The miR-183/Taok1 target pair is implicated in cochlear responses to acoustic trauma.

Acoustic trauma, one of the leading causes of sensorineural hearing loss, induces sensory hair cell damage in the cochlea. Identifying the molecular mechanisms involved in regulating sensory hair cell death is critical towards developing effective treatments for preventing hair cell damage. Recently, microRNAs (miRNAs) have been shown to participate in the regulatory mechanisms of inner ear development and homeostasis. However, their involvement in cochlear sensory cell degeneration following acoustic trauma is unknown. Here, we profiled the expression pattern of miRNAs in the cochlear sensory epithelium, defined miRNA responses to acoustic overstimulation, and explored potential mRNA targets of miRNAs that may be responsible for the stress responses of the cochlea. Expression analysis of miRNAs in the cochlear sensory epithelium revealed constitutive expression of 176 miRNAs, many of which have not been previously reported in cochlear tissue. Exposure to intense noise caused significant threshold shift and apoptotic activity in the cochleae. Gene expression analysis of noise-traumatized cochleae revealed time-dependent transcriptional changes in the expression of miRNAs. Target prediction analysis revealed potential target genes of the significantly downregulated miRNAs, many of which had cell death- and apoptosis-related functions. Verification of the predicted targets revealed a significant upregulation of Taok1, a target of miRNA-183. Moreover, inhibition of miR-183 with morpholino antisense oligos in cochlear organotypic cultures revealed a negative correlation between the expression levels of miR-183 and Taok1, suggesting the presence of a miR-183/Taok1 target pair. Together, miRNA profiling as well as the target analysis and validation suggest the involvement of miRNAs in the regulation of the degenerative process of the cochlea following acoustic overstimulation. The miR-183/Taok1 target pair is likely to play a role in this regulatory process

Reduction in noise-induced functional loss of the cochleae in mice with pre-existing cochlear dysfunction due to genetic interference of prestin.

Various cochlear pathologies, such as acoustic trauma, ototoxicity and age-related degeneration, cause hearing loss. These pre-existing hearing losses can alter cochlear responses to subsequent acoustic overstimulation. So far, the knowledge on the impacts of pre-existing hearing loss caused by genetic alteration of cochlear genes is limited. Prestin is the motor protein expressed exclusively in outer hair cells in the mammalian cochlea. This motor protein contributes to outer hair cell motility. At present, it is not clear how the interference of prestin function affects cochlear responses to acoustic overstimulation. To address this question, a genetic model of prestin dysfunction in mice was created by inserting an internal ribosome entry site (IRES)-CreERT2-FRT-Neo-FRT cassette into the prestin locus after the stop codon. Homozygous mice exhibit a threshold elevation of auditory brainstem responses with large individual variation. These mice also display a threshold elevation and a shift of the input/output function of the distortion product otoacoustic emission, suggesting a reduction in outer hair cell function. The disruption of prestin function reduces the threshold shifts caused by exposure to a loud noise at 120 dB (sound pressure level) for 1 h. This reduction is positively correlated with the level of pre-noise cochlear dysfunction and is accompanied by a reduced change in Cdh1 expression, suggesting a reduction in molecular responses to the acoustic overstimulation. Together, these results suggest that prestin interference reduces cochlear stress responses to acoustic overstimulation

Expression levels of miRNA target genes in cochlear organotypic cultures treated with antisense morpholinos.

<p>Expression changes in (A) <i>Egr1</i>, (B) <i>Irs1</i> and (C) <i>Taok1</i> after miR-183* transfection. *<i>p</i><0.05, Students <i>t</i>-test.</p

Downregulation of miR-183 in cochlear organotypic cultures treated with antisense morpholinos.

<p>Changes in miR-183 expression in the cochlear explants treated with miR-183^* antisense morpholino. *<i>p</i><0.05, Students <i>t-</i>test.</p

Fold Changes in miRNA Expression Following Acoustic Overstimulation.

<p>NOTE: Significance of analysis of microarrays (SAM), False discovery rate (FDR) <0% at 2 h, FDR<4% at 1 d.</p

Venn diagram showing the number of miRNAs detected in either noise-exposed cochleae, control-cochleae, or both.

<p>(<b>A</b>) The numbers of miRNAs detected in the normal samples (pre-noise exposure), the noise-samples (collected at 2 h post-noise exposure) and both. Cut off C_T value ≤34. (<b>B</b>) The numbers of miRNAs detected in the normal samples (pre-noise exposure), the noise-samples (collected at 1 d post-noise exposure) and both. Cut off C_T value ≤34.</p

miRNA/mRNA targeting pathway map.

<p>Targeting interactions between a total of seven miRNAs (light pink ellipses) and seven mRNAs (light blue rectangles) are depicted. These seven miRNAs which were downregulated 1 d post-noise exposure were found to have a list of target genes in the TargetScan database. The remaining 14 genes (13 downregulated at 1 d post-noise exposure and 1 upregulated at 2 h post-noise exposure) were either not included in the TargetScan database or had limited number of target genes to be sufficient for subsequent target analysis.</p

Immunolabeling of Taok1 protein in the cochlear sensory epithelium.

<p>(<b>A</b>) A typical image taken using confocal microscopy showing Taok1 immunoreactivity (green fluorescence) in IHCs, OHCs, and Hensen cells (HC). (<b>B</b>) The merged image from (A) and nuclear staining (propidium iodide, red fluorescence) in the same region. (<b>C</b>) Image showing Taok1 immunoreactivity in pillar cells (PC) and Deiters cells (DC). Bar: 10 µm. (<b>D</b>) The merged image from (C) and nuclear staining in the same region. (<b>E</b>) The image of negative control tissue tested without the primary antibody exhibiting nuclear propidium iodide staining in the OHCs with no clear green fluorescence in the same region. (<b>F</b>) Western blot analysis of Taok1 expression in the rat cochlea. Gapdh was used as a cochlea tissue-specific loading control. A single band around 150 kDa corresponds to Taok1 and the 36 kDa band corresponds to Gapdh.</p

Changes in expression levels of five target mRNAs examined 1 d after noise exposure.

<p><i>Nfat5</i> and <i>Map3k2</i> are downregulated and <i>Taok1</i>, <i>Xiap</i> and <i>Bach2</i> are upregulated in the noise-exposed cochleae compared to the normal cochleae. *<i>p</i><0.05, Student’s <i>t</i>-test.</p

Typical images of cochlear organotypic explants treated with fluorescein-conjugated morpholino oligo.

<p>(<b>A</b>) A typical cross-section image of an organotypic cochlear explant cultured with fluorescein-conjugated morpholino (24 h) taken using confocal microscopy. Arrows indicate the presence of the fluorescein-conjugated morpholino oligo (green fluorescence) in the cytoplasm of the cells. OHC1, OHC2 and OHC3 indicate the first, second, and third rows of OHCs. HC indicates Hensen cells. (<b>B</b>) DIC image showing the three rows of OHCs, the single row of IHC and the HC in the same region as (A). (<b>C</b>) A typical cross-section image of an organotypic cochlear explant cultured without fluorescein-conjugated morpholino (24 h) to demonstrate the level of auto-fluorescence. Scale bar: 10 µm.</p