Michio Murakoshi

Prestin binding peptides as ligands for targeted polymersome mediated drug delivery to outer hair cells in the inner ear

Targeted delivery of treatment agents to the inner ear using nanoparticles is an advanced therapeutic approach to cure or alleviate hearing loss. Designed to target the outer hair cells of the cochlea, two 12-mer peptides (A(665) and A(666)) with affinity to prestin were identified following 3 rounds of sequential phage display. Two-round display with immobilized prestin protein was used to enrich the library for full-length prestin. The last round was performed using Cos-7 cells transiently transfected with a cCFP-prestin plasmid to display phages expressing peptides restrictive to the extracellular loops of prestin. The binding properties of A(665) and A(666) shown by flow cytometry demonstrated selectivity to prestin-expressing Chinese hamster ovary cells. PEG6K-b-PCL19K polymersomes covalently labelled with these peptides demonstrated effective targeting to outer hair cells in a rat cochlear explant study.

Salicylate restores transport function and anion exchanger activity of missense pendrin mutations.

The SLC26A4 gene encodes the transmembrane protein pendrin, which is involved in the homeostasis of the ion concentration of the endolymph of the inner ear, most likely by acting as a chloride/bicarbonate transporter. Mutations in the SLC26A4 gene cause sensorineuronal hearing loss. However, the mechanisms responsible for such loss have remained unknown. Therefore, in this study, we focused on the function of ten missense pendrin mutations (p.P123S (Pendred syndrome), p.M147V (NSEVA), p.K369E (NSEVA), p.A372V (Pendred syndrome/NSEVA), p.N392Y (Pendred syndrome), p.C565Y (NSEVA), p.S657N (NSEVA), p.S666F (NSEVA), p.T721M (NSEVA) and p.H723R (Pendred syndrome/NSEVA)) reported in Japanese patients, and analyzed their cellular localization and anion exchanger activity using HEK293 cells transfected with each mutant gene. Immunofluorescent staining of the cellular localization of the pendrin mutants revealed that p.K369E and p.C565Y, as well as wild-type pendrin, were transported to the plasma membrane, while 8 other mutants were retained in the cytoplasm. Furthermore, we analyzed whether salicylate, as a pharmacological chaperone, restores normal plasma membrane localization of 8 pendrin mutants retained in the cytoplasm to the plasma membrane. Incubation with 10 mM of salicylate of the cells transfected with the mutants induced the transport of 4 pendrin mutants (p.P123S, p.M147V, p.S657Y and p.H723R) from the cytoplasm to the plasma membrane and restored the anion exchanger activity. These findings suggest that salicylate might contribute to development of a new method of medical treatment for sensorineuronal hearing loss caused by the mutation of the deafness-related proteins, including pendrin.

Immune atomic force microscopy of prestin-transfected CHO cells using quantum dots

Prestin, a membrane protein of the outer hair cells (OHCs), is known to be the motor which drives OHC somatic electromotility. Electron microscopic studies showed the lateral membrane of the OHCs to be densely covered with 10-nm particles, they being believed to be a motor protein. Imaging by atomic force microscopy (AFM) of prestin-transfected Chinese hamster ovary (CHO) cells revealed 8- to 12-nm particle-like structures to possibly be prestin. However, since there are many kinds of intrinsic membrane proteins other than prestin in the plasma membranes of OHCs and CHO cells, it was impossible to clarify which structures observed in such membranes were prestin. In the present study, an experimental approach combining AFM with quantum dots (Qdots), used as topographic surface markers, was carried out to detect individual prestin molecules. The inside-out plasma membranes were isolated from the prestin-transfected and untransfected CHO cells. Such membranes were then incubated with antiprestin primary antibodies and Qdot-conjugated secondary antibodies. Fluorescence labeling of the prestin-transfected CHO cells but not of the untransfected CHO cells was confirmed. The membranes were subsequently scanned by AFM, and Qdots were clearly seen in the prestin-transfected CHO cells. Ring-like structures, each with four peaks and one valley at its center, were observed in the vicinity of the Qdots, suggesting that these structures are prestin expressed in the plasma membranes of the prestin-transfected CHO cells.

Dynamic characteristics of the middle ear in neonates.

OBJECTIVE Early diagnosis and treatment of hearing disorders in neonates is highly effective for realization of linguistic competence and intellectual development. To objectively and quickly evaluate the dynamic characteristics of the middle ear, a sweep frequency impedance (SFI) meter was developed, which allowed the diagnosis of middle-ear dysfunctions in adults and children. However, this SFI meter was not applicable to neonates since the size of the measurement probe was too large. In the present study, therefore, the SFI meter was improved, i.e., the diameter of the probe was reduced to that of the neonatal external ear canal. By using this newly designed SFI meter, SFI tests were performed in healthy neonates. METHODS A sound of the sweeping sinusoidal frequency between 0.1 kHz and 2.0 kHz in 0.02-kHz step intervals is presented to the ear canal by an SFI probe while the static pressure of the ear canal is kept constant. During this procedure, the sound pressure level (SPL) is measured. The measurements are performed at 50-daPa intervals of static pressure from 200 daPa to -200 daPa. RESULTS Measurements were conducted in 10 ears of 9 neonates. The SPL showed two variations at 0.26 ± 0.03 kHz and 1.13 ± 0.12 kHz. Since the SPL is known to show a variation at frequencies from 1.0 kHz to 1.6 kHz due to the resonance of the middle ear in adults and children with normal hearing, the second variation is probably related to such resonance in neonates. The measurement of gel models, which mimics the neonatal external ear canal, showed a variation in SPL at around 0.5 kHz. This implies that the source of the first variation may possibly be related to the resonance of the external ear canal wall. CONCLUSIONS SFI tests revealed that there were two variations in the SPL curve in neonates, one at 0.26 ± 0.03 kHz and the other at 1.13 ± 0.12 kHz, the former and the latter being possibly related to the resonance of the external ear canal wall and that of the middle ear, respectively. This result suggests that the dynamic characteristics of the middle ear in neonates are different from those in adults.

Atomic force microscopy imaging of the structure of the motor protein prestin reconstituted into an artificial lipid bilayer

Prestin is the motor protein of cochlear outer hair cells and is essential for mammalian hearing. The present study aimed to clarify the structure of prestin by atomic force microscopy (AFM). Prestin was purified from Chinese hamster ovary cells which had been modified to stably express prestin, and then reconstituted into an artificial lipid bilayer. Immunofluorescence staining with anti‐prestin antibody showed that the cytoplasmic side of prestin was possibly face up in the reconstituted lipid bilayer. AFM observation indicated that the cytoplasmic surface of prestin was ring‐like with a diameter of about 11 nm.

Mutation-induced reinforcement of prestin-expressing cells

The motor protein prestin in cochlear outer hair cells is a member of the solute carrier 26 family, but among the proteins of that family, only prestin can confer the cells with nonlinear capacitance (NLC) and motility. In the present study, to clarify contributions of unique amino acids of prestin, namely, Met-122, Met-225 and Thr-428, to the characteristics of prestin, mutations were introduced into those amino acids. As a result, NLC remained unchanged by both replacement of Met-122 by isoleucine and that of Thr-428 by leucine, suggesting that those amino acids were not important for the generation of NLC. Surprisingly, the replacement of Met-225 by glutamine statistically increased NLC as well as the motility of prestin-expressing cells without an increase in the amount of prestin expression in the plasma membrane. This indicates that Met-225 in prestin somehow adjusts NLC and the motility of prestin-expressing cells.

Effects of heat stress on Young's modulus of outer hair cells in mice.

Intense sound exposure causes permanent hearing loss due to hair cell and cochlear damage. Prior conditioning with sublethal stressors, such as nontraumatic sound, heat stress and restraint protects the ear from acoustic injury. However, the mechanisms underlying conditioning-related cochlear protection remain unknown. In this paper, Youngs modulus and the amount of filamentous actin (F-actin) of outer hair cells (OHCs) with/without heat stress were investigated by atomic force microscopy and confocal laser scanning microscopy, respectively. Conditioning with heat stress resulted in a statistically significant increase in Youngs modulus of OHCs at 3-6 h after application, and such modulus then began to decrease by 12 h and returned to pre-conditioning level at 48 h after heat stress. The amount of F-actin began to increase by 3 h after heat stress and peaked at 12 h. It then began to decrease by 24 h and returned to the pre-conditioning level by 48-96 h after heat stress. These time courses are consistent with a previous report in which heat stress was shown to suppress permanent threshold shift (PTS). In addition, distortion product otoacoustic emissions (DPOAEs) were confirmed to be enhanced by heat stress. These results suggest that conditioning with heat stress structurally modifies OHCs so that they become stiffer due to an increase in the amount of F-actin. As a consequence, OHCs possibly experience less strain when they are exposed to loud noise, resulting in protection of mammalian hearing from traumatic noise exposure.

Normative sweep frequency impedance measures in healthy neonates

BACKGROUND Diagnosing middle ear disorders in neonates is a challenging task for both audiologists and otolaryngologists. Although high-frequency (1000 Hz) tympanometry and acoustic stapedial reflex tests are useful in diagnosing middle ear problems in this age group, they do not provide information about the dynamics of the middle ear in terms of its resonance frequency (RF) and mobility. The sweep frequency impedance (SFI) test can provide this information, which may assist in the diagnosis of middle ear dysfunction in neonates. PURPOSE This study aimed to investigate the feasibility of testing neonates using the SFI technique, establish normative SFI data for RF and mobility of the middle ear in terms of changes in sound pressure level (∆SPL in dB), and describe the dynamics of the middle ear in healthy Australian neonates. STUDY SAMPLE A prospective sample of 100 neonates (58 males, 42 females) with a mean gestational age of 39.3 wk (SD = 1.3 wk; range = 38-42 wk), who passed all three tests, namely, automated auditory brainstem response, transient evoked otoacoustic emissions, and 1000 Hz tympanometry, were included in this study. DATA COLLECTION AND ANALYSIS A SFI research prototype was used to collect the data. First, the SPL in the ear canal was measured as a probe-tone frequency was swept from 100-2000 Hz with the ear canal static pressure held constant at 200 daPa. Then, this measurement was repeated with the static pressure reduced in 50 daPa steps to -200 daPa. Additional measurement was also performed at the static pressure, where the peak of the 1000 Hz tympanogram occurred. A graph showing the variation of SPL against frequency at all static pressures was plotted. From this graph, the RF and ∆SPL at tympanometric peak pressure (TPP) were determined. Descriptive statistics and an analysis of variance (ANOVA) were applied to the RF and ∆SPL data with gender and ear as independent variables. RESULTS The results showed two resonance regions of the outer/middle ear with the high RF (mean = 1236 Hz; 90% range: 830-1518 Hz) being approximately equal to four times that of the low RF (mean = 287 Hz; 90% range = 209-420 Hz). The low RF was more easily identifiable than the high RF. The ∆SPL at the low RF (mean = 8.2 dB; 90% range = 3.4-13 dB) was greater than that at the high RF (mean = 5.0 dB; 90% range = 1.5-8.1 dB). There were no significant differences or interactions between genders and ears. CONCLUSION The study showed that the SFI is a feasible test of middle ear function in neonates. The SFI results revealed two regions of resonance with the lower resonance (287 Hz) possibly related to the movements of the outer ear canal wall and higher resonance (1236 Hz) related to the resonance of the middle ear. The normative data developed in this study will be useful in evaluating outer and middle ear function in neonates.

An apparatus for diagnosis of ossicular chain mobility in humans

Confirmation of the conditions of the ossicles is essential for tympanoplasty. However, at present, ossicular mobility is experimentally estimated with palpation by a surgeon, and the results depend on the surgeons skill. In this study, a new apparatus for quantitatively measuring ossicular mobility was developed. With this apparatus, the ossicles were displaced and the reaction force from the ossicles, (i.e. the load applied to the ossicles) was simultaneously detected. Ossicular mobility of three patients with otosclerosis or chronic otitis media was measured to evaluate the usability of the apparatus. The apparatus can distinguish the differences in ossicular mobility between normal and fixed ossicles, and it makes estimating the change of mobility between pre- and post-treatments for ossicular fixation possible. Positive correlation was seen between ossicular mobility and hearing level. Sumario La confirmación de la movilidad osicular es esencial para la timpanoplastia; sin embargo, actualmente se estima experimentalmente por medio de palpación por parte del cirujano y el resultado depende de la destreza del mismo. En este estudio se desarrolló un nuevo aparato para medir cuantitativamente la movilidad osicular. Con este aparato, los huesecillos son desplazados y su fuerza de reacción, p.e. la carga que se les aplica, es detectada simultáneamente. Se midió la movilidad osicular de tres pacientes con otoesclerosis u otitis media crónica para evaluar la utilidad del aparato. El aparato puede distinguir entre movilidad normal o huesecillos fijos, y da un estimado de cambio de movilidad pre y post-tratamiento. Se observó una correlación positiva entre la movilidad osicular y el nivel de audición.

Effects of ear canal static pressure on the dynamic behaviour of outer and middle ear in newborns

OBJECTIVE The present study investigated the effect of ear canal pressure on the dynamic behaviour of the outer and middle ear in newborns with and without a conductive condition using the sweep frequency impedance (SFI) technology. METHODS A test battery consisting of automated auditory brainstem response (AABR), transient evoked otoacoustic emission (TEOAE) and 1000-Hz tympanometry (HFT) was performed on 122 ears of 86 healthy newborns and 10 ears of 10 newborns with a conductive condition (failed TEOAE and HFT). The dynamic behaviour of the outer and middle ear, when the pressure applied to the ear canal was varied from 200 to -200daPa, was evaluated in terms of the sound pressure level (SPL) in the ear canal, resonance frequency (RF) and displacement (ΔSPL). RESULTS Application of either a positive or negative static pressure to the ear canal of healthy newborns increased the resonance frequency of the outer (RF1) and middle ear (RF2), but decreased the displacements of the outer (ΔSPL1) and middle ear (ΔSPL2). Positive static pressures resulted in lower SPL while negative static pressures resulted in higher SPL than that at ambient pressure (0daPa). At -200daPa, more than 90% of ears showed signs of collapsed ear canal. The dynamic behaviour under various positive and negative static pressures for newborn ears with a conductive condition indicated similar pattern of SPL, RF1 and ΔSPL1 responses for the outer ear as per healthy ears, but abnormal responses for the middle ear. CONCLUSIONS While both positive and negative pressures applied to the ear canal have the same effect of stiffening the outer and middle ear, negative pressure of up to -200daPa resulted in more than 90% of ears with a collapsed ear canal. The results of the present study do not only offer useful clinical information for differentiating healthy ears from ears with a conductive condition, but also provide information on the maturation aspects of the outer and middle ear in newborns.