Small GTPases mediate transmembrane signaling and regulate the actin cytoskeleton in eukaryotic cells. Here, we characterize the auditory pathology of adult male CBA/J mice exposed to traumatic noise (2–20 kHz; 106 dB; 2 h). Loss of outer hair cells was evident 1 h after noise exposure in the basal region of the cochlea and spread apically with time, leading to permanent threshold shifts of 35, 60, and 65 dB at 8, 16, and 32 kHz. Several biochemical and molecular changes correlated temporally with the loss of cells. Immediately after exposure, the concentration of ATP decreased in cochlear tissue and reached a minimum after 1 h while the immunofluorescent signal for p-AMPKα significantly increased in sensory hair cells at that time. Levels of active Rac1 increased, whereas those of active RhoA decreased significantly 1 h after noise attaining a plateau at 1 to 3 h; the formation of a RhoA-p140mDia complex was consistent with an activation of Rho GTPase pathways. Also at 1 to 3 h after exposure, the caspase-independent cell death marker, endonuclease G, translocated to the nuclei of outer hair cells. Finally, experiments with the inner ear HEI-OC1 cell line demonstrated that the energy-depleting agent oligomycin enhanced both Rac1 activity and cell death. The sum of the results suggests that traumatic noise induces transient cellular ATP depletion and activates Rho GTPase pathways, leading to death of outer hair cells in the cochlea.

Post-translational modification of histones is an important form of chromatin regulation impacting transcriptional activation. Histone acetyltransferases (HATs), for example, acetylate lysine residues on histone tails enhancing gene transcription, while histone deacetylases (HDACs) remove those acetyl groups and repress gene transcription. Deficient histone acetylation is associated with pathologies, and histone deacetylase inhibitors have been studied in the treatment of cancer and neurodegenerative diseases. Here we explore histone acetylation in cochlear sensory cells following a challenge with gentamicin, an aminoglycoside antibiotic known to cause loss of auditory hair cells and hearing. The addition of the drug to organotypic cultures of the mouse organ of Corti decreased the acetylation of histone core proteins (H2A Ack5, H2B Ack12, H3 Ack9, and H4 Ack8) followed by a loss of sensory cells. Protein levels of HDAC1, HDAC3 and HDAC4 were increased while the HATs CBP and p300 remained unchanged. We next hypothesized that protecting histone acetylation should prevent cell death and tested the effects of HDAC-inhibitors on the actions of gentamicin. Co-treatment with trichostatin-A maintained near-normal levels of acetylation of histone core proteins in cochlear outer hair cells and attenuated gentamicin-induced cell death. The addition of sodium butyrate also rescued hair cells from damage by gentamicin. The results are consistent with an involvement of deficient histone acetylation in aminoglycoside-induced hair cell death by and point to the potential value of HDAC-inhibitors in protection from the side effects of these drugs.

We have previously demonstrated that oxidative stress increases in the inner ear of aging CBA/J mice and might contribute to the loss of function of the sensory system. We now investigate the activation of cell death pathways in the cochlea of these animals. Middle-aged (12 months) and old (18-26 months) mice with hearing deficits displayed outer hair cell nuclei with apoptotic and, to a lesser extent, necrotic features. Both intrinsic and extrinsic cell death pathways were activated by translocation or post-translational modification of proteins in the aging cochlea as compared to young (3 months) animals. Cytosolic cytochrome C increased, formed a complex with, and activated caspase 9. Endonuclease G translocated to the nuclei of aging outer hair cells suggesting its function as an apoptotic DNase. The cleaved (and hence active) forms of calpain I and calpain II increased while active cathepsin D was transiently elevated in middle-aged but not old animals. Finally, increases in the phosphorylation of p38 MAPK and JNK implicated the additional involvement of the MAPK pathway. The results suggest that multiple cell death pathways, all potentially linked to oxidative stress, are activated in hair cells of the auditory organ in aging mice.

It has been reported that the degeneration of cochlear hair cells is the typical cause of presbycusis (or age-related hearing loss). However, the molecular mechanisms that mediate cochlear hair cell apoptosis are not yet fully understood and there is no effective treatment for this disorder. MicroRNAs (miRNAs or miRs) have been increasingly shown to be associated with age-related diseases and are emerging as promising therapeutic targets. In this study, we investigated whether miR-29b is involved in the degeneration of cochlear hair cells. To examine our hypothesis, nuclear staining and terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) were used to quantify the hair cell counts. RT-qPCR and western blot analysis were used to examine miR-29b/sirtuin 1 (SIRT1)/proliferator-activated receptor-gamma coactivator 1α (PGC-1α) signaling in cochlear hair cells. We found that there was a significant degeneration of cochlear hair cells and a higher expression of miR-29b in aged C57BL/6 mice compared with young mice. There was also an age-related decrease in the expression of SIRT1 and PGC-1α. In the inner ear cell line, HEI-OC1, miR-29b overexpression (by transfection with miR-29b mimic) inhibited SIRT1 and PGC-1α expression, leading to an increase in mitochondrial dysfunction and apoptosis. Moreover, the inhibition of miR-29b (by transfection with miR-29b inhibitor) increased SIRT1 and PGC-1α expression, while it decreased apoptosis. Taken together, our findings support a link between age-related cochlear hair cell apoptosis and miR-29b/SIRT1/PGC-1α signaling, which may present an attractive pharmacological target for the development of novel drugs for the treatment of age-related hearing loss.

The wild species related to the cultivated tomato, Lycopersicon esculentum Mill., are a rich source of useful genes for germplasm improvement and varietal development. Characterization and utilization of exotic germplasm can be accelerated by the use of molecular linkage maps. Recently, we have identified an accession (LA722) within L. pimpinellifolium Jusl., a closely-related, red-fruited wild species of tomato, which exhibits a number of desirable agricultural characteristics including salt tolerance, disease resistance, and high fruit quality. The limited DNA marker polymorphism between L. esculentum and L. pimpinellifolium, however, restricts the use of the high-density molecular map of tomato, which was previously constructed based on a cross between L. esculentum and L. pennellii Corr., for marker-assisted identification and utilization of useful genes in LA722. To overcome this problem, we have constructed a linkage map based on restriction fragment length polymorphisms (RFLPs) which were identified between LA722 and a fresh-market tomato breeding line (NC84173). The mapping population (consisting of 119 BC 1 individuals) was genotyped for 151 RFLP markers, including 17 germination related and 2 potassium transporter cDNAs. The DNA markers spanned approximately 1192 cM of the tomato genome with an average distance of 7.9 cM between markers. The length of the map and the linear order of the markers were in good agreement with those of the previously published molecular maps of tomato, however, there were considerable differences in the distribution of recombinations along the chromosomes. Comparison of all seven published molecular maps of tomato, which were constructed based on different inter-and intraspecific crosses, indicated that some chromosomal regions were more stable than others in terms of the frequency of recombinations. Similarities and differences among tomato molecular maps are discussed in relation to phylogenetic relationships between parents of the mapping populations. In comparison, a L. esculentum × L. pimpinellifolium map should be more useful than other interspecific maps for marker-assisted exploitation of genetic variation that exists within L. pimpinellifolium or L. esculentum; the latter is because of the extensive introgression of genes from L. pimpinellifolium into L. esculentum, which occurred over time by natural means or through plant breeding. 103Résumé : Les espèces sauvages apparentées à la tomate cultivée, Lycopersicon esculentum Mill., constituent un riche réservoir de gènes utiles pour l'amélioration du germoplasme et la création variétale. La caractérisation et l'exploitation du germoplasme exotique peut être accélérée par l'emploi de cartes génétiques composées de marqueurs moléculaires. Récemment, les auteurs ont identifié une accession (LA722) intéressante parmi le L. pimpinellifolium Jusl., une espèce sauvage à fruits rouges qui est très apparentée à la tomate. Cette accession possède plusieurs caractéristiques avantageuses dont la tolérance à la salinité, la résist...

We have previously reported the activation of cell death pathways in the sensory cells of the aging cochlea. Here we investigate age-associated changes in survival mechanisms focusing on phosphatidylinositol 3,4,5-trisphosphate (PIP 3 )/Akt signaling. The animal model is the CBA/J mouse of 18 months of age prior to the onset of major functional loss (ABR thresholds, 26 8 dB SPL) which is compared to young animals of 3 months of age (ABR thresholds, 19 7 dB SPL). Immunostaining on cochlear cryosections revealed a wide-spread distribution of PIP 3 in the cochlea which was markedly attenuated in old animals in inner and outer hair cells, Deiters cells and pillar cells. Protein levels of the lipid phosphatase PTEN which regulates PIP 3 increased in those cells with aging while its mRNA did not, suggesting an age-related reduction of PTEN degradation. Furthermore, staining intensity of phosphorylated PTEN (ser380) and its nuclear localization increased. Consistent with a reduction of PIP 3 , the phosphorylation of the downstream target Akt at threonine 308 significantly decreased in outer hair cells. The results suggest a decline of the survival capacity of aging outer hair cells due to a decrease in PIP 3 /Akt signaling caused by an increase of PTEN.

Trans-tympanic injection into the middle ear has long been the standard for local delivery of compounds in experimental studies. Here we demonstrate the advantages of the novel method of intra-tympanic injection through the otic bone for the delivery of compounds or siRNA into the adult mouse cochlea. First, a fluorescently-conjugated scrambled siRNA probe was applied via intra-tympanic injection into the middle ear cavity and was detected in sensory hair cells and nerve fibers as early as 6 h after the injection. The fluorescent probe was also detected in other cells of the organ of Corti, the lateral wall, and in spiral ganglion cells 48 h after the injection. Furthermore, intra-tympanic delivery of Nox3 siRNA successfully reduced immunofluorescence associated with Nox3 in outer hair cells 72 h after injection by 20%. Drug or siRNA delivery via intra-tympanic injection does not compromise the tympanic membrane or interfere with noise-induced hearing loss, while trans-tympanic injections significantly altered the cochlear response to noise exposure. In summary, intra-tympanic injection through the otic bone into the middle ear cavity provides a promising approach for delivery of compounds or siRNA to cochlear hair cells of adult mice, relevant for the study of mechanisms underlying inner ear insults and, specifically, noise-induced hearing loss.

Aminoglycoside antibiotics and cisplatin (CDDP) are the major ototoxins of clinical medicine due to their capacity to cause significant, as well as permanent hearing loss by targeting the mammalian sensory cells. Understanding the pathogenesis of damage is the first step in designing effective prevention of drug-induced hearing loss. In-vitro systems greatly enhance the efficiency of biochemical and molecular investigations through ease of access and manipulation. HEI-OC1, an inner ear cell line derived from the immortomouse, expresses markers for auditory sensory cells and, therefore, is a potential tool to study the ototoxic mechanisms of drugs like aminoglycoside antibiotics and CDDP. HEI-OC1 cells (and also HeLa cells) efficiently take up fluorescently tagged gentamicin and respond to drug treatment with changes in cell death and survival signaling pathways. Within hours, the C-jun N-terminal kinase pathway and the transcription factor AP-1 were activated and at later times, the “executioner caspase”, caspase-3. These responses were robust and elicited by both gentamicin and kanamycin. However, despite the initiation of apoptotic pathways and transient changes in nuclear morphology, cell death was not observed following aminoglycoside treatment, while administration of CDDP lead to significant cell death as determined by flow cytometric measurements; β-galactosidase analysis ruled out senescence in gentamicin-treated cells. The ability to withstand treatment with aminoglycosides but not with CDDP suggests that this cell line might be helpful in providing some insight into the differential actions of the two ototoxic drugs.