Prestin-Mediated Frequency Selectivity Does not Cover Ultrahigh Frequencies in Mice / 神经科学通报·英文版

In mammals, the piezoelectric protein, Prestin, endows the outer hair cells (OHCs) with electromotility (eM), which confers the capacity to change cellular length in response to alterations in membrane potential. Together with basilar membrane resonance and possible stereociliary motility, Prestin-based OHC eM lays the foundation for enhancing cochlear sensitivity and frequency selectivity. However, it remains debatable whether Prestin contributes to ultrahigh-frequency hearing due to the intrinsic nature of the cell's low-pass features. The low-pass property of mouse OHC eM is based on the finding that eM magnitude dissipates within the frequency bandwidth of human speech. In this study, we examined the role of Prestin in sensing broad-range frequencies (4-80 kHz) in mice that use ultrasonic hearing and vocalization (to >100 kHz) for social communication. The audiometric measurements in mice showed that ablation of Prestin did not abolish hearing at frequencies >40 kHz. Acoustic associative behavior tests confirmed that Prestin-knockout mice can learn ultrahigh-frequency sound-coupled tasks, similar to control mice. Ex vivo cochlear Ca2+ imaging experiments demonstrated that without Prestin, the OHCs still exhibit ultrahigh-frequency transduction, which in contrast, can be abolished by a universal cation channel blocker, Gadolinium. In vivo salicylate treatment disrupts hearing at frequencies <40 kHz but not ultrahigh-frequency hearing. By pharmacogenetic manipulation, we showed that specific ablation of the OHCs largely abolished hearing at frequencies >40 kHz. These findings demonstrate that cochlear OHCs are the target cells that support ultrahigh-frequency transduction, which does not require Prestin.

ROS Scavenger, Ebselen, Has No Preventive Effect in New Hearing Loss Model Using a Cholesterol-Chelating Agent

BACKGROUND AND OBJECTIVES: The antioxidant ebselen will be able to limit or prevent the ototoxicity arising from 2-hydroxypropyl-β-cyclodextrin (HPβCD). Niemann-Pick Type C (NPC) disease is a disorder of lysosomal storage manifested in sphingolipidosis. Recently, it was noted that experimental use of HPβCD could partially resolve the symptoms in both animals and human patients. Despite its desirable effect, HPβCD can induce hearing loss, which is the only major side effect noted to date. Understanding of the pathophysiology of hearing impairment after administration of HPβCD and further development of preventive methods are essential to reduce the ototoxic side effect. The mechanisms of HPβCD-induced ototoxicity remain unknown, but the resulting pathology bears some resemblance to other ototoxic agents, which involves oxidative stress pathways. To indirectly determine the involvement of oxidative stress in HPβCD-induced ototoxicity, we tested the efficacy of an antioxidant reagent, ebselen, on the extent of inner ear side effects caused by HPβCD. MATERIALS AND METHODS: Ebselen was applied prior to administration of HPβCD in mice. Auditory brainstem response thresholds and otopathology were assessed one week later. Bilateral effects of the drug treatments also were examined. RESULTS: HPβCD-alone resulted in bilateral, severe, and selective loss of outer hair cells from base to apex with an abrupt transition between lesions and intact areas. Ebselen co-treatment did not ameliorate HPβCD-induced hearing loss or alter the resulting histopathology. CONCLUSIONS: The results indirectly suggest that cochlear damage by HPβCD is unrelated to reactive oxygen species formation. However, further research into the mechanism(s) of HPβCD otopathology is necessary.

Effect of melatonin on expression of Prestin protein in the inner ear of mice following radiotherapy / 中华耳鼻咽喉头颈外科杂志

Objective@#To investigate the effect of melatonin on the expression of prestin protein in the inner ear of mice following a single dose radiation therapy, so as to provide the basis for the mechanism study of radiation induced inner ear injury and its prevention.@*Methods@#Sixty 4-week-old male mice were randomly divided into six groups, including the control group (A group), 50 mg/kg MLT group (B group), 5 mg/kg MLT group (C group), 50 mg/kg MLT + radiotherapy group (D group), 5 mg/kg MLT+ radiotherapy group (E group), and 16 Gy radiotherapy group (F group). Each experimental group was randomly subdivided into two subgroups, which were killed to harvest the cochlea on the 3rd and 7th days following 16 Gy radiation. The specimens were used for immunostaining and Western blot to detect the expression of prestin protein. SPSS 19.0 software was used for statistical analysis.@*Results@#Prestin protein mainly distributed in the lateral membrane above the outer hair cell nucleus. When compared with A, B and C group, the expression of prestin protein in the inner ear was significantly up-regulated in F group (P<0.05). However, D and E group reduced the abnormal expression of prestin following radiotherapy when compared with F group, the difference was statistically significant (P<0.05), and the effect of D group was more significant than E group (P<0.05).@*Conclusions@#The prestin protein of cochlea is mainly distributed in the lateral membrane above the outer hair cell nucleus. Following the high-dose radiotherapy, the prestin expression is upregulated, and melatonin can control the abnormal expression of prestin protein induced by radiotherapy with dose dependent.

Identification and analysis of the proteins interacted with Prestin in cochlear outer hair cells of guinea pig / 中华耳鼻咽喉头颈外科杂志

Objective@#To explore the regulation and mechanism of Prestin protein by identifying the proteins interacted with Prestin in cochlear outer hair cell(OHC) and analyzing their biological function.@*Methods@#Co-immunoprecipitation combined mass spectrometry technology was used to isolate and identify the proteins interacted with Prestin protein of OHC, bioinformatics was used to construct Prestin protein interaction network. The proteins interacted with Prestin in OHC of guinea pig were determined by matching primary interaction mass spectrometry with protein interaction network, and annotated their functions.@*Results@#The results of co-immunoprecipitation combined with mass spectrometry showed that 116 kinds of credible proteins could interact with Prestin. By constructing Prestin protein interaction network, matching the results of mass spectrometry and analyzing of sub-cellular localization, eight kinds of proteins were confirmed that they interacted with Prestin directly, namely EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1, and ERH, respectively, which were mainly involved in the synthesis and transportation, transmembrane folding and localization, structural stability and signal transduction of Prestin protein.@*Conclusion@#EEF2, HSP90AB1, FN1, FLNA, EEF1A1, HSP90B1, ATP5A1 and ERH provide molecular basis for sensory amplification function of OHCs by participating in biotransformation, transmembrane folding and localization, signal transduction and other biological processes of Prestin protein.

Prestin Expression in HEI-OC1 Cells With Oxidative Stress Damage / 听力学及言语疾病杂志

Objective To examine the effects of oxidative stress induced damage to the Prestin expression in HEI-OC1 cells,and to study the mechanism of sensory deafness.Methods We used different concentrations (50μM,100μM,200μM)of hydrogen peroxide canister to cultivate HEI-OC1 cells,and to detect the activity of su-peroxide dismutase(SOD).The quantitative real-time PCR and immunofluorescence were used to detect the prestin expression of mRNA.Results The SOD activity decreased in the HEI-OC1 cells damaged by oxidative stress.The high concentration of the infected group decreased more significantly(F= 9926.293,P<0.01).The expressions of Prestin mRNA and Prestin protein were decreased obviously in the HEI-OC1 cells.The high concentration of in-fected group decreased more significantly (F= 4065.046and7657.217,P<0.01).Conclusion Oxidative stress in-ducing damage inhibits the expression of prestin.Prestin protein may be used as a molecular marker of sensory deafness.

Prestin and Motility of the Cochlear Outer Hair Cell

The main objective of this study is to describe the role and function of prestin on cochlear amplification based on the relationship of electromotility and prestin in the outer hair cells (OHCs). After the finding of cochlear active process or amplification, OHCs have been received a lot of attention as a source of the cochlear amplification. In response to acoustic signals, the OHCs produce the receptor potentials resulting in changes in the length of the OHCs called electromotility. The electromotility originates within the lateral wall of the OHCs and relates to the unique structures of the OHCs. The OHC electromotility depends on particles of the lateral plasma membrane due to an area motor in the lateral plasma membrane. Recently, it has been reported that the electromotility requires a voltage-dependent membrane based motor protein, prestin. Prestin means fast in Italian. The presence of prestin is essential for cochlear amplification and electromotility. Prestin is a member of solute carrier 26 anion transporter family. Prestin is associated with the unique structure of the lateral wall of the OHCs. Prestin forms motor complexes with other proteins and lipids of the lateral wall sensing the transmembrane potential and generating force by changing its surface area. Recently, prestin knockout mice have been used to prove the presence of prestin. Prestin is required for electromotility of the OHCs and for cochlear amplification in normal hearing because targeted depletion of prestin in mice leads to loss of OHC electromotility and loss of hearing sensitivity up to 60 dB. In addition, recent studies have shown that the loss of cochlear amplification after intense noise exposure can result from damage to prestin and prestin involves in the process of aminoglycoside-induced apoptosis in OHCs. These show that prestin plays an important role in transducing apoptosis signals in response to antibiotics. Therefore, the presence of prestin is mandatory for cochlear active process and amplification in normal hearing.

The Distribution of Prestin on the Whole Basolateral Surface of Outer Hair Cells / 听力学及言语疾病杂志

Objective The prestin,a motor protein responsible for outer hair cell(OHC)electromotility,is expressed on the OHC surface.Previous experiments revealed that OHC electromotility and its associated nonlinear capacitance was mainly located at the OHC lateral wall and was absent at the apical cuticular plate and the basal nucleus pole.Immunofluorescent staining for prestin failed to demonstrate the prestin expression at the OHC basal ends in whole-mount preparation of the organ of Corti.The aim of this study is to investigate the distribution of prestin at OHC.Methods In this experiment,the localization of prestin protein in single dissociated OHCs from cochlea of normal mouse,rat and guinea pig,were examined by immunofluorescent staining and confocal microscopy.Results We found that prestin was uniformly expressed on the OHC basolateral surface,including its basal pole.No staining was observed on the cuticular plate and stereocilia.The OHC lateral wall had a trilaminate organization and was composed of the plasma membrane,cortical lattice,and subsurface cisternae.By with co-staining with a membrane marker di-8-ANEPPS,prestin-labeling was found locating at the outer layer of the OHC lateral wall.Further separating the plasma membrane from the underlying subsurface cisternae,using a hypotonic extracellular solution,prestin-labeling was shown locating at the plasma membrane instead of the subsurface cisternae.Conclusion The data revealed that prestin is expressed in the plasma membrane on the whole OHC basolateral surface.

CONSTRUCTION OF cDNA LIBRARY OF GERBIL′S COCHLEA / 解放军医学杂志

To construct a cDNA library of gerbil′s cochlea, the mRNA was separated from the cochlea of gerbil and the first strand cDNA was synthesized through reverse transcription by a modified oligo(dt) primer. The double strand cDNA was amplified by Ld pcR. After cDNA size fractionation , the ds cDNA was ligated in the ? TripIEx2 vector . The cDNA library was identified with special primers of prestin genes of cochlea by PCR .The results showed that the titer of library was 1.8?10 6 pfu and the percentage of recombinant clones was 80% . Prestin gene was contained in the library , the size of the fragment was 863bp . The results suggest that the established cDNA library has a high titer, recombinant percentage and large insert fragments of genes . The study lays the foundation of molecular biological study of the cochlea.