2 standard deviations of the background activity. The ABR thresholds were obtained for sound frequencies of 8, 16, and 32 kHz in this study.
The DPOAEs of 3×Tg-AD mice were recorded using TDTs (Alachua, FL) System 2/System 3 digital signal processing hardware and software. All DPOAE stimuli were created using TDT SigGen software, and ear canal recordings were conducted using TDT BioSig software using an Intel microprocessor-based computer. The DPOAEs were generated by simultaneously two tones differing in frequency (the lower frequency labeled f1 and the higher frequency f2) into the sealed ear canal of HD and WT mice. The primary tones (f1 and f2) are presented simultaneously into the sealed ear canal. The primary tones are always presented at a fixed ratio (f2/f1 = 1.2). The distortion product at the 2f1–f2 frequency is a reliable indicator of outer hair cell function.[33] The place of distortion generation in the cochlea appears to be close to the frequency place of f2. Consequently, we plotted the distortion threshold curve as a function of f2. DPOAE threshold curves were measured for f2 frequencies from 8 to 16 and then 32 kHz. By keeping f2 constant and varying f1, the optimum stimulus separation (best ratio f2/f1) was determined, which produced maximum DPOAE levels at low stimulus levels. With the primary tones set at the best ratio, growth functions of the 2f1−f2 distortion were measured by stepwise increasing the stimulus levels. The level of f2 sufficient to elicit a DPOAE of −10 dB SPL was defined as the threshold criterion.
Morphological analysis in the cochlea of 3×Tg-AD mice
After anesthetization, inner ear tissues of 3×Tg-AD mice at 3, 6, 9, and 12 months of age were fixed by perilymphatic perfusion with phosphate-buffered 4% paraformaldehyde (Sigma-Aldrich, USA) through the round window after removing the stapes and puncturing the round window membrane. The dissected temporal bone was then immersed in fixative for 24 h at 4°C and rinsed in 0.1 M sodium phosphate buffer (pH 7.4). For the light microscopic study, the temporal bones were subsequently decalcified in 0.35 MEDTA and embedded in glycol methacrylate. Samples were washed in phosphate buffer, pH 7.2, four times for 20 min, and dehydrated in a graded series of ethanol concentrations (30%, 50%, 70%, and 100%), each for 15 min. Once the samples had been dehydrated, cochleae were dissected in 100% ethanol under a high-power dissecting microscope, embedded in paraffin, and sectioned serially at a thickness of 3–5 μm and stained with eosin and hematoxylin (H and E staining) with a kit-based approach (Sigma-Aldrich Corporation).
Quantification of spiral ganglion neuron density and hair cells in the cochlea of 3×Tg-AD mice
The SGN and hair cells were examined from the cochlear section with H and E staining. Using mid-modiolus sections, the survival SGN was calculated as the number of SGN per mm2. The hair cells were calculated as the number of intact inner hair cells and outer hair cells that should be observed in each turn of one cochlea in tissue sections of 3×Tg-AD mice. The corresponding area of the Rosenthal canal was measured on digital photomicrographs of each canal profile. The computer then calculated the area within the outline.
Immunocytochemistry analysis in the cochlea of 3×Tg-AD mice
For immunostaining studies, following the heat-induced epitope retrieval method, cochlear tissue sections were separately stained at room temperature for 1 h with antibodies for Aβ (Cell Signaling, USA), p-Tau 181 (Cell Signaling, USA), p-GSK-3α (Cell Signaling, USA), p-GSK-3 β (Cell Signaling, USA), neurofilament (Cell Signaling, USA), and p53 (Cell Signaling, USA) polyclonal rabbit antibody (1:1000 diluted, Cell Signaling Technology). Then, immunostaining was detected through incubation with biotinylated secondary antibodies (Novolink™ Polymer Detection System l, Leica Biosystems Newcastle Ltd., Newcastle, UK) for 30 min and the avidin–biotin–horseradish peroxidase (HRP) complex (Novolink™ Polymer Detection System l, Leica Biosystems Newcastle Ltd.) for additional 30 min. Immunostaining was visualized using DAB Chromogen (Novolink™ Polymer Detection System l, Leica Biosystems Newcastle Ltd.), and the slides were counterstained with hematoxylin (Novolink™ Polymer Detection System l, Leica Biosystems Newcastle Ltd.).
Protein analysis and western blotting in the cochlea of 3×Tg-AD mice
Total proteins were extracted from the cochlea of 3×Tg-AD mice following the treatment described. The removed cochlea tissue was homogenized in a buffer solution that included 0.05 M tris(hydroxymethyl)aminomethane (Tris, pH 8.0, Bionovas, USA), 0.15 M sodium chloride (NaCl, Bionovas, USA), 0.02 M ethylenediaminetetraacetic acid (EDTA, Bionovas, USA), 1% deoxycholic acid (Bionovas, USA), 1% nonidet P40 (Bionovas, USA), 0.1% sodium dodecyl sulfate (SDS, Bionovas, USA), 1% protease inhibitor cocktail for full range (Bionovas, USA), 1% serine/threonine phosphatase inhibitor cocktail (Bionovas, USA), and 1% tyrosine phosphatase inhibitor cocktail (Bionovas, USA). The homogenized buffer solution was placed on ice for an hour and then centrifuged at 4°C for 13,000 rpm for another 20 min, and the supernatant solution was separated. The separated solution was quantitated by using BCA protein assay kit (Thermo, USA). Thirty micrograms of the total protein was denatured at 95°C for 5 min with 5X sample dye, that included 0.25 M tris(hydroxymethyl)aminomethane hydrochloride (Tris-HCl, pH 6.8, Bionovas, USA), 10% sodium dodecyl sulfate (SDS, Bionovas, USA), 0.5% bromophenol blue (Bionovas, USA), 50% glycerol (Bionovas, USA) and 5% beta-mercaptoethanol (Bionovas, USA). The electrophoresis was done on a 12.5% discontinuous sodium dodecylsulfate (SDS-PAGE)-polyacrylamide gel. The proteins were then electroblotted onto a 0.2 μm polyvinylidenedifluoride (PVDF, GE Healthcare Life Sciences, USA) membrane for 120 min at 100 V. The membranes were reacted with blocking buffer (5% skim milk in TBS-T buffer) for an hour at the ambient temperature and then they were blocked.
The beta-actin (Thermo, USA), p-GSK-3α (Cell Signaling, USA), p-GSK-3 β (Cell Signaling, USA), Aβ (Cell Signaling, USA), p-Tau 181 (Cell Signaling, USA), neurofilament (Cell Signaling, USA), p53 (Cell Signaling, USA), Calpain (Cell Signaling, USA), B-cell lymphoma 2 (Bcl-2)-associated X protein (Bax) (Thermo, USA), Bcl-2 (Santa Cruz, USA), cytochrome C (Epitomics, USA), caspases 12 (Cell Signaling, USA) and caspase 3 (Cell Signaling, USA) antibodies were reacted for 2 h at the ambient temperature (or overnight at 4 centigrade) and the membrane was washed 3 times using TBS-T at intervals of 10 min. As the secondary antibodies, anti-rabbit IgG-HRP (1:5000 dilution, PerkinElmer, USA), anti-mouse IgG-HRP (1:5000 dilution, PerkinElmer, USA), and anti-goat IgG-HRP (1:5000 dilution, Enzo Life Sciences, USA) were reacted at the ambient temperature for 1 h and the membrane was washed again with TBS-T 3 times with an interval of 10 min between each washing. Each band was visualized by utilizing ECL western blotting detection reagents (GE Healthcare Life Sciences, USA), and the chemiluminescence was detected using LAS-4000 (GE Healthcare Life Sciences, USA). Densitometric assessment of the bands was performed using ImageJ software (version 1.48t, Wayne Rasband, USA).
Statistical analysis
Statistical evaluations were done by multiple comparisons test. Hence, one-way or two-way ANOVA was first performed and then Student-Newman-Keuls was completed as the posteriori test (SigmaPlot 12.5 software, Systat Software Inc.).[25] All average values are presented as means ± standard error of the mean (SEM). Significance was set at P < 0.05.
| Results | | |
Body weight and motor deficits of the 3×Tg-AD mouse model at different lifespan stage
In this study, 8 of 3×Tg-AD mice were used to assess body weight, motor deficits, and auditory impairment (n = 8), and 16 3×Tg-AD mice were used to assess morphological, immunostaining, and western blotting analysis in the cochlear tissue at the lifespan stage of 3, 6, 9, and 12 months (n = 4 for each group). We examined and found that body weight among 3×Tg-AD mice at 6–12 months of age were quite similar and not much difference [[Figure 1]a, P > 0.05]. By using rotarod testing, we observed that the latencies to fall of 3×Tg-AD mice became significantly worse from 9 months of age and thereafter [[Figure 1]b, P < 0.05]. | Figure 1: Body weight and motor deficits in the 3×Tg-AD mouse model at different lifespan stage. (a) Body weight became significantly heavier from 9 months of age and thereafter. (b) Latencies to fall of 3×Tg-AD mice became significantly worse from 9 months of age and thereafter as observed by rotarod testing. Values are mean ± SEM (n = 8, One-way ANOVA, followed by a Student-Newman-Keuls multiple comparisons posttest). SEM: Standard error of the mean.
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Auditory impairment in 3×Tg-AD mice
In this study, we assessed the hearing sensitivity of 3×Tg-AD mice by ABR and DPOAE examination. A representative ABR of 3×Tg-AD mouse elicited by 16 kHz tone burst stimuli at different lifespan stages is shown in [Figure 2]a. The ABR threshold of a 3-month 3×Tg-AD mouse was 30 dB SPL, while the ABR threshold of a 6-month 3×Tg-AD mouse was 40 dB SPL. The ABR threshold was obviously increased to 70 dB SPL in a 9-month 3×Tg-AD mouse and then increased to 80 dB SPL in a 12-month 3×Tg-AD mouse. We compared the ABR thresholds elicited by tone bursts of 8 kHz (lower pitch), 16 kHz (middle pitch), and 32 kHz (higher pitch) in 3×Tg-AD mice. The average ABR thresholds elicited by tone bursts of 8, 16, and 32 kHz for 9- and 12-month 3×Tg-AD mice were significantly elevated approximately 30–45 dB in comparison to 3-month 3×Tg-AD mice [[Figure 2]b, P < 0.01–0.05]. To clarify the possible role of cochlear hair cells in hearing loss of 3×Tg-AD mice, we further compared the DPOAE thresholds in 3×Tg-AD mice. The DPOAE threshold curves were measured and compared for f2 frequencies from 8 to 16 and then to 32 kHz in 3×Tg-AD mice. The DPOAE thresholds for 6-, 9-, and 12-month 3×Tg-AD mice were approximately the same as the DPOAE thresholds in 3-month 3×Tg-AD mice [[Figure 2]c, P > 0.05]. | Figure 2: Hearing thresholds of 3×Tg-AD mice at different lifespan stage. (a) Representative ABR elicited by 16 kHz tone burst stimuli of 3×Tg-AD mice at 3, 6, 9, and 12 months of age. (b) The average ABR thresholds elicited by 8, 16, and 32 kHz tone burst stimuli. Significant elevation of ABR thresholds was observed from 9 months of age and thereafter in 3×Tg-AD mice. Values are mean ± SEM (n = 8, **P < 0.01, *P < 0.05, one-way ANOVA. followed by a Student-Newman-Keuls multiple comparisons posttest). (c) The average DPOAE thresholds elicited by two-tone burst stimuli that f2 frequency was set at 8, 16, and 32 kHz. DPOAE thresholds to 2f1-f2 were similar among 3×Tg-AD mice at 6, 9, and 12 months of age. Values are mean ± SEM (n = 6, P > 0.05). An example of DPOAE was shown in figure. DPOAE: Distortion product otoacoustic emission, SEM: Standard error of the mean, ABR: Auditory brainstem response.
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Morphological neurodegeneration in the cochlea of 3×Tg-AD mice
Morphological degeneration of auditory neurons in the cochlea of 3×Tg-AD mice at different lifespan stages is examined in [Figure 3]. We observed obvious loss of SGN in cochlear of 3×Tg-AD mice from 9 months of age and thereafter, while IHCs and OHCs are quite intact in cochlear of 3×Tg-AD mice in 3, 6, 9, and 12 months of age [Figure 3]a and [Figure 3]b. We found that the SGN density in cochlear of 3×Tg-AD mice was significantly reduced from 9 months of age and thereafter [[Figure 3]c, P < 0.01–0.05]. Unlike SGN loss, there is no loss of hair cells in the cochlea of 3×Tg-AD mice [[Figure 3]d, P > 0.05]. Although we observed that the loss of SGN density in the basal portion was a little greater than that in the apical portion, there is no significant difference between the SGN density in basal and apical portions of the cochlea in 3×Tg-AD mice [[Figure 3]c and [Figure 3]d, apical vs. basal portion, P > 0.05]. | Figure 3: Cochlear SGN and hair cells among 3×Tg-AD mice at different lifespan stage. (a) Representative H and E staining of mid-modiolus sections of the cochlear tissue of a 3×Tg-AD mouse at 3 months of age (scale bars: 60 μm). AP and BP showed AP and BP of cochlear regions. (b) Enlarged sections of AP and BP of cochlear regions of 3×Tg-AD mice at 3, 6, 9, and 12 months of age (scale bars: 30 μm). (c) Comparison of SGN density in cochlear apical and basal portions among 3×Tg-AD mice at 3, 6, 9, and 12 months of age. The significant reduction of SGN density was observed in 3×Tg-AD mice at 9 and 12 months of age. Values are mean ± SEM (n = 8, **P < 0.01, *P < 0.05, Two-way ANOVA, followed by a Student-Newman-Keuls multiple comparisons posttest). (d) Comparison of relative percentage (%) of hair cells in cochlear apical and basal portions among 3×Tg-AD mice at 3, 6, 9, and 12 months of age. Values are mean ± SEM (n = 4 for each group, P > 0.05, Two-way ANOVA, followed by a Student-Newman-Keuls multiple comparisons posttest). SGN: Spiral ganglion neurons, AP: Apical portion, BP: Basal portion, SEM: Standard error of the mean.
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Aβ and phosphorylated tau protein in the cochlea of 3×Tg-AD mice
It has been reported that the presence of Aβ and p-Tau is involved in the toxic process of the AD brain.[6] Thus, we further examined whether Aβ and p-Tau expression was existed in the cochlear region of 3×Tg-AD mice. Our immunostaining data showed only the p-Tau, but not the Aβ, is developed and existed in the cochlear region of 3×Tg-AD mice [Figure 4]a. Our western blotting data further showed positive p-Tau expression, but not Aβ expression, is expressed in the cochlear tissue of 3×Tg-AD mice [Figure 4]b. The relative intensity of p-Tau expression in cochlea tissue of 3×Tg-AD mice is significantly increased with ages [[Figure 4]b, P < 0.01–0.05], while the relative intensity of Aβ expression is not examined from western blotting data in [Figure 4]c. | Figure 4: Cochlear p-Tau expressions among 3×Tg-AD mice at different lifespan stage. (a) Positive immunostaining of p-Tau expressions (dark brown in color) in SGN of 3×Tg-AD mice at 3, 6, 9, and 12 months of age (scale bars: 30 μm). (b) The representative western blotting and densitometry analysis of p-Tau and Aβ proteins in the cochlea of 3×Tg-AD mice. Negative western blotting of Aβ expressions was shown in the cochlea of 3×Tg-AD mice. (c) Statistical comparison of quantified p-Tau expressions of cochlear tissue among 3×Tg-AD mice at 3, 6, 9, and 12 months of age. Values are mean ± SEM (n = 4 for each group, **P < 0.01, *P < 0.05, One-way ANOVA, followed by a Student-Newman-Keuls multiple comparisons posttest). SEM: Standard error of the mean, SGN: Spiral ganglion neurons.
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Upregulation of p-glycogen synthase kinase 3 in the cochlea of 3×Tg-AD mice
Both p-GSK-3α and p-GSK-3β may induce the hyperphosphorylation of Tau.[26] Thus, we examined whether p-GSK-3α and p-GSK-3β expressions were existed in the cochlear region of 3×Tg-AD mice. Our immunostaining data showed obvious p-GSK-3α and p-GSK-3β expressions in SGN of 3×Tg-AD mice from 3 months of age and thereafter [Figure 5]a and [Figure 5]b. Our western blotting data further showed gradually increased p-GSK-3α and p-GSK-3β expressions in cochlea tissue of 3×Tg-AD mice with ages [Figure 5]c. The relative intensity of p-GSK-3α and p-GSK-3β expressions in cochlea tissue of 3×Tg-AD mice was significantly increased with ages [[Figure 5]d, P < 0.01–0.05]. | Figure 5: Cochlear p-GSK-3 expressions among 3×Tg-AD mice at different lifespan stage. Positive immunostaining of (a) p-GSK-3α and (b) p-GSK-3 β expressions (dark brown in color) in SGN and hair cells of 3×Tg-AD mice at 3, 6, 9, and 12 months of age (scale bars: 30 μm). For observing conveniently, partially twice enlarged sections of SGN and hair cells were presented in the upper left and right corners, respectively. (c) Cochlear p-GSK-3α and p-GSK-3β expression levels of 3×Tg-AD mice at 3, 6, 9, and 12 months of age through western blotting. (d) Statistical comparison of quantified p-GSK-3α and p-GSK-3β expressions of cochlear tissue among 3×Tg-AD mice at 3, 6, 9, and 12 months of age. Values are mean ± SEM (n = 4 for each group, **P < 0.01, *P < 0.05, one-way ANOVA, followed by a Student-Newman-Keuls multiple comparisons posttest). p-GSK-3: Phospho-glycogen synthase kinase 3, SEM: Standard error of the mean, SGN: Spiral ganglion neurons.
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Neurofilament in the cochlea of 3×Tg-AD mice
As reported previously, neurofilament is one of the main components of NFTs.[27] We examined whether neurofilament expression was existed in the cochlear region of 3×Tg-AD mice at different lifespan stage. Obvious neurofilament immunostaining is observed in SGN of the cochlea of 3×Tg-AD mice from 6 months of age and thereafter [Figure 6]a. Our immunoblot data showed gradually increased neurofilament expression in the cochlea tissue of 3×Tg-AD mice with ages [Figure 6]c. The relative intensity of neurofilament expression in cochlea tissue of 3×Tg-AD mice is significantly increased with ages ([Figure 6]d, P < 0.01). | Figure 6: Cochlear neurofilament and p53 expressions among 3×Tg-AD mice at different lifespan stage. Positive immunostaining of (a) neurofilament and (b) p53 expressions (dark brown in color) in SGN and hair cells of 3×Tg-AD mice at 3, 6, 9, and 12 months of age (scale bars: 30 μm). For observing conveniently, partially twice enlarged sections of SGN and hair cells were presented in the upper left and right corners, respectively. (c) Cochlear neurofilament and p53 expression levels of 3×Tg-AD mice at 3, 6, 9, and 12 months of age through western blotting. (d) Statistical comparison of quantified neurofilament and p53 expressions of cochlear tissue among 3×Tg-AD mice at 3, 6, 9, and 12 months of age. Values are mean ± SEM (n = 4 for each group, **P < 0.01, One-way ANOVA, followed by a Student-Newman-Keuls multiple comparisons posttest). SEM: Standard error of the mean, SGN: Spiral ganglion neurons.
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Upregulation of p53 in the cochlea of 3×Tg-AD mice
p53 is associated with apoptosis in neurodegenerative diseases such as AD.[28] We examined whether p53 expression was existed in the cochlear region of 3×Tg-AD mice at different lifespan stage. We observed notable p53 immunostaining in SGN of the cochlear of 3×Tg-AD mice from 6 months of age and thereafter [Figure 6]b. Our immunoblot data showed gradually increased p53 expression in the cochlea tissue of 3×Tg-AD mice with ages [Figure 6]c. The relative intensity of p53 expression in the cochlea tissue of 3×Tg-AD mice was significantly increased with ages ([Figure 6]d, P < 0.01).
Upregulation of apoptosis-related proteins in the cochlea of 3×Tg-AD mice
It has been suggested that the p53-dependent apoptosis pathway involves Bcl-2/Bax decrease, cytochrome c release, and caspase activation.[29] Thus, we examined whether Bcl-2/Bax decrease, cytochrome c release, and caspase activation were existed in the cochlear region of 3×Tg-AD mice at different lifespan stage. Our immunoblot data showed gradually decreased Bcl-2/Bax and increased cytochrome c, caspases 9 and 3 in the cochlea tissue of 3×Tg-AD mice with ages [Figure 7]a. The ratio of Bcl-2/Bax in the cochlea tissue of 3×Tg-AD mice was significantly decreased with ages [Figure 7]b, P < 0.01]. While the relative intensity of cytochrome c, caspases 9 and 3 expressions in the cochlea tissue of 3×Tg-AD mice was significantly increased with ages [[Figure 7]b, P < 0.01–0.05]. | Figure 7: Cochlear mitochondria- and endoplasmic reticulum stress-mediated apoptosis-related proteins expressions among 3×Tg-AD mice at different lifespan stage. (a) Cochlear Bcl-2, Bax, Cyto-C, Calpain-2, Caspase-9, and Caspase-3 expression levels of 3×Tg-AD mice at 3, 6, 9, and 12 months of age through western blotting. (b) Statistical comparison of quantified Bcl-2, Bax, Cyto-C, Calpain-2, Caspase-9, and Caspase-3 expressions of cochlear tissue among 3×Tg-AD mice at 3, 6, 9, and 12 months of age. Values are mean ± SEM (n = 4 for each group, **P < 0.01, *P < 0.05, One-way ANOVA, followed by a Student-Newman-Keuls multiple comparisons posttest). SEM: Standard error of the mean.
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In addition, calpains have been suggested that might be involved in ER stress-mediated apoptosis, as well as in mitochondria-mediated apoptosis.[30] Thus, we also examined whether calpains activation were existed in the cochlear region of 3×Tg-AD mice at different lifespan stage. Our immunoblot data showed gradually increased calpain 2 in the cochlea tissue of 3×Tg-AD mice with ages [Figure 7]a. The relative intensity of calpain 2 expression in the cochlea tissue of 3×Tg-AD mice was significantly increased with ages [[Figure 7]b, P < 0.01].
| Discussion | | |
The present study is the first to show possible molecular mechanisms responsible for auditory pathology in 3×Tg-AD mice. The 3×Tg-AD mice express PS1, APP, and tau mutations, and develop Aβ and Tau pathology that was similar to human AD in terms of the hippocampal regions in which the Aβ and p-Tau developed.[21] Although both mutant transgenes were expressed at the same time, similar to the Aβ and Tau genes in humans with AD, we observed the Aβ deposits developed at 3 months of age before tau hyperphosphorylation and before NFT aggregation at 9 months of age, indicating that Aβ precedes tau pathology. Our observation is similar to the findings of LaFerla and Oddo that Aβ triggers or facilitates the accumulation of tau pathology in the hippocampal regions of 3×Tg-AD mice.[31]
Similar to the abnormal auditory processing in AD patients, we found that the thresholds of ABR, but not DPOAE, were significantly increased in AD mice from 9 months of age and thereafter [Figure 2]. AD patients need to accommodate for the complex processing of speech and as a consequence, working memory is sacrificed to allow for more cognitive reserve.[32] If there is a peripheral hearing loss in AD patients, difficulties are expected in the detection and discrimination of signals. Poor peripheral and central auditory deficits can occupy and exhaust information processing resources in the central neuron systems and not allow working memory to reach its capacity and the information to be stored correctly for cognitive purposes. When AD patients hearing thresholds are compared to their peers, their thresholds are greater than their peers.[32]
As reported previously, only ABR thresholds, but not DPOAE thresholds, were significantly elevated in 9- and 12-month 3×Tg-AD mice [Figure 2]. The DPOAEs are distorted sounds generated by the cochlear outer hair cells in response to two tones that are close in frequency. The presence of a DPOAE response is an indication that the outer hair cells are functioning properly.[33] Generally, auditory impairment can be caused by primary degeneration of SGN or by secondary degeneration of SGN after hair cell loss. Sensorineural hearing loss can be caused by a loss of hair cells, the sound transducing sensory cells of the cochlea, or by loss of SGN that connects the hair cells to the cochlear nucleus in the brainstem. In agreement with the DPOAE test, our H and E staining data showed that only the cochlear SGN, but not the cochlear hair cells, was significantly reduced in 3×Tg-AD mice from 9 months of age and thereafter [Figure 3]. The histological staining results provide us a reasonable explanation concerning why only the ABR thresholds, but not the DPOAE thresholds, are elevated obviously in 3×Tg-AD mice from 9 months of age and thereafter.
By immunostaining and western blotting, we observed hallmark characteristics, p-Tau, p-GSK-3α, p-GSK-3β, and neurofilament were gradually increased with age in the cochlea of 3×Tg-AD mice [Figure 4], [Figure 5], [Figure 6]. It is worth noting that only tau hyperphosphorylation, but not the Aβ, is critically involved in the cochlear neurodegeneration of 3×Tg-AD mice. The molecular mechanisms responsible for auditory cochlear pathology were quite different from the brain pathogenesis in 3×Tg-AD mice. The p-Tau and NFTs formation have been extensively reported in the AD literature.[34],[35],[36] This convincing evidence came from research with 3×Tg-AD mice. Tau is abundantly present in the central nervous system and is predominantly expressed in neuronal axons.[37] In addition, an earlier study also reported that tau is expressed in the SGN of the mammalian cochlea.[38] Tau is a major microtubule-associated protein that plays a large role in the outgrowth of neuronal processes and the development of neuronal polarity. In addition, tau promotes microtubule assembly, stabilizes microtubules, and affects the dynamics of microtubules in neurons.[35],[39],[40] Many studies have suggested that hyperphosphorylated tau is critically involved in AD pathogenesis, particularly in impairing axonal transport of subcellular organelles including mitochondria in neurons affected by AD.[41],[42] Further, N-terminal fragmented tau is associated with mitochondria and causing mitochondrial dysfunction and synaptic damage.[43],[44] The tau phosphorylation regulates microtubule binding and assembly.[45] In contrast, pathological tau becomes hyperphosphorylated, which destabilizes microtubules by decreased binding to microtubules, resulting in the aggregation of p-Tau and NFTs are associated with auditory neurodegeneration in the cochlea of 3×Tg-AD mice [Figure 4] and [Figure 6]. Neurofilament is one of the main components of NFTs; their abnormality in AD is a hallmark of neuronal dysfunction, especially marking axonal degeneration.[27] Furthermore, it has been suggested that both p-GSK-3α and p-GSK-3β may induce the tau hyperphosphorylation.[26] Our immunostaining and western blotting results provide a better understanding of a possible contribution of p-GSK-3α and p-GSK-3β in tau hyperphosphorylation for neurofilament aggregation and auditory neurodegeneration in the cochlea of 3×Tg-AD mice [Figure 5].
In this study, we observed that the apoptosis-related proteins of p53, Bax, cytochrome c, caspase-9, and caspase-3 were gradually increased [Figure 6] and [Figure 7], while Bcl-2 was gradually decreased with age in the cochlea of 3×Tg-AD mice [Figure 7]. In p53-dependent neuronal apoptosis, the Bax has been identified as a major mediator of cell death.[46],[47] Concentrated Bax expression, possibly through an apoptotic mechanism, has been found in the AD hippocampus.[48] The proteins of the Bcl-2 family consist of proapoptotic and antiapoptotic regulators of apoptosis or programmed cell death. Activation of Bax or inhibition of Bcl-2 promotes the release of downstream factors such as cytochrome c which leads to the activation of caspase-3 which are the key executioners of p53-induced apoptosis. Our observations indicate the probability of the induction of apoptosis of SGN through the p53 pathway in the cochlea of 3×Tg-AD mice. Furthermore, the cochlear neurodegeneration in 3×Tg-AD mice might be involved in ER stress-mediated apoptosis because we observed gradually increasing calpain-2 and caspase-9 in SGN of the cochlea with age. Activation of calpains is initiated by Ca2+ and shows autocleavage of both large and small subunits. Several calpain substrates have been identified including p53.[49] There is evidence to show that cleavage of caspase-9 by calpain leads to the inhibition of cytochrome c-dependent caspase-3 cleavage.[50] They suggest an alternative way to modulate ER stress-mediated apoptosis by intracellular calcium signals. Several evidence has shown that calpain substrates include caspase-9, caspase-3, Bax, and Bcl-2.[51],[52] Calpains have been verified to enhance the constitutive apoptotic death program.[30] Thus. we suggest that the cochlear neurodegeneration in 3×Tg-AD mice might be involved in ER stress-mediated apoptosis, as well as in mitochondria-mediated apoptosis.
As mentioned before, many pathological indications, including p-Tau in SGN, were significantly increased in 6-month 3×Tg-AD mice, while their ABR thresholds [Figure 2]b were comparable to those in mice at age of 3 months. However, these pathological conditions did not cause hearing impairments in 6-month 3×Tg-AD mice. We found that cochlear SGN density, but not the cochlear hair cells, was significantly reduced in 3×Tg-AD mice from 9 months of age and thereafter [Figure 3]. In addition, caspase-9 activation stimulates the caspase cascade culminating in activation of executioner caspases-3. These caspase-9 and caspases-3 are responsible for the cleavage of numerous cellular proteins, leading to the biochemical and morphological hallmarks of apoptosis. As suggested from [Figure 7], both caspase-9 and caspases-3 expressions show a significant increase from 9-month 3×Tg-AD mice. This evidence may partially explain why 3×Tg-AD mice cause hearing impairments from 9 months of age and thereafter.
| Conclusions | | |
The results of the present study indicate that 3×Tg-AD mice show progressively abnormal ABR, but not the DPOAE, and SGN degeneration in the cochlea with age growth. Although Aβ and p-Tau are histopathological landmarks of AD in the association cortex and subcortical nuclei, we observed that only tau hyperphosphorylation and p-Tau aggregation, but not the Aβ, were developed in the cochlea of 3×Tg-AD mice. We further observed that neurofilament was gradually increased with age in the cochlea of 3×Tg-AD mice. We suggest that overexpressed p-GSK-3α and p-GSK-3β in the cochlea of 3×Tg-AD mice may induce tau hyperphosphorylation and p-Tau aggregation and then promote neurofilament formation of SGN. Neurofilament formation of SGN might participate in auditory neurodegeneration in the cochlea of 3×Tg-AD mice. In addition to tau, we demonstrate that apoptosis-related proteins of p53, Bax, cytochrome c, caspase-9, and caspase-3 were gradually increased, while Bcl-2 was gradually decreased with age in the cochlea of 3×Tg-AD mice. Furthermore, we observed calpain-2 and caspase-9 were gradually increased with age in the cochlea of 3×Tg-AD mice. Thus, we suggest that mitochondria- and ER stress-mediated apoptosis may also play a role in auditory neurodegeneration in the cochlear SGN of 3×Tg-AD mice. Gradually, damage and loss of the cochlear SGN provides a reasonable explanation for hearing loss of 3×Tg-AD mice from 9 months of age and thereafter.
Acknowledgment
The invaluable support by Wallace Academic Editing in reviewing and proofreading of this manuscript is enormously appreciated.
Financial support and sponsorship
The research was partially supported by grants from the Ministry of Science and Technology, Taiwan (MOST 107-2321-B-003-001, MOST 108-2321-B-003-001, MOST 109-2321-B-003-001, and MOST 107-2320-B-003-003-MY3).
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6], [Figure 7]
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