Changes In Stereocilia Research Articles

The integrity of cochlear hair cells is established and maintained through the localization of Dia1 at apical junctional complexes and stereocilia

Dia1, which belongs to the diaphanous-related formin family, influences a variety of cellular processes through straight actin elongation activity. Recently, novel DIA1 mutants such as p.R1213X (p.R1204X) and p.A265S, have been reported to cause an autosomal dominant sensorineural hearing loss (DFNA1). Additionally, active DIA1 mutants induce progressive hearing loss in a gain-of-function manner. However, the subcellular localization and pathological function of DIA1(R1213X/R1204X) remains unknown. In the present study, we demonstrated the localization of endogenous Dia1 and the constitutively active DIA1 mutant in the cochlea, using transgenic mice expressing FLAG-tagged DIA1(R1204X) (DIA1-TG). Endogenous Dia1 and the DIA1 mutant were regionally expressed at the organ of Corti and the spiral ganglion from early life; alongside cochlear maturation, they became localized at the apical junctional complexes (AJCs) between hair cells (HCs) and supporting cells (SCs). To investigate HC vulnerability in the DIA1-TG mice, we exposed 4-week-old mice to moderate noise, which induced temporary threshold shifts with cochlear synaptopathy and ultrastructural changes in stereocilia 4 weeks post noise exposure. Furthermore, we established a knock-in (KI) mouse line expressing AcGFP-tagged DIA1(R1213X) (DIA1-KI) and confirmed mutant localization at AJCs and the tips of stereocilia in HCs. In MDCKAcGFP-DIA1(R1213X) cells with stable expression of AcGFP-DIA1(R1213X), AcGFP-DIA1(R1213X) revealed marked localization at microvilli on the apical surface of cells and decreased localization at cell-cell junctions. The DIA1-TG mice demonstrated hazy and ruffled circumferential actin belts at AJCs and abnormal stereocilia accompanied with HC loss at 5 months of age. In conclusion, Dia1 plays a pivotal role in the development and maintenance of AJCs and stereocilia, ensuring cochlear and HC integrity. Subclinical/latent vulnerability of HCs may be the cause of progressive hearing loss in DFNA1 patients, thus suggesting new therapeutic targets for preventing HC degeneration and progressive hearing loss associated with DFNA1.

Assembly of hair bundles, an amazing problem for cell biology.

The hair bundle—the sensory organelle of inner-ear hair cells of vertebrates—exemplifies the ability of a cell to assemble complex, elegant structures. Proper construction of the bundle is required for proper mechanotransduction in response to external forces and to transmit information about sound and movement. Bundles contain tightly controlled numbers of actin-filled stereocilia, which are arranged in defined rows of precise heights. Indeed, many deafness mutations that disable hair-cell cytoskeletal proteins also disrupt bundles. Bundle assembly is a tractable problem in molecular and cellular systems biology; the sequence of structural changes in stereocilia is known, and a modest number of proteins may be involved.

Lower Dose of Aminoglycoside Ototoxic Exposure Causes Presynaptic Alterations Assoicated with Hearing Loss

ObjectiveTo study presynaptic alternations of cochlear ribbons arising from aminoglycoside ototoxic stimuli in C57BL/6J mice. MethodsAnimals were injected with low dose gentamicin (100 mg/kg/day) for 14 days, From the 14th to 28th days, the mice were maintained free of gentamicin treatment. Immunohistochemistry labeling was employed to trace RIBEYE, a major presynaptic componment of ribbon synapses. RIBEYE/CtBP2 expression levels were assessed and compared with hearing threshold shifts. Auditory function was assessed by auditory brainstem responses. The stereocilia of outer hair cells (OHCs) and IHCs was examined by scanning electron microscopy (SEM). ResultsHearing thresholds were elevated with peak hearing loss observed on the 7th day after gentamicin exposure, followed by improvement after the 7th day. RIBEYE/CtBP2 expression directly correlated with observed hearing threshold shifts. Strikingly, we did not see any obvious changes in stereocilia in both OHCs and IHCs until the 28th day. Mild changes in stereocilia were only observed in OHCs on the 28th day. ConclusionsThese findings indicate that presynapse cochlear ribbons, rather than stereocilia, may be sensitive to aminoglycoside ototoxic exposure in mice cochleae. A pattern of RIBEYE/CtBP2 expression changes seems to parallel hearing threshold shifts and suggests presynaptic response properties to lower dosage of aminoglycoside ototoxic stimuli.

Dynamic changes in hair cell stereocilia and cochlear transduction after noise exposure

The structures of cochlear transduction include stereocilia at the apical surface of hair cells and their connection to the tectorial membrane. The transduction site is one of the loci for noise-induced cochlear damage. Although stereocilia are susceptible to noise, it has been found that in the inner ears of avians, this fragile structure is largely self-repairing and is associated with recovery of hearing sensitivity after noise exposure, as observed in the difference between the temporal threshold shift (TTS) and the permanent threshold shift (PTS). In the mammalian cochleae, however, threshold shifts measured in the auditory brainstem responses (ABR) did not parallel the chronological changes in the stereocilia on hair cells. It is unclear how the morphological recovery of the stereocilia on the mammalian hair cells is correlated with the changes in cochlear transduction that can be assessed by measuring receptor potential. In the present study, guinea pigs were exposed to a broadband noise of 110dBSPL for 2h. Auditory sensitivity was evaluated using ABR and cochlear transduction was assessed using cochlear microphonics (CM). Stereocilia morphology was quantified at different time points after the noise and compared with the control. The noise produced a TTS of 55.69±14.13dB in frequency-averaged ABR thresholds. The threshold shift was reduced to 9.58±11.75dBSPL 1month later with virtually no loss of hair cells. Damage to the stereocilia immediately after noise exposure was found to be associated with depression of CM amplitude. Virtually no abnormal stereocilia were observed 1month after the noise in association with a fully recovered CM.

Intercellular contacts between chick stereocilia after acoustic overstimulation

The goal of this study was to analyze the distribution of actin and the shape of stereocilia of chick hair cells that survive acoustic trauma. Chicks were exposed to intense octave band noise for 4 h. They were killed either immediately after the exposure, after 6 or after 72 h. The basilar papillae were examined using scanning electron microscopy and fluorescence microscopy, with phalloidin as an actin-specific probe. Injured hair cells which survived the trauma displayed disorganized stereocilia bundles, elongated stereocilia, and supernumerary stereocilia bundles. Tips of stereocilia in the damaged region of the basilar papilla appeared to be in contact with tips of stereocilia of neighboring hair cells. These contacts may represent ‘stress links’ which appear in traumatized hair cells. These results show that substantial changes in stereocilia occur within hours of exposure to intense noise. We speculate that surviving hair cells may play a role in the process of repair of the basilar papilla after noise trauma and that the changes in stereocilia structure described here are related to this role.

3-D analysis of F-actin in stereocilia of cochlear hair cells after loud noise exposure

Fluorescence microscopy can be a useful tool in the early detection of pathological changes in the stereocilia of outer hair cells which have undergone acoustic overstimulation. Fluorescent phalloidin, a highly specific F-actin stain, can be used to label F-actin in stereocilia. In this study, phalloidin label is used to determine quantitative changes of F-actin in the stereocilia of guinea pigs exposed to loud noise (117 dB; octave band noise, centered at 1 kHz; 4 h). Reliably determining three-dimensional (3-D) structural changes in stereocilia is a challenging problem in optical microscopy since stereocilia diameter is close to the optical resolution limit. In order to alleviate the problem, a computational 3-D microscopy technique is used (Avinash et al., 1992). Whole-mounts of the cochlear second and third turns were examined in a Leitz Orthoplan microscope through a Leitz Plan Apo objective lens (100 ×; 1.32 N. A.; 170 0.17 ). Images were acquired with a charge-coupled device camera where the focus was shifted in 0.2 μm steps using a piezoelectric translator. Images were processed with the appropriate point spread function of the optical system. Analysis of control cochleas indicate that our technique can resolve single stereocilia and distinguish between various intensities of label along each stereocilia. In noise-exposed cochleas, our data show length and intensity changes in the phalloidin label. These results suggest that both depolymerization and polymerization of F-actin can occur in stereocilia of outer hair cells after acoustic overstimulation. Our findings demonstrate the applicability of computational 3-D microscopy to quantitative and qualitative analysis of stereocilia.

Effect of Furosemide Upon Morphology of Hair Bundles in Guinea Pig Cochlear Hair Cells

Furosemide was administered by intraperitoneal injection and intracochlear perfusion. The peak-to-peak amplitude of the cochlear microphonic at high sound intensities was reduced however the drug was administered. After a period of time ranging from 1-4 h following drug application, cochleae were fixed and prepared for scanning electron microscopy to examine the hair cells. Changes in the hair bundles from basal turns tended to be more extensive than those of more apical turns. Initially there was an increase in the granularity of the surface of the stereociliary membrane and a tendency for cross-links to swell, stretch and break. Later, the surface texture of stereocilia became smoother than normal, and hair cells in the basal turns showed extensive erosion and fracture of cross-links. Tip links could survive even when extensively stretched. It is possible that the morphological changes in stereocilia reported here do not arise from direct actions of furosemide, but indirectly, from perturbations of the ionic composition of cochlear fluids induced by effects of the drug upon the stria vascularis.

Scanning electron microscopy of the cochlea in rats with Streptococcus pneumoniae otitis media.

This investigation was performed to determine the morphologic effects of bacterial otitis media on the organ of Corti. The middle ear cavities of rats were inoculated with Streptococcus pneumoniae or saline and the animals were killed on days 1, 4, 7, 10, 14, and 21 after inoculation. Middle ear cultures were obtained and the cochleas were examined using scanning electron microscopy. All animals killed on day 1 had positive cultures, but by day 21, all cultures were negative. Cochlear changes observed were (1) damage to supporting cells (Deiters' cells), (2) morphologic changes of hair cell stereocilia, and (3) loss of inner and outer hair cell stereocilia and cell bodies (to a lesser extent), especially in the lower middle and basal turns. These changes appeared to occur in a definite sequence; ie, damage to the supporting cells, changes in stereocilia, and, finally, hair cell loss. These data show that cochlear damage and hair loss can be associated with bacterial otitis media.