Indispensable role of Mdm2/p53 interaction during the embryonic and postnatal inner ear development

p53 is a key component of a signaling network that protects cells against various stresses. As excess p53 is detrimental to cells, its levels are tightly controlled by several mechanisms. The E3 ubiquitin ligase Mdm2 is a major negative regulator of p53. The significance of balanced p53 levels in normal tissues, at different stages of lifetime, is poorly understood. We have studied in vivo how the disruption of Mdm2/p53 interaction affects the early-embryonic otic progenitor cells and their descendants, the auditory supporting cells and hair cells. We found that p53 accumulation, as a consequence of Mdm2 abrogation, is lethal to both proliferative progenitors and non-proliferating, differentiating cells. The sensitivity of postmitotic supporting cells to excess p53 decreases along maturation, suggesting that maturation-related mechanisms limit p53's transcriptional activity towards pro-apoptotic factors. We have also investigated in vitro whether p53 restricts supporting cell's regenerative capacity. Unlike in several other regenerative cellular models, p53 inactivation did not alter supporting cell's proliferative quiescence nor transdifferentiation capacity. Altogether, the postmitotic status of developing hair cells and supporting cells does not confer protection against the detrimental effects of p53 upregulation. These findings might be linked to auditory disturbances observed in developmental syndromes with inappropriate p53 upregulation.Peer reviewe

Differentiation of the Lateral Compartment of the Cochlea Requires a Temporally Restricted FGF20 Signal

FGF20 signaling in mice is required specifically for the differentiation of cochlear outer hair cells, the cells most often damaged during age-related hearing loss

Neurod1 Suppresses Hair Cell Differentiation in Ear Ganglia and Regulates Hair Cell Subtype Development in the Cochlea

Background: At least five bHLH genes regulate cell fate determination and differentiation of sensory neurons, hair cells and supporting cells in the mammalian inner ear. Cross-regulation of Atoh1 and Neurog1 results in hair cell changes in Neurog1 null mice although the nature and mechanism of the cross-regulation has not yet been determined. Neurod1, regulated by both Neurog1 and Atoh1, could be the mediator of this cross-regulation. Methodology/Principal Findings: We used Tg(Pax2-Cre) to conditionally delete Neurod1 in the inner ear. Our data demonstrate for the first time that the absence of Neurod1 results in formation of hair cells within the inner ear sensory ganglia. Three cell types, neural crest derived Schwann cells and mesenchyme derived fibroblasts (neither expresses Neurod1) and inner ear derived neurons (which express Neurod1) constitute inner ear ganglia. The most parsimonious explanation is that Neurod1 suppresses the alternative fate of sensory neurons to develop as hair cells. In the absence of Neurod1, Atoh1 is expressed and differentiates cells within the ganglion into hair cells. We followed up on this effect in ganglia by demonstrating that Neurod1 also regulates differentiation of subtypes of hair cells in the organ of Corti. We show that in Neurod1 conditional null mice there is a premature expression of several genes in the apex of the developing cochlea and outer hair cells are transformed into inner hair cells. Conclusions/Significance: Our data suggest that the long noted cross-regulation of Atoh1 expression by Neurog1 migh

Disorganized Innervation and Neuronal Loss in the Inner Ear of Slitrk6-Deficient Mice

Slitrks are type I transmembrane proteins that share conserved leucine-rich repeat domains similar to those in the secreted axonal guidance molecule Slit. They also show similarities to Ntrk neurotrophin receptors in their carboxy-termini, sharing a conserved tyrosine residue. Among 6 Slitrk family genes in mammals, Slitrk6 has a unique expression pattern, with strong expression in the sensory epithelia of the inner ear. We generated Slitrk6-knockout mice and investigated the development of their auditory and vestibular sensory organs. Slitrk6-deficient mice showed pronounced reduction in the cochlear innervation. In the vestibule, the innervation to the posterior crista was often lost, reduced, or sometimes misguided. These defects were accompanied by the loss of neurons in the spiral and vestibular ganglia. Cochlear sensory epithelia from Slitrk6-knockout mice have reduced ability in promoting neurite outgrowth of spiral ganglion neurons. Indeed the Slitrk6-deficient inner ear showed a mild but significant decrease in the expression of Bdnf and Ntf3, both of which are essential for the innervation and survival of sensory neurons. In addition, the expression of Ntrk receptors, including their phosphorylated forms was decreased in Slitrk6-knockout cochlea. These results suggest that Slitrk6 promotes innervation and survival of inner ear sensory neurons by regulating the expression of trophic and/or tropic factors including neurotrophins from sensory epithelia

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63

Rosmarinic Acid, Active Component of Dansam-Eum Attenuates Ototoxicity of Cochlear Hair Cells through Blockage of Caspase-1 Activity

Cisplatin causes auditory impairment due to the apoptosis of auditory hair cells. There is no strategy to regulate ototoxicity by cisplatin thus far. Dansam-Eum (DSE) has been used for treating the central nerve system injury including hearing loss in Korea. However, disease-related scientific investigation by DSE has not been elucidated. Here, we demonstrated that DSE and its component rosmarinic acid (RA) were shown to inhibit apoptosis of the primary organ of Corti explants as well as the auditory cells. Administration of DSE and RA reduced the thresholds of the auditory brainstem response in cisplatin-injected mice. A molecular docking simulation and a kinetic assay show that RA controls the activity of caspase-1 by interaction with the active site of caspase-1. Pretreatment of RA inhibited caspase-1 downstream signal pathway, such as the activation of caspase-3 and 9, release of cytochrome c, translocation of apoptosis-inducing factor, up-regulation of Bax, down-regulation of Bcl-2, generation of reactive oxygen species, and activation of nuclear factor-κB. Anticancer activity by cisplatin was not affected by treatment with RA in SNU668, A549, HCT116, and HeLa cells but not B16F10 cells. These findings show that blocking a critical step by RA in apoptosis may be useful strategy to prevent harmful side effects of ototoxicity in patients with having to undergo chemotherapy

CLIMP-63 is a gentamicin-binding protein that is involved in drug-induced cytotoxicity

Aminoglycoside-induced nephrotoxicity and ototoxicity is a major clinical problem. To understand how aminoglycosides, including gentamicin, induce cytotoxicity in the kidney proximal tubule and the inner ear, we identified gentamicin-binding proteins (GBPs) from mouse kidney cells by pulling down GBPs with gentamicin–agarose conjugates and mass spectrometric analysis. Among several GBPs specific to kidney proximal tubule cells, cytoskeleton-linking membrane protein of 63 kDa (CLIMP-63) was the only protein localized in the endoplasmic reticulum, and was co-localized with gentamicin-Texas Red (GTTR) conjugate after cells were treated with GTTR for 1 h. In western blots, kidney proximal tubule cells and cochlear cells, but not kidney distal tubule cells, exhibited a dithiothreitol (DTT)-resistant dimer band of CLIMP-63. Gentamicin treatment increased the presence of DTT-resistant CLIMP-63 dimers in both kidney proximal (KPT11) and distal (KDT3) tubule cells. Transfection of wild-type and mutant CLIMP-63 into 293T cells showed that the gentamicin-dependent dimerization requires CLIMP-63 palmitoylation. CLIMP-63 siRNA transfection enhanced cellular resistance to gentamicin-induced toxicity, which involves apoptosis, in KPT11 cells. Thus, the dimerization of CLIMP-63 is likely an early step in aminoglycoside-induced cytotoxicity in the kidney and cochlea. Gentamicin also enhanced the binding between CLIMP-63 and 14-3-3 proteins, and we also identified that 14-3-3 proteins are involved in gentamicin-induced cytotoxicity, likely by binding to CLIMP-63

Celastrol inhibits aminoglycoside-induced ototoxicity via heat shock protein 32

Hearing loss is often caused by death of the mechanosensory hair cells of the inner ear. Hair cells are susceptible to death caused by aging, noise trauma, and ototoxic drugs, including the aminoglycoside antibiotics and the antineoplastic agent cisplatin. Ototoxic drugs result in permanent hearing loss for over 500 000 Americans annually. We showed previously that induction of heat shock proteins (HSPs) inhibits both aminoglycoside- and cisplatin-induced hair cell death in whole-organ cultures of utricles from adult mice. In order to begin to translate these findings into a clinical therapy aimed at inhibiting ototoxic drug-induced hearing loss, we have now examined a pharmacological HSP inducer, celastrol. Celastrol induced upregulation of HSPs in utricles, and it provided significant protection against aminoglycoside-induced hair cell death in vitro and in vivo. Moreover, celastrol inhibited hearing loss in mice receiving systemic aminoglycoside treatment. Our data indicate that the major heat shock transcription factor HSF-1 is not required for celastrol-mediated protection. HSP32 (also called heme oxygenase-1, HO-1) is the primary mediator of the protective effect of celastrol. HSP32/HO-1 inhibits pro-apoptotic c-Jun N-terminal kinase (JNK) activation and hair cell death. Taken together, our data indicate that celastrol inhibits aminoglycoside ototoxicity via HSP32/HO-1 induction

Variable expressivity of FGF3 mutations associated with deafness and LAMM syndrome

<p>Abstract</p> <p>Background</p> <p>Recessive mutations of fibroblast growth factor 3 (FGF3) can cause LAMM syndrome (OMIM 610706), characterized by fully penetrant complete labyrinthine aplasia, microtia and microdontia.</p> <p>Methods</p> <p>We performed a prospective molecular genetic and clinical study of families segregating hearing loss linked to <it>FGF3 </it>mutations. Ten affected individuals from three large Pakistani families segregating <it>FGF3 </it>mutations were imaged with CT, MRI, or both to detect inner ear abnormalities. We also modeled the three dimensional structure of FGF3 to better understand the structural consequences of the three missense mutations.</p> <p>Results</p> <p>Two families segregated reported mutations (p.R104X and p.R95W) and one family segregated a novel mutation (p.R132GfsX26) of <it>FGF3</it>. All individuals homozygous for p.R104X or p.R132GfsX26 had fully penetrant features of LAMM syndrome. However, recessive p.R95W mutations were associated with nearly normal looking auricles and variable inner ear structural phenotypes, similar to that reported for a Somali family also segregating p.R95W. This suggests that the mild phenotype is not entirely due to genetic background. Molecular modeling result suggests a less drastic effect of p.R95W on FGF3 function compared with known missense mutations detected in fully penetrant LAMM syndrome. Since we detected significant intrafamilial variability of the inner ear structural phenotype in the family segregating p.R95W, we also sequenced <it>FGF10 </it>as a likely candidate for a modifier. However, we did not find any sequence variation, pointing out that a larger sample size will be needed to map and identify a modifier. We also observed a mild to moderate bilateral conductive hearing loss in three carriers of p.R95W, suggesting either a semi-dominant effect of this mutant allele of <it>FGF3</it>, otitis media, or a consequence of genetic background in these three family members.</p> <p>Conclusions</p> <p>We noted a less prominent dental and external ear phenotype in association with the homozygous p.R95W. Therefore, we conclude that the manifestations of recessive <it>FGF3 </it>mutations range from fully penetrant LAMM syndrome to deafness with residual inner ear structures and, by extension, with minimal syndromic features, an observation with implications for cochlear implantation candidacy.</p

AKT Signaling Mediates IGF-I Survival Actions on Otic Neural Progenitors

Background: Otic neurons and sensory cells derive from common progenitors whose transition into mature cells requires the coordination of cell survival, proliferation and differentiation programmes. Neurotrophic support and survival of post-mitotic otic neurons have been intensively studied, but the bases underlying the regulation of programmed cell death in immature proliferative otic neuroblasts remains poorly understood. The protein kinase AKT acts as a node, playing a critical role in controlling cell survival and cell cycle progression. AKT is activated by trophic factors, including insulin-like growth factor I (IGF-I), through the generation of the lipidic second messenger phosphatidylinositol 3-phosphate by phosphatidylinositol 3-kinase (PI3K). Here we have investigated the role of IGF-dependent activation of the PI3K-AKT pathway in maintenance of otic neuroblasts. Methodology/Principal Findings: By using a combination of organotypic cultures of chicken (Gallus gallus) otic vesicles and acoustic-vestibular ganglia, Western blotting, immunohistochemistry and in situ hybridization, we show that IGF-I-activation of AKT protects neural progenitors from programmed cell death. IGF-I maintains otic neuroblasts in an undifferentiated and proliferative state, which is characterised by the upregulation of the forkhead box M1 (FoxM1) transcription factor. By contrast, our results indicate that post-mitotic p27Kip-positive neurons become IGF-I independent as they extend their neuronal processes. Neurons gradually reduce their expression of the Igf1r, while they increase that of the neurotrophin receptor, TrkC. Conclusions/Significance: Proliferative otic neuroblasts are dependent on the activation of the PI3K-AKT pathway by IGF-I for survival during the otic neuronal progenitor phase of early inner ear development