Exacerbated age-related hearing loss in mice lacking the p43 mitochondrial T3 receptor.

BACKGROUND: Age-related hearing loss (ARHL), also known as presbycusis, is the most common sensory impairment seen in elderly people. However, the cochlear aging process does not affect people uniformly, suggesting that both genetic and environmental (e.g., noise, ototoxic drugs) factors and their interaction may influence the onset and severity of ARHL. Considering the potential links between thyroid hormone, mitochondrial activity, and hearing, here, we probed the role of p43, a N-terminally truncated and ligand-binding form of the nuclear receptor TRα1, in hearing function and in the maintenance of hearing during aging in p43-/- mice through complementary approaches, including in vivo electrophysiological recording, ultrastructural assessments, biochemistry, and molecular biology. RESULTS: We found that the p43-/- mice exhibit no obvious hearing loss in juvenile stages, but that these mice developed a premature, and more severe, ARHL resulting from the loss of cochlear sensory outer and inner hair cells and degeneration of spiral ganglion neurons. Exacerbated ARHL in p43-/- mice was associated with the early occurrence of a drastic fall of SIRT1 expression, together with an imbalance between pro-apoptotic Bax, p53 expression, and anti-apoptotic Bcl2 expression, as well as an increase in mitochondrial dysfunction, oxidative stress, and inflammatory process. Finally, p43-/- mice were also more vulnerable to noise-induced hearing loss. CONCLUSIONS: These results demonstrate for the first time a requirement for p43 in the maintenance of hearing during aging and highlight the need to probe the potential link between human THRA gene polymorphisms and/or mutations and accelerated age-related deafness or some adult-onset syndromic deafness.

Epithelial-mesenchymal transition, and collective and individual cell migration regulate epithelial changes in the amikacin-damaged organ of Corti.

Characterizing the microenvironment of a damaged organ of Corti and identifying the basic mechanisms involved in subsequent epithelial reorganization are critical for improving the outcome of clinical therapies. In this context, we studied the expression of a variety of cell markers related to cell shape, cell adhesion and cell plasticity in the rat organ of Corti poisoned with amikacin. Our results indicate that, after severe outer hair cell losses, the cytoarchitectural reorganization of the organ of Corti implicates epithelial-mesenchymal transition mechanisms and involves both collective and individual cell migratory processes. The results also suggest that both root cells and infiltrated fibroblasts participate in the homeostasis of the damaged epithelium, and that the flat epithelium that may emerge offers biological opportunities for late regenerative therapies.

High mobility group box 1 (HMGB1): dual functions in the cochlear auditory neurons in response to stress?

High mobility group box 1 (HMGB1) is a DNA-binding protein that facilitates gene transcription and may act extracellularly as a late mediator of inflammation. The roles of HMGB1 in the pathogenesis of the spiral ganglion neurons (SGNs) of the cochlea are currently unknown. In the present study, we tested the hypothesis that early phenotypical changes in the SGNs of the amikacin-poisoned rat cochlea are mediated by HMGB1. Our results showed that a marked downregulation of HMGB1 had occurred by completion of amikacin treatment, coinciding with acute damage at the dendrite extremities of the SGNs. A few days later, during the recovery of the SGN dendrites, the protein was re-expressed and transiently accumulated within the nuclei of the SGNs. The phosphorylated form of the transcription factor c-Jun (p-c-Jun) was concomitantly detected in the nuclei of the SGNs where it often co-localized with HMGB1, while the anti-apoptotic protein BCL2 was over-expressed in the cytoplasm. In animals co-treated with amikacin and the histone deacetylase inhibitor trichostatin A, both HMGB1 and p-c-Jun were exclusively found within the cytoplasm. The initial disappearance of HMGB1 from the affected SGNs may be due to its release into the external medium, where it may have a cytokine-like function. Once re-expressed and translocated into the nucleus, HMGB1 may facilitate the transcriptional activity of p-c-Jun, which in turn may promote repair mechanisms. Our study therefore suggests that HMGB1 can positively influence the survival of SGNs following ototoxic exposure via both its extracellular and intranuclear functions.

Contribution of auditory nerve fibers to compound action potential of the auditory nerve.

Sound-evoked compound action potential (CAP), which captures the synchronous activation of the auditory nerve fibers (ANFs), is commonly used to probe deafness in experimental and clinical settings. All ANFs are believed to contribute to CAP threshold and amplitude: low sound pressure levels activate the high-spontaneous rate (SR) fibers, and increasing levels gradually recruit medium- and then low-SR fibers. In this study, we quantitatively analyze the contribution of the ANFs to CAP 6 days after 30-min infusion of ouabain into the round window niche. Anatomic examination showed a progressive ablation of ANFs following increasing concentration of ouabain. CAP amplitude and threshold plotted against loss of ANFs revealed three ANF pools: 1) a highly ouabain-sensitive pool, which does not participate in either CAP threshold or amplitude, 2) a less sensitive pool, which only encoded CAP amplitude, and 3) a ouabain-resistant pool, required for CAP threshold and amplitude. Remarkably, distribution of the three pools was similar to the SR-based ANF distribution (low-, medium-, and high-SR fibers), suggesting that the low-SR fiber loss leaves the CAP unaffected. Single-unit recordings from the auditory nerve confirmed this hypothesis and further showed that it is due to the delayed and broad first spike latency distribution of low-SR fibers. In addition to unraveling the neural mechanisms that encode CAP, our computational simulation of an assembly of guinea pig ANFs generalizes and extends our experimental findings to different species of mammals. Altogether, our data demonstrate that substantial ANF loss can coexist with normal hearing threshold and even unchanged CAP amplitude.

Supporting cells regulate the remodelling of aminoglycoside-injured organ of Corti, through the release of high mobility group box 1.

To examine whether an inflammatory process occurs in the amikacin-poisoned cochlea, we investigated the presence of the cytokines tumour necrosis factor-α (TNF-α), interleukin (IL)-1ß, and IL-10. No TNF-α, IL-1ß or IL-10 was detected in the cochlear perilymph after the loss of most auditory hair cells, indicating the absence of severe inflammation. In contrast, we observed a significant and temporary increase in the level of extracellular high mobility group box 1 (HMGB1), a late mediator of inflammation that also functions as a signal of tissue damage. This increase coincided with epithelial remodelling of the injured organ of Corti, and occurred concomitantly with robust and transient cytoplasmic expression of acetylated HMGB1 within the non-sensory supporting cells, Deiters cells. Here, HMGB1 was found to be enclosed within vesicles, a number of which carried the secretory vesicle-associated membrane-bound protein Rab 27A. In addition, transient upregulation of receptor for advanced glycation end-products (RAGE), an HMGB1 membrane receptor, was found in most epithelial cells of the scarring organ of Corti when extracellular levels of HMGB1 were at their highest. Altogether, these results strongly suggest that, in stressful conditions, Deiters cells liberate HMGB1 to regulate the epithelial reorganization of the injured organ of Corti through engagement of RAGE in neighbouring epithelial cells.

Caspase inhibitors, but not c-Jun NH2-terminal kinase inhibitor treatment, prevent cisplatin-induced hearing loss.

Cisplatin (CDDP) is a highly effective chemotherapeutic agent but with significant ototoxic side effects. Apoptosis is an important mechanism of cochlear hair cell loss following exposure to an ototoxic level of CDDP. This study examines intracellular pathways involved in hair cell death induced by CDDP exposure in vivo to develop effective therapeutic strategies to protect the auditory receptor from CDDP-initiated hearing loss. Guinea pigs were treated with systemic administration of CDDP. Cochlear hair cells from CDDP-treated animals exhibited classic apoptotic alterations in their morphology. Several important signaling events that regulate the death of CDDP-injured cochlear hair cells were identified. CDDP treatment induced the activation and redistribution of cytosolic Bax and the release of cytochrome c from injured mitochondria. Activation of caspase-9 and caspase-3, but not caspase-8, was detected after treatment with CDDP, and the cleavage of fodrin by activated caspase-3 was observed within damaged hair cells. Intracochlear perfusions with caspase-3 inhibitor (z-DEVD-fmk) and caspase-9 inhibitor (z-LEHD-fmk) prevent hearing loss and loss of sensory cells, but caspase-8 inhibitor (z-IETD-fmk) and cathepsin B inhibitor (z-FA-fmk) do not. Although the stress-activated protein kinase/c-Jun NH(2)-terminal kinase (JNK) signaling pathway is activated in response to CDDP toxicity, intracochlear perfusion of d-JNKI-1, a JNK inhibitor, did not protect against CDDP ototoxicity but instead potentiated the ototoxic effects of CDDP. The results of the present study show that blocking a critical step in apoptosis may be a useful strategy to prevent harmful side effects of CDDP ototoxicity in patients having to undergo chemotherapy.

Changes in MAP2 and tyrosinated alpha-tubulin expression in cochlear inner hair cells after amikacin treatment in the rat.

The expression of MAP2 (microtubule-associated protein 2) and of tyrosinated alpha-tubulin was investigated immunocytochemically in the cochleas of normal and amikacin-treated rats. For MAP2, two different antibodies were used: anti-MAP2ab, against the high molecular weight forms, and anti-MAP2abc, additionally against the embryonic form c. In the cochlea of the normal rat, the outer (OHCs) and inner (IHCs) hair cells were labeled for MAP2abc. The labeling was weaker in IHCs than in OHCs. The hair cells were rarely labeled for MAPab. Both OHCs and IHCs were labeled for tyrosinated alpha-tubulin. In the cochlea of the amikacin-treated rat, aggregates of anti-MAP2abc and anti-tyrosinated alpha-tubulin antibodies were seen in the apical region of the IHCs as early as the end of the antibiotic treatment. In rats investigated during the following week, the cell body of most of the surviving IHCs were not labeled for MAP2abc and tyrosinated alpha-tubulin. Then, labeling for these two antibodies reappeared in the surviving IHCs, including their giant stereocilia. Fewer surviving IHCs were labeled for tyrosinated alpha-tubulin than for MAP2abc. The amikacin-poisoned IHCs were rarely labeled for MAP2ab. These results suggest that cochlear hair cells essentially express form c of MAP2. In the amikacin-damaged cochlea, the apical aggregation of MAP2c and tyrosinated alpha-tubulin within the poisoned IHCs could be implicated in a cell degenerative process. By contrast, the extinction and recovery of MAP2c and tyrosinated alpha-tubulin labeling in the remaining IHCs suggest the occurrence of a limited repair process. A possible role of MAP2 and tubulin in hair cell survival is discussed.

Calpain activity in the amikacin-damaged rat cochlea.

The principal aim of this study was to investigate the involvement of calpain in the degeneration of hair cells and ganglion neurons in the amikacin-poisoned rat cochlea. An antibody designed against fodrin-breakdown products (FBDP), which result exclusively from cleavage by calpain, was used. In addition, the involvement of both caspases and protein kinase C (PKC) was studied using, respectively, antibodies against activated caspase 3 and PKCgamma. The results demonstrate the accumulation of FBDP in the degenerating hair cells, in some supporting cells such as Deiters cells, and, later, in the affected ganglion neurons that had been deprived of their sensory targets. Activated caspase 3 was evidenced in a few dying hair cells and ganglion neurons. PKCgamma was highly expressed in all ganglion neurons, sometimes after the loss of hair cells. We conclude that calpain plays a role in the degradation of both the sensory cells and neurons after amikacin ototoxicity. In the poisoned hair cells, calpain and caspase 3 may have synergistic effects in the process of apoptosis. In the ganglion neurons deprived of their sensory elements, calpain may have a prominent role in cell degradation. By contrast, in these ganglion neurons PKCgamma may be implicated in a survival process. Finally, we suggest that calpain is involved in the remodeling of Deiters cells during the scarring process that follows hair cell loss.

Microtubule-associated protein 2 (MAP2) expression during synaptic plasticity in the guinea pig cochlea.

The expression of different isoforms of microtubule-associated proteins 2 (MAP2), including the low molecular weight form MAP2c present mainly in developing neurons, was investigated in the primary auditory neurons after alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) perfusion in the guinea pig cochlea. MAP2 expression appeared to be tightly regulated in the repairing neurons. Neurite regrowth seems to involve the MAP2c isoform. In cochlear neurons, mechanisms involved in the period of development might be reactivated after excitotoxic injury in the mature cochlea.

Oxidative stress, inflammation, and autophagic stress as the key mechanisms of premature age-related hearing loss in SAMP8 mouse Cochlea.

AIMS: In our aging society, age-related hearing loss (ARHL) or presbycusis is increasingly important. Here, we study the mechanism of ARHL using the senescence-accelerated mouse prone 8 (SAMP8) which is a useful model to probe the effects of aging on biological processes. RESULTS: We found that the SAMP8 strain displays premature hearing loss and cochlear degeneration recapitulating the processes observed in human presbycusis (i.e., strial, sensory, and neural degeneration). The molecular mechanisms associated with premature ARHL in SAMP8 mice involve oxidative stress, altered levels of antioxidant enzymes, and decreased activity of Complexes I, II, and IV, which in turn lead to chronic inflammation and triggering of apoptotic cell death pathways. In addition, spiral ganglion neurons (SGNs) also undergo autophagic stress and accumulated lipofuscin. INNOVATION AND CONCLUSION: Our results provide evidence that targeting oxidative stress, chronic inflammation, or apoptotic pathways may have therapeutic potential. Modulation of autophagy may be another strategy. The fact that autophagic stress and protein aggregation occurred specifically in SGNs also offers promising perspectives for the prevention of neural presbycusis.

Macrophage contribution to the response of the rat organ of Corti to amikacin.

Transdifferentiation of nonsensory supporting cells into sensory hair cells occurs naturally in the damaged avian inner ear. Such transdifferentiation was achieved experimentally in the cochlea of deaf guinea pigs through Atoh 1 gene transfection. Supporting cells may therefore serve as targets for transdifferentiation therapy. Supporting cells rapidly degenerate after hair cell disappearance, however, limiting the therapeutic window for gene transfer. We studied the time course of ultrastructural and phenotypical changes occurring in Deiters cells (hair cell supporting cells) after ototoxic treatment in the rat. The presence of macrophages in the cochlea was also investigated, to study any deleterious effects they may have on pathologic tissues. One week after treatment most hair cells had disappeared. Deiters cells no longer expressed the glial marker vimentin but instead displayed typical hair cell markers, the calcium binding proteins calbindin and parvalbumin. This suggests that a process of transdifferentiation of Deiters cells into hair cells was activated. By 3 weeks post-treatment, however, the Deiters cells began to degenerate and by 10 weeks post-treatment the organ of Corti was degraded fully. Interestingly, a marked increase in macrophage density was seen after the end of amikacin treatment to 10 weeks post-treatment. This suggests chronic inflammation is involved in epithelium degeneration. Consequently, early treatments with anti-inflammatory factors might promote supporting cell survival, thus improving the efficacy of more specific strategies aimed to regenerate hair cells from nonsensory cells.

Inhibition of the c-Jun N-terminal kinase-mediated mitochondrial cell death pathway restores auditory function in sound-exposed animals.

We tested and characterized the therapeutic value of round window membrane-delivered (RWM) d-JNKI-1 peptide (Bonny et al., 2001) against sound trauma-induced hearing loss. Morphological characteristics of sound-damaged hair cell nuclei labeled by Hoechst staining show that apoptosis is the predominant mode of cell death after sound trauma. Analysis of the events occurring after sound trauma demonstrates that c-Jun N-terminal kinase (JNK)/stress-activated protein kinase activates a mitochondrial cell death pathway (i.e., activation of Bax, release of cytochrome c, activation of procaspases, and cleavage of fodrin). Fluorescein isothiocyanate (FITC)-conjugated d-JNKI-1 peptide applied onto an intact cochlear RWM diffuses through this membrane and penetrates cochlear tissues with the exception of the stria vascularis. A time sequence of fluorescence measurements demonstrates that FITC-labeled d-JNKI-1 remains in cochlear tissues for as long as 3 weeks. In addition to blocking JNK-mediated activation of a mitochondrial cell death pathway, RWM-delivered d-JNKI-1 prevents hair cell death and development of a permanent shift in hearing threshold that is caused by sound trauma in a dose-dependent manner (EC50 = 2.05 microM). The therapeutic window for protection of the cochlea from sound trauma with RWM delivery of d-JNKI-1 extended out to 12 h after sound exposure. These results show that the mitogen-activated protein kinase/JNK signaling pathway plays a crucial role in sound trauma-initiated hair cell death. Blocking this signaling pathway with RWM delivery of d-JNKI-1 may have significant therapeutic value as a therapeutic intervention to protect the human cochlea from the effects of sound trauma.