Yukako Asai

Mechanotransduction in mouse inner ear hair cells requires transmembrane channel–like genes

Inner ear hair cells convert the mechanical stimuli of sound, gravity, and head movement into electrical signals. This mechanotransduction process is initiated by opening of cation channels near the tips of hair cell stereocilia. Since the identity of these ion channels is unknown, and mutations in the gene encoding transmembrane channel-like 1 (TMC1) cause hearing loss without vestibular dysfunction in both mice and humans, we investigated the contribution of Tmc1 and the closely related Tmc2 to mechanotransduction in mice. We found that Tmc1 and Tmc2 were expressed in mouse vestibular and cochlear hair cells and that GFP-tagged TMC proteins localized near stereocilia tips. Tmc2 expression was transient in early postnatal mouse cochlear hair cells but persisted in vestibular hair cells. While mice with a targeted deletion of Tmc1 (Tmc1(Δ) mice) were deaf and those with a deletion of Tmc2 (Tmc2(Δ) mice) were phenotypically normal, Tmc1(Δ)Tmc2(Δ) mice had profound vestibular dysfunction, deafness, and structurally normal hair cells that lacked all mechanotransduction activity. Expression of either exogenous TMC1 or TMC2 rescued mechanotransduction in Tmc1(Δ)Tmc2(Δ) mutant hair cells. Our results indicate that TMC1 and TMC2 are necessary for hair cell mechanotransduction and may be integral components of the mechanotransduction complex. Our data also suggest that persistent TMC2 expression in vestibular hair cells may preserve vestibular function in humans with hearing loss caused by TMC1 mutations.

TMC1 and TMC2 Are Components of the Mechanotransduction Channel in Hair Cells of the Mammalian Inner Ear

Sensory transduction in auditory and vestibular hair cells requires expression of transmembrane channel-like (Tmc) 1 and 2 genes, but the function of these genes is unknown. To investigate the hypothesis that TMC1 and TMC2 proteins are components of the mechanosensitive ion channels that convert mechanical information into electrical signals, we recorded whole-cell and single-channel currents from mouse hair cells that expressed Tmc1, Tmc2, or mutant Tmc1. Cells that expressed Tmc2 had high calcium permeability and large single-channel currents, while cells with mutant Tmc1 had reduced calcium permeability and reduced single-channel currents. Cells that expressed Tmc1 and Tmc2 had a broad range of single-channel currents, suggesting multiple heteromeric assemblies of TMC subunits. The data demonstrate TMC1 and TMC2 are components of hair cell transduction channels and contribute to permeation properties. Gradients in TMC channel composition may also contribute to variation in sensory transduction along the tonotopic axis of the mammalian cochlea.

Dehydroepiandrosterone retards atherosclerosis formation through its conversion to estrogen: the possible role of nitric oxide.

Dehydroepiandrosterone (DHEA) is speculated to have an antiatherosclerotic effect, although the mechanism of action remains unclear. The objective of the current study was to determine whether the antiatherosclerotic effect of DHEA is related to its conversion to estrogen and to define the role of nitric oxide (NO) in the antiatherosclerotic effect of DHEA. Forty-eight oophorectomized rabbits were divided into 5 groups and fed the following diets for 10 weeks: group 1, a regular rabbit diet plus 1% cholesterol (a high-cholesterol diet [HCD]); group 2, an HCD plus 0.3% DHEA; group 3, an HCD plus 0.3% DHEA and fadrozole (2.0 mg x kg(-1) x d(-1)), a specific aromatase inhibitor; group 4, an HCD plus 17beta-estradiol (20 microg x kg(-1) x d(-1)); and group 5, a regular diet. Atherosclerotic lesions, lipid deposition in aortic vessels, and basal and stimulated NO release were measured in the aforementioned groups of rabbits. NO release was measured by using an NO-selective electrode as well as by measuring vascular responses and the plasma NO metabolites nitrite and nitrate. The plasma total cholesterol level was increased, but there were no significant differences in lipid profile in the 4 groups of rabbits that were fed the HCD. The area occupied by atherosclerosis in the thoracic aorta was diminished by approximately 60% in the DHEA-treated rabbits (group 2) compared with the HCD group of rabbits (group 1); there was a corresponding 80% decrease in the estradiol group (group 4) but only a 30% decrease in the DHEA plus fadrozole group (group 3). In the aortas of rabbits from groups 1 and 3, the acetylcholine-induced and tone-related basal NO-mediated relaxations were diminished compared with those of the controls (group 5). However, these relaxations were restored in the aortas of group 2 and 4 rabbits, and an increase in NO release was observed in groups 2 and 4 compared with groups 1 and 3, as measured by an NO-selective electrode. Injection of neither solvent (20% ethanol/distilled water) nor fadrozole significantly affected the atherosclerotic area or the NO-related responses described above. We conclude that approximately 50% of the total antiatherosclerotic effect of DHEA was achieved through the conversion of DHEA to estrogen. NO may also play a role in the antiatherosclerotic effect of DHEA and 17beta-estradiol.

Tonotopic Gradient in the Developmental Acquisition of Sensory Transduction in Outer Hair Cells of the Mouse Cochlea

Inner ear hair cells are exquisite mechanosensors that transduce nanometer scale deflections of their sensory hair bundles into electrical signals. Several essential elements must be precisely assembled during development to confer the unique structure and function of the mechanotransduction apparatus. Here we investigated the functional development of the transduction complex in outer hair cells along the length of mouse cochlea acutely excised between embryonic day 17 (E17) and postnatal day 8 (P8). We charted development of the stereociliary bundle using scanning electron microscopy; FM1-43 uptake, which permeates hair cell transduction channels, mechanotransduction currents evoked by rapid hair bundle deflections, and mRNA expression of possible components of the transduction complex. We demonstrated that uptake of FM1-43 first occurred in the basal portion of the cochlea at P0 and progressed toward the apex over the subsequent week. Electrophysiological recordings obtained from 234 outer hair cells between E17 and P8 from four cochlear regions revealed a correlation between the pattern of FM1-43 uptake and the acquisition of mechanotransduction. We found a spatiotemporal gradient in the properties of transduction including onset, amplitude, operating range, time course, and extent of adaptation. We used quantitative RT-PCR to examine relative mRNA expression of several hair cell myosins and candidate tip-link molecules. We found spatiotemporal expression patterns for mRNA that encodes cadherin 23, protocadherin 15, myosins 3a, 7a, 15a, and PMCA2 that preceded the acquisition of transduction. The spatiotemporal expression patterns of myosin 1c and PMCA2 mRNA were correlated with developmental changes in several properties of mechanotransduction.

Ion-coupling Determinants of Na+-driven and H+-driven Flagellar Motors

The bacterial flagellar motor is a tiny molecular machine that uses a transmembrane flux of H(+) or Na(+) ions to drive flagellar rotation. In proton-driven motors, the membrane proteins MotA and MotB interact via their transmembrane regions to form a proton channel. The sodium-driven motors that power the polar flagellum of Vibrio species contain homologs of MotA and MotB, called PomA and PomB. They require the unique proteins MotX and MotY. In this study, we investigated how ion selectivity is determined in proton and sodium motors. We found that Escherichia coli MotA/B restore motility in DeltapomAB Vibrio alginolyticus. Most hypermotile segregants isolated from this weakly motile strain contain mutations in motB. We constructed proteins in which segments of MotB were fused to complementary portions of PomB. A chimera joining the N terminus of PomB to the periplasmic C terminus of MotB (PotB7(E)) functioned with PomA as the stator of a sodium motor, with or without MotX/Y. This stator (PomA/PotB7(E)) supported sodium-driven motility in motA or motB E.coli cells, and the swimming speed was even higher than with the original stator of E.coli MotA/B. We conclude that the cytoplasmic and transmembrane domains of PomA/B are sufficient for sodium-driven motility. However, MotA expressed with a B subunit containing the N terminus of MotB fused to the periplasmic domain of PomB (MomB7(E)) supported sodium-driven motility in a MotX/Y-dependent fashion. Thus, although the periplasmic domain of PomB is not necessary for sodium-driven motility in a PomA/B motor, it can convert a MotA/B proton motor into a sodium motor.

Tmc gene therapy restores auditory function in deaf mice

Injection of AAV vectors that encoded wild-type Tmc1 or Tmc2 restores auditory function in mouse models of human deafnesses DFNB7/11 and DFNA36. Can you hear me now? Because genetics is a major cause of deafness, Askew and colleagues developed a therapeutic approach to replace mutant genes associated with the mechanotransduction machinery of the inner ear. The gene encoding transmembrane channel–like 1, Tmc1, or its ortholog, Tmc2, was packaged in adeno-associated viral vectors and delivered to mice with mutations in Tmc1 or Beethoven—models representative of autosomal recessive and dominant human deafness, respectively. Both vectors were able to transduce inner hair cells of the mouse cochlea, and partially restore hearing, as determined by auditory brainstem responses and startle reflexes. More than 30 TMC1 mutations have been implicated in recessive prelingual deafness, so it is hoped that this gene therapeutic approach will work long-term to maintain hearing recovery, perhaps supplementary existing technologies such as cochlear implants and hearing aids. Genetic hearing loss accounts for up to 50% of prelingual deafness worldwide, yet there are no biologic treatments currently available. To investigate gene therapy as a potential biologic strategy for restoration of auditory function in patients with genetic hearing loss, we tested a gene augmentation approach in mouse models of genetic deafness. We focused on DFNB7/11 and DFNA36, which are autosomal recessive and dominant deafnesses, respectively, caused by mutations in transmembrane channel–like 1 (TMC1). Mice that carry targeted deletion of Tmc1 or a dominant Tmc1 point mutation, known as Beethoven, are good models for human DFNB7/11 and DFNA36. We screened several adeno-associated viral (AAV) serotypes and promoters and identified AAV2/1 and the chicken β-actin (Cba) promoter as an efficient combination for driving the expression of exogenous Tmc1 in inner hair cells in vivo. Exogenous Tmc1 or its closely related ortholog, Tmc2, were capable of restoring sensory transduction, auditory brainstem responses, and acoustic startle reflexes in otherwise deaf mice, suggesting that gene augmentation with Tmc1 or Tmc2 is well suited for further development as a strategy for restoration of auditory function in deaf patients who carry TMC1 mutations.

Coupling ion specificity of chimeras between H+‐ and Na+‐driven motor proteins, MotB and PomB, in Vibrio polar flagella

We have shown that a hybrid motor consisting of proton‐type Rhodobacter sphaeroides MotA and sodium‐type Vibrio alginolyticus PomB, MotX and MotY, can work as a sodium‐driven motor in Vibrio cells. In this study, we tried to substitute the B subunits, which contain a putative ion‐binding site in the transmembrane region. Rhodobacter sphaeroides MotB did not work with either MotA or PomA in Vibrio cells. Therefore, we constructed chimeric proteins (MomB), which had N‐terminal MotB and C‐terminal PomB. MomB proteins, with the entire transmembrane region derived from the H+‐type MotB, gave rise to an Na+ motor with MotA. The other two MomB proteins, in which the junction sites were within the transmembrane region, also formed Na+ motors with PomA, but were changed for Na+ or Li+ specificity. These results show that the channel part consisting of the transmembrane regions from the A and B subunits can interchange Na+‐ and H+‐type subunits and this can affect the ion specificity. This is the first report to have changed the specificity of the coupling ions in a bacterial flagellar motor.

Physiological Concentration of 17β-Estradiol Retards the Progression of Severe Atherosclerosis Induced by a High-Cholesterol Diet Plus Balloon Catheter Injury Role of NO

The molecular mechanisms of the antiatherosclerotic effects of estrogen are not yet known. We evaluated the effects of 17beta-estradiol (E(2)) on high cholesterol diet- (HCD; standard diet and 1% cholesterol) and balloon injury-induced atherosclerosis in female New Zealand White rabbits. The abdominal aortas of 40 oophorectomized (Groups 1 through 5) and 8 nonoophorectomized (Group 6) rabbits were injured by balloon catheter, and the animals were then divided into the following groups and treated for 10 weeks: Group 1, standard diet; Group 2, standard diet plus a moderate dose of E(2) (100 microg x kg(-1) x d(-1)); Group 3, HCD; Group 4, HCD plus a moderate dose of E(2); Group 5, HCD plus a low dose of E(2) (20 microg x kg(-1) x d(-1)); and Group 6, HCD in nonoophorectomized rabbits. After the treatment phase, plasma E(2) was increased up to 282.2+/-45.5 pg/mL in Group 2, 263.0+/-41.5 pg/mL in Group 4, 87. 9+/-18.8 pg/mL in Group 5, and 45.6+/-7.3 pg/mL in Group 6. HCD-mediated increases in plasma lipid levels were not changed by E(2) treatment, whereas E(2) decreased the aortic intimal thickening in Group 2 animals compared with those in Group 1 and reduced atherosclerosis in the thoracic and abdominal aortas of Group 4, 5, and 6 rabbits compared with those in Group 3. E(2) restored the impaired abdominal aortic endothelium-dependent relaxation of balloon-injured and HCD-supplemented rabbits, and E(2) increased basal nitric oxide (NO) release. The basal NO-releasing effect showed a significant, inverse relation with the severity of atherosclerosis. Plasma E(2) concentration also showed a significant, inverse relation with atherosclerotic area. In conclusion, physiological concentrations of E(2) can retard the progression of severe atherosclerosis and stabilize atheromas induced by HCD and balloon injury. The retardation may be partially mediated by endothelial NO function in vessels treated with E(2).

In vivo imaging of adenovirus-mediated over-expression of dopamine D2 receptors in rat striatum by positron emission tomography.

PET was used to provide in vivo imaging of the over-expression of dopamine D2 receptor (D2R) induced by adenovirus vector-mediated gene transfer in rat striatum. The uptake of three kinds of D2R-specific ligands, [11C]raclopride, [11C]nemonapride and [11C]N-methylspiperone, measured by PET was higher in the striatum injected with the vectors for D2R than the contralateral striatum injected with a control vector 2–3 days after injection. However, the uptake of [11C]SCH 23390, a dopamine D1 receptor specific ligand, or [11C]β-CIT-FP, a dopamine transporter specific tracer, was not different between bilateral striata. Co-injection of excess unlabeled raclo-pride inhibited the uptake of [11C]raclopride. At day 16 the increased uptake of [11C]raclopride declined to basal level, consistent with past in vitro assessment of this vector. In vivo imaging of D2R will permit longitudinal assessment of the eficiency of this and similar vectors in rat brain that can be related to functional changes being observed.

Gene therapy restores auditory and vestibular function in a mouse model of Usher syndrome type 1c

Because there are currently no biological treatments for hearing loss, we sought to advance gene therapy approaches to treat genetic deafness. We focused on Usher syndrome, a devastating genetic disorder that causes blindness, balance disorders and profound deafness, and studied a knock-in mouse model, Ush1c c.216G>A, for Usher syndrome type IC (USH1C). As restoration of complex auditory and balance function is likely to require gene delivery systems that target auditory and vestibular sensory cells with high efficiency, we delivered wild-type Ush1c into the inner ear of Ush1c c.216G>A mice using a synthetic adeno-associated viral vector, Anc80L65, shown to transduce 80–90% of sensory hair cells. We demonstrate recovery of gene and protein expression, restoration of sensory cell function, rescue of complex auditory function and recovery of hearing and balance behavior to near wild-type levels. The data represent unprecedented recovery of inner ear function and suggest that biological therapies to treat deafness may be suitable for translation to humans with genetic inner ear disorders.