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1.  SLC26A4 Targeted to the Endolymphatic Sac Rescues Hearing and Balance in Slc26a4 Mutant Mice 
PLoS Genetics  2013;9(7):e1003641.
Mutations of SLC26A4 are a common cause of human hearing loss associated with enlargement of the vestibular aqueduct. SLC26A4 encodes pendrin, an anion exchanger expressed in a variety of epithelial cells in the cochlea, the vestibular labyrinth and the endolymphatic sac. Slc26a4Δ/Δ mice are devoid of pendrin and develop a severe enlargement of the membranous labyrinth, fail to acquire hearing and balance, and thereby provide a model for the human phenotype. Here, we generated a transgenic mouse line that expresses human SLC26A4 controlled by the promoter of ATP6V1B1. Crossing this transgene into the Slc26a4Δ/Δ line restored protein expression of pendrin in the endolymphatic sac without inducing detectable expression in the cochlea or the vestibular sensory organs. The transgene prevented abnormal enlargement of the membranous labyrinth, restored a normal endocochlear potential, normal pH gradients between endolymph and perilymph in the cochlea, normal otoconia formation in the vestibular labyrinth and normal sensory functions of hearing and balance. Our study demonstrates that restoration of pendrin to the endolymphatic sac is sufficient to restore normal inner ear function. This finding in conjunction with our previous report that pendrin expression is required for embryonic development but not for the maintenance of hearing opens the prospect that a spatially and temporally limited therapy will restore normal hearing in human patients carrying a variety of mutations of SLC26A4.
Author Summary
Mutations of SLC26A4 are the most common cause for hearing loss associated with a swelling of the inner ear. This human disease is largely recapitulated in a mutant mouse model. Mutant mice lack Slc26a4 expression and their inner ears swell during embryonic development, which leads to failure of the cochlea and the vestibular organs resulting in deafness and loss of balance. SLC26A4 is normally found in the cochlea and vestibular organs of the inner ear as well as in the endolymphatic sac, which is a non-sensory part of the inner ear. The multitude of sites where SLC26A4 is located made the goal to restore function through restoration look futile, unless some sites were more important than others. Here, we generated a new mutant mouse that expresses SLC26A4 in the endolymphatic sac but not in the cochlea or the vestibular organs of the inner ear. Fantastically, this mouse did not develop the detrimental swelling of the inner ear and even more exciting, the mouse developed normal hearing and balance. Our study provides the proof-of-concept that a therapy aimed at repairing the endolymphatic sac during embryonic development is sufficient to restore a life-time of normal hearing and balance.
doi:10.1371/journal.pgen.1003641
PMCID: PMC3708829  PMID: 23874234
2.  Endolymphatic Na+ and K+ Concentrations during Cochlear Growth and Enlargement in Mice Lacking Slc26a4/pendrin 
PLoS ONE  2013;8(5):e65977.
Slc26a4Δ/Δ mice are deaf, develop an enlarged membranous labyrinth, and thereby largely resemble the human phenotype where mutations of SLC26A4 cause an enlarged vestibular aqueduct and sensorineural hearing loss. The enlargement is likely caused by abnormal ion and fluid transport during the time of embryonic development, however, neither the mechanisms of ion transport nor the ionic composition of the luminal fluid during this time of development are known. Here we determine the ionic composition of inner ear fluids at the time at which the enlargement develops and the onset of expression of selected ion transporters. Concentrations of Na+ and K+ were measured with double-barreled ion-selective electrodes in the cochlea and the endolymphatic sac of Slc26a4Δ/+, which develop normal hearing, and of Slc26a4Δ/Δ mice, which fail to develop hearing. The expression of specific ion transporters was examined by quantitative RT-PCR and immunohistochemistry. High Na+ (∼141 mM) and low K+ concentrations (∼11 mM) were found at embryonic day (E) 16.5 in cochlear endolymph of Slc26a4Δ/+ and Slc26a4Δ/Δ mice. Shortly before birth the K+ concentration began to rise. Immediately after birth (postnatal day 0), the Na+ and K+ concentrations in cochlear endolymph were each ∼80 mM. In Slc26a4Δ/Δ mice, the rise in the K+ concentration occurred with a ∼3 day delay. K+ concentrations were also found to be low (∼15 mM) in the embryonic endolymphatic sac. The onset of expression of the K+ channel KCNQ1 and the Na+/2Cl−/K+ cotransporter SLC12A2 occurred in the cochlea at E19.5 in Slc26a4Δ/+ and Slc26a4Δ/Δ mice. These data demonstrate that endolymph, at the time at which the enlargement develops, is a Na+-rich fluid, which transitions into a K+-rich fluid before birth. The data suggest that the endolymphatic enlargement caused by a loss of Slc26a4 is a consequence of disrupted Na+ transport.
doi:10.1371/journal.pone.0065977
PMCID: PMC3669272  PMID: 23741519
3.  NOS Inhibition Enhances Myogenic Tone by Increasing Rho-Kinase Mediated Ca2+ Sensitivity in the Male but Not the Female Gerbil Spiral Modiolar Artery 
PLoS ONE  2013;8(1):e53655.
Cochlear blood flow regulation is important to prevent hearing loss caused by ischemia and oxidative stress. Cochlear blood supply is provided by the spiral modiolar artery (SMA). The myogenic tone of the SMA is enhanced by the nitric oxide synthase (NOS) blocker L-NG-Nitro-Arginine (LNNA) in males, but not in females. Here, we investigated whether this gender difference is based on differences in the cytosolic Ca2+ concentration and/or the Ca2+ sensitivity of the myofilaments. Vascular diameter, myogenic tone, cytosolic Ca2+, and Ca2+ sensitivity were evaluated in pressurized SMA segments isolated from male and female gerbils using laser-scanning microscopy and microfluorometry. The gender difference of the LNNA-induced tone was compared, in the same vessel segments, to tone induced by 150 mM K+ and endothelin-1, neither of which showed an apparent gender-difference. Interestingly, LNNA-induced tone in male SMAs was observed in protocols that included changes in intramural pressure, but not when the intramural pressure was held constant. LNNA in male SMAs did not increase the global Ca2+ concentration in smooth muscle cells but increased the Ca2+ sensitivity. This increase in the Ca2+ sensitivity was abolished in the presence of the guanylyl cyclase inhibitor ODQ or by extrinsic application of either the nitric oxide (NO)-donor DEA-NONOate or the cGMP analog 8-pCPT-cGMP. The rho-kinase blocker Y27632 decreased the basal Ca2+ sensitivity and abolished the LNNA-induced increase in Ca2+ sensitivity in male SMAs. Neither LNNA nor Y27632 changed the Ca2+ sensitivity in female SMAs. The data suggest that the gender difference in LNNA-induced tone is based on a gender difference in the regulation of rho-kinase mediated Ca2+ sensitivity. Rho-kinase and NO thus emerge as critical factors in the regulation of cochlear blood flow. The larger role of NO-dependent mechanisms in male SMAs predicts greater restrictions on cochlear blood flow under conditions of impaired endothelial cell function.
doi:10.1371/journal.pone.0053655
PMCID: PMC3536759  PMID: 23301097
4.  Developmental expression of SLC26A4 (Pendrin) during amelogenesis in developing rodent teeth 
European journal of oral sciences  2011;119(Suppl 1):185-192.
Ameloblasts need to regulate pH during formation of enamel crystals, a process that generates protons. Solute carrier family 26A member 4 (SLC26A4, or pendrin) is an anion exchanger for chloride, bicarbonate, iodine and formate. It is expressed in apical membranes of ion-transporting epithelia in kidney, inner ear and thyroid where it regulates luminal pH and fluid transport. We hypothesized that maturation ameloblasts express SLC26A4 to neutralize acidification of enamel fluid in forming enamel. In rodents, secretory and maturation ameloblasts were immunopositive for SLC26A4. Staining was particularly strong in apical membranes of maturation ameloblasts facing forming enamel. RT-PCR confirmed the presence of mRNA transcripts for Slc26a4 in enamel organs. SLC26A4 immunostaining was also found in mineralizing connective tissues including odontoblasts, osteoblasts, osteocytes, osteoclasts, bone lining cells, cellular cementoblasts and cementocytes. However, Slc26a4-null mutant mice had no overt dental phenotype. The presence of SLC26A4 in apical plasma membranes of maturation ameloblasts is consistent with a potential function as pH regulator. SLC26A4 does not appear critical for ameloblast functioning and is likely compensated by other pH regulators.
doi:10.1111/j.1600-0722.2011.00901.x
PMCID: PMC3496853  PMID: 22243245
ameloblasts; mineralization; pH regulation; immunolocalization; null mutant; bone cells; odontoblasts
5.  Hearing Loss Associated with Enlargement of the Vestibular Aqueduct: Mechanistic Insights from Clinical Phenotypes, Genotypes, and Mouse Models 
Hearing research  2011;281(1-2):11-17.
Enlargement of the vestibular aqueduct (EVA) is one of the most common inner ear malformations associated with sensorineural hearing loss in children. The delayed onset and progressive nature of this phenotype offer a window of opportunity to prevent or retard progression of hearing loss. EVA is not the direct cause of hearing loss in these patients, but rather is a radiologic marker for some underlying pathogenetic defect. Mutations of the SLC26A4 gene are a common cause of EVA. Studies of an Slc26a4 knockout mouse demonstrate that enlargement of the scala media is a key event in the pathogenesis of deafness. The enlargement is driven by fluid secretion in the vestibular labyrinth and a failure of fluid absorption in the embryonic endolymphatic sac. Elucidating the mechanism of hearing loss may offer clues to potential therapeutic strategies.
doi:10.1016/j.heares.2011.05.009
PMCID: PMC3183377  PMID: 21669267
6.  SLC26A4 Genotypes and Phenotypes Associated with Enlargement of the Vestibular Aqueduct 
Enlargement of the vestibular aqueduct (EVA) is the most common inner ear anomaly detected in ears of children with sensorineural hearing loss. Pendred syndrome (PS) is an autosomal recessive disorder characterized by bilateral sensorineural hearing loss with EVA and an iodine organification defect that can lead to thyroid goiter. Pendred syndrome is caused by mutations of the SLC26A4 gene. SLC26A4 mutations may also be identified in some patients with nonsyndromic EVA (NSEVA). The presence of two mutant alleles of SLC26A4 is correlated with bilateral EVA and Pendred syndrome, whereas unilateral EVA and NSEVA are correlated with one (M1) or zero (M0) mutant alleles of SLC26A4. Thyroid gland enlargement (goiter) appears to be primarily dependent on the presence of two mutant alleles of SLC26A4 in pediatric patients, but not in older patients. In M1 families, EVA may be associated with a second, undetected SLC26A4 mutation or epigenetic modifications. In M0 families, there is probably etiologic heterogeneity that includes causes other than, or in addition to, monogenic inheritance.
doi:10.1159/000335119
PMCID: PMC3709178  PMID: 22116369
SLC26A4; Pendred syndrome; Hearing; Deafness; Inner ear; Genotype-phenotype correlation
7.  TNFα enhances microvascular tone and reduces blood flow in the cochlea via enhanced S1P signaling 
Objective
To demonstrate that TNFα, via sphingosine-1-phosphate (S1P) signaling, has the potential to alter cochlear blood flow and thus, cause ischemic hearing loss.
Methods and Results
TNFα induced a pro-constrictive state throughout the cochlear microvasculature, which reduced capillary diameter and cochlear blood flow in vivo. In vitro isolated preparations of the spiral modiolar artery and spiral ligament capillaries confirmed these observations. Antagonizing S1P receptor 2 subtype signaling (1µmol/L JTE013) attenuated the effects of TNFα in all models. TNFα activated Sk1 and induced its translocation to the smooth muscle cell membrane. Expression of a dominant-negative Sk1 mutant (Sk1G82D) eliminated both baseline spiral modiolar artery calcium sensitivity and TNFα effects, while a non-phosphorylatable Sk1 mutant (Sk1S225A) only blocked the effects of TNFα. A small group of etanercept-treated hearing loss patients recovered with a one-phase exponential decay (t½=1.56±0.20 weeks), which matched a kinetic predicted for a vascular origin.
Conclusions
TNFα indeed reduces cochlear blood flow via the activation of vascular S1P signaling. This integrates hearing loss into the family of ischemic microvascular pathologies, with implications for risk stratification, diagnosis and treatment.
doi:10.1161/STROKEAHA.110.593327
PMCID: PMC3404620  PMID: 20930159
Signal transduction; transfection; etanercept; sphingosine kinase 1; cochlear microcirculation
8.  IsK Channel in Strial Marginal Cells. Voltage-Dependence, Ion-Selectivity, Inhibition by 293B and Sensitivity to Clofilium 
Auditory neuroscience  1997;3(3):215-230.
Strial marginal cells (SMC) and vestibular dark cells (VDC) are known to secrete K+ into endolymph. Slowly-activating, voltage-dependent K+ channels (KCNQ1/KCNE1; IsK; min K) have been identified in the apical membrane of these cells. Several experimental maneuvers known to increase or decrease transepithelial K+ secretion have been found in VDC to change the current through these channels in the same ways. In both SMC and VDC the kinetics of activation and deactivation resemble those of the IsK channel exogenously expressed in Xenopus oocytes and endogenous to heart myocytes. The present study sought evidence that this current is indeed carried by IsK channels and that this current is the basis for transepithelial K+ secretion. Both on-cell macro-patch recordings of the apical membrane and perforated-patch whole-cell recordings were made on SMC from gerbil in order to measure macroscopic cell currents. The on-cell current was found to 1) be K+-selective, 2) have a cation permeability sequence of K+ ~ Rb+ > Cs+ >> Li+ = Na+, 3) be activated with a time constant of 1764 ± 413 ms by voltage steps from 0 to +40 mV, 4) be deactivated with a time constant of 324 ± 57 ms by voltage steps from 0 to -40 mV and 5) be reduced 84 ± 5% by bumetanide (10-5 M), an inhibitor of K+ secretion. The single-channel conductance of the apical currents in the homologous VDC was estimated by fluctuation analysis to be 1.6 pS. The potent inhibitor of IsK channels, chromanol 293B (10-5 M), reduced the whole-cell current in SMC by 72 ± 10 %. Clofilium (10-4 M), a putative IsK channel inhibitor known to have additional non-specific effects, led to a stimulation of both on-cell (by 598 ± 177%) and whole-cell (by 162 ± 18%) currents in gerbil SMC but to a decrease of whole-cell currents (by 39 ± 12%) in rat SMC. Taken together with other findings reviewed here, these results strongly argue that the slowly-activating, voltage-dependent conductance in the apical membrane of SMC is the IsK channel and provide additional evidence for the poor specificity of clofilium.
PMCID: PMC3348584  PMID: 22582020
K+ channel; perforated-patch whole-cell voltage clamp; fluctuation analysis; KCNQ1/KCNE1 K channel; gerbil; rat
9.  N-Ethylmaleimide Stimulates and Inhibits Ion Transport in Vestibular Dark Cells of Gerbil 
Auditory neuroscience  1994;1:101-109.
Vestibular dark cell epithelium was isolated from the semicircular canal of gerbils to test the proposal that the sulfhydryl alkylating agent N-ethylmaleimide (NEM) inhibits K+ secretion by this tissue and does so by reacting with a site in or near the apical membrane. Dark cell epithelium was mounted in a micro-Ussing chamber for measurements of transepithelial voltage (Vt) and resistance (Rt) or in a perfused bath on the stage of a microscope for measurement of cell height as an index of cell volume. Perfusion of the apical or basolateral side with 10−3 M NEM caused an increase in Vt superimposed upon a slower decrease of Vt, resulting in a triphasic response. There were only small changes in Rt. Under this condition, Vt is proportional to short circuit current and to K+ secretion. Both the stimulatory and the inhibitory responses of Vt were dose-dependent between 10−6 and 10−3 M NEM and the inhibition was irreversible. The specificity of the reaction of NEM with sulfhydryl groups was confirmed by the use of the reducing agent dithiothreitol (DTT). Perfusion of 5×10−4 M DTT on the apical side caused no significant changes in Vt but completely prevented both stimulation and inhibition of Vt by NEM (10−3 M). The amplitudes of the stimulation and the inhibition of Vt were greater for basolateral than for apical perfusion of NEM. Similarly, the response times for each effect were faster from the basolateral side, suggesting that the primary sites of action are at or near the basolateral membrane. The site of action of NEM was further explored by subjecting the tissue to a membrane-impermeant sulfhydryl reagent, stilbenedisulfonate maleimide (SDM). Apical perfusion of 10−3 M SDM had no effect on Vt or Rt, whereas basolateral perfusion caused a reversible increase of Vt (5.2 ± 0.5 to initially 6.8 ± 0.5 mV which relaxed after 60 s to 5.8 ± 0.5 mV) and to an initial decrease in Rt by 4%. No inhibitory phase was observed. Elevation of basolateral [K+] from 3.6 to 25 mM is known to increase Vt and reduce Rt via direct stimulation of basolateral K+ uptake and indirect stimulation of the apical membrane conductance. Basolateral perfusion of 10−3 M NEM fully inhibited the increase of Vt due to 25 mM K+. Elevation of basolateral [K+] from 3.6 to 25 mM is known to increase reversibly cell volume. NEM was found to inhibit cell swelling in a dose-dependent manner but did not initially affect the rate of shrinking after K+-induced swelling, pointing to action only on basolateral transport pathways. The effects of NEM on K+-induced cell swelling were completely prevented by 5×10−4 M DTT, demonstrating that the inhibitory effect of NEM was on sulfhydryl groups. In contrast to interpretations of NEM action in frog semicircular canal, we have found that NEM appears to stimulate an ion transport process in mammalian dark cells at an extracellular site in the basolateral membrane and inhibits another ion transport process in the basolateral membrane at another site. Inhibition by NEM from the apical side occurs most likely by diffusion of the agent to a site at or near the cytosolic side of the basolateral membrane.
PMCID: PMC3291124  PMID: 22389574
Vestibular labyrinth; epithelial transport; cell volume; dark cells; sulfhydryl reagents
10.  Mouse model of enlarged vestibular aqueducts defines temporal requirement of Slc26a4 expression for hearing acquisition  
The Journal of Clinical Investigation  2011;121(11):4516-4525.
Mutations in human SLC26A4 are a common cause of hearing loss associated with enlarged vestibular aqueducts (EVA). SLC26A4 encodes pendrin, an anion-base exchanger expressed in inner ear epithelial cells that secretes HCO3– into endolymph. Studies of Slc26a4-null mice indicate that pendrin is essential for inner ear development, but have not revealed whether pendrin is specifically necessary for homeostasis. Slc26a4-null mice are profoundly deaf, with severe inner ear malformations and degenerative changes that do not model the less severe human phenotype. Here, we describe studies in which we generated a binary transgenic mouse line in which Slc26a4 expression could be induced with doxycycline. The transgenes were crossed onto the Slc26a4-null background so that all functional pendrin was derived from the transgenes. Varying the temporal expression of Slc26a4 revealed that E16.5 to P2 was the critical interval in which pendrin was required for acquisition of normal hearing. Lack of pendrin during this period led to endolymphatic acidification, loss of the endocochlear potential, and failure to acquire normal hearing. Doxycycline initiation at E18.5 or discontinuation at E17.5 resulted in partial hearing loss approximating the human EVA auditory phenotype. These data collectively provide mechanistic insight into hearing loss caused by SLC26A4 mutations and establish a model for further studies of EVA-associated hearing loss.
doi:10.1172/JCI59353
PMCID: PMC3204851  PMID: 21965328
11.  Gender Differences in Myogenic Regulation along the Vascular Tree of the Gerbil Cochlea 
PLoS ONE  2011;6(9):e25659.
Regulation of cochlear blood flow is critical for hearing due to its exquisite sensitivity to ischemia and oxidative stress. Many forms of hearing loss such as sensorineural hearing loss and presbyacusis may involve or be aggravated by blood flow disorders. Animal experiments and clinical outcomes further suggest that there is a gender preference in hearing loss, with males being more susceptible. Autoregulation of cochlear blood flow has been demonstrated in some animal models in vivo, suggesting that similar to the brain, blood vessels supplying the cochlea have the ability to control flow within normal limits, despite variations in systemic blood pressure. Here, we investigated myogenic regulation in the cochlear blood supply of the Mongolian gerbil, a widely used animal model in hearing research. The cochlear blood supply originates at the basilar artery, followed by the anterior inferior cerebellar artery, and inside the inner ear, by the spiral modiolar artery and the radiating arterioles that supply the capillary beds of the spiral ligament and stria vascularis. Arteries from male and female gerbils were isolated and pressurized using a concentric pipette system. Diameter changes in response to increasing luminal pressures were recorded by laser scanning microscopy. Our results show that cochlear vessels from male and female gerbils exhibit myogenic regulation but with important differences. Whereas in male gerbils, both spiral modiolar arteries and radiating arterioles exhibited pressure-dependent tone, in females, only radiating arterioles had this property. Male spiral modiolar arteries responded more to L-NNA than female spiral modiolar arteries, suggesting that NO-dependent mechanisms play a bigger role in the myogenic regulation of male than female gerbil cochlear vessels.
doi:10.1371/journal.pone.0025659
PMCID: PMC3183064  PMID: 21980520
12.  Calcium sparks in the intact gerbil spiral modiolar artery 
BMC Physiology  2011;11:15.
Background
Calcium sparks are ryanodine receptor mediated transient calcium signals that have been shown to hyperpolarize the membrane potential by activating large conductance calcium activated potassium (BK) channels in vascular smooth muscle cells. Along with voltage-dependent calcium channels, they form a signaling unit that has a vasodilatory influence on vascular diameter and regulation of myogenic tone. The existence and role of calcium sparks has hitherto been unexplored in the spiral modiolar artery, the end artery that controls blood flow to the cochlea. The goal of the present study was to determine the presence and properties of calcium sparks in the intact gerbil spiral modiolar artery.
Results
Calcium sparks were recorded from smooth muscle cells of intact arteries loaded with fluo-4 AM. Calcium sparks occurred with a frequency of 2.6 Hz, a rise time of 17 ms and a time to half-decay of 20 ms. Ryanodine reduced spark frequency within 3 min from 2.6 to 0.6 Hz. Caffeine (1 mM) increased spark frequency from 2.3 to 3.3 Hz and prolonged rise and half-decay times from 17 to 19 ms and from 20 to 23 ms, respectively. Elevation of potassium (3.6 to 37.5 mM), presumably via depolarization, increased spark frequency from 2.4 to 3.2 Hz. Neither ryanodine nor depolarization changed rise or decay times.
Conclusions
This is the first characterization of calcium sparks in smooth muscle cells of the spiral modiolar artery. The results suggest that calcium sparks may regulate the diameter of the spiral modiolar artery and cochlear blood flow.
doi:10.1186/1472-6793-11-15
PMCID: PMC3170618  PMID: 21871098
13.  Non-linear classification for on-the-fly fractional mass filtering and targeted precursor fragmentation in mass spectrometry experiments 
Bioinformatics  2010;26(6):791-797.
Motivation: Mass spectrometry (MS) has become the method of choice for protein/peptide sequence and modification analysis. The technology employs a two-step approach: ionized peptide precursor masses are detected, selected for fragmentation, and the fragment mass spectra are collected for computational analysis. Current precursor selection schemes are based on data- or information-dependent acquisition (DDA/IDA), where fragmentation mass candidates are selected by intensity and are subsequently included in a dynamic exclusion list to avoid constant refragmentation of highly abundant species. DDA/IDA methods do not exploit valuable information that is contained in the fractional mass of high-accuracy precursor mass measurements delivered by current instrumentation.
Results: We extend previous contributions that suggest that fractional mass information allows targeted fragmentation of analytes of interest. We introduce a non-linear Random Forest classification and a discrete mapping approach, which can be trained to discriminate among arbitrary fractional mass patterns for an arbitrary number of classes of analytes. These methods can be used to increase fragmentation efficiency for specific subsets of analytes or to select suitable fragmentation technologies on-the-fly. We show that theoretical generalization error estimates transfer into practical application, and that their quality depends on the accuracy of prior distribution estimate of the analyte classes. The methods are applied to two real-world proteomics datasets.
Availability: All software used in this study is available from http://software.steenlab.org/fmf
Contact: hanno.steen@childrens.harvard.edu
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq036
PMCID: PMC3202308  PMID: 20134030
14.  Epithelial Cell Stretching and Luminal Acidification Lead to a Retarded Development of Stria Vascularis and Deafness in Mice Lacking Pendrin 
PLoS ONE  2011;6(3):e17949.
Loss-of-function mutations of SLC26A4/pendrin are among the most prevalent causes of deafness. Deafness and vestibular dysfunction in the corresponding mouse model, Slc26a4−/−, are associated with an enlargement and acidification of the membranous labyrinth. Here we relate the onset of expression of the HCO3− transporter pendrin to the luminal pH and to enlargement-associated epithelial cell stretching. We determined expression with immunocytochemistry, cell stretching by digital morphometry and pH with double-barreled ion-selective electrodes. Pendrin was first expressed in the endolymphatic sac at embryonic day (E) 11.5, in the cochlear hook-region at E13.5, in the utricle and saccule at E14.5, in ampullae at E16.5, and in the upper turn of the cochlea at E17.5. Epithelial cell stretching in Slc26a4−/− mice began at E14.5. pH changes occurred first in the cochlea at E15.5 and in the endolymphatic sac at E17.5. At postnatal day 2, stria vascularis, outer sulcus and Reissner's membrane epithelial cells, and utricular and saccular transitional cells were stretched, whereas sensory cells in the cochlea, utricle and saccule did not differ between Slc26a4+/− and Slc26a4−/− mice. Structural development of stria vascularis, including vascularization, was retarded in Slc26a4−/− mice. In conclusion, the data demonstrate that the enlargement and stretching of non-sensory epithelial cells precedes luminal acidification in the cochlea and the endolymphatic sac. Stretching and luminal acidification may alter cell-to-cell communication and lead to the observed retarded development of stria vascularis, which may be an important step on the path to deafness in Slc26a4−/− mice, and possibly in humans, lacking functional pendrin expression.
doi:10.1371/journal.pone.0017949
PMCID: PMC3056798  PMID: 21423764
15.  Failure of Fluid Absorption in the Endolymphatic Sac Initiates Cochlear Enlargement that Leads to Deafness in Mice Lacking Pendrin Expression 
PLoS ONE  2010;5(11):e14041.
Mutations of SLC26A4 are among the most prevalent causes of hereditary deafness. Deafness in the corresponding mouse model, Slc26a4−/−, results from an abnormally enlarged cochlear lumen. The goal of this study was to determine whether the cochlear enlargement originates with defective cochlear fluid transport or with a malfunction of fluid transport in the connected compartments, which are the vestibular labyrinth and the endolymphatic sac. Embryonic inner ears from Slc26a4+/− and Slc26a4−/− mice were examined by confocal microscopy ex vivo or after 2 days of organ culture. Culture allowed observations of intact, ligated or partially resected inner ears. Cochlear lumen formation was found to begin at the base of the cochlea between embryonic day (E) 13.5 and 14.5. Enlargement was immediately evident in Slc26a4−/− compared to Slc26a4+/− mice. In Slc26a4+/− and Slc26a4−/− mice, separation of the cochlea from the vestibular labyrinth by ligation at E14.5 resulted in a reduced cochlear lumen. Resection of the endolymphatic sacs at E14.5 led to an enlarged cochlear lumen in Slc26a4+/− mice but caused no further enlargement of the already enlarged cochlear lumen in Slc26a4−/− mice. Ligation or resection performed later, at E17.5, did not alter the cochlea lumen. In conclusion, the data suggest that cochlear lumen formation is initiated by fluid secretion in the vestibular labyrinth and temporarily controlled by fluid absorption in the endolymphatic sac. Failure of fluid absorption in the endolymphatic sac due to lack of Slc26a4 expression appears to initiate cochlear enlargement in mice, and possibly humans, lacking functional Slc26a4 expression.
doi:10.1371/journal.pone.0014041
PMCID: PMC2984494  PMID: 21103348
16.  Expression of epithelial calcium transport system in rat cochlea and vestibular labyrinth 
BMC Physiology  2010;10:1.
Background
The low luminal Ca2+ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We recently reported the expression of mRNA for a Ca2+-absorptive transport system in primary cultures of semicircular canal duct (SCCD) epithelium.
Results
We now identify this system in native vestibular and cochlear tissues by qRT-PCR, immunoblots and confocal immunolocalization. Transcripts were found and quantified for several isoforms of epithelial calcium channels (TRPV5, TRPV6), calcium buffer proteins (calbindin-D9K, calbindin-D28K), sodium-calcium exchangers (NCX1, NCX2, NCX3) and plasma membrane Ca2+-ATPase (PMCA1, PMCA2, PMCA3, and PMCA4) in native SCCD, cochlear lateral wall (LW) and stria vascularis (SV) of adult rat as well as Ca2+ channels in neonatal SCCD. All components were expressed except TRPV6 in SV and PMCA2 in SCCD. 1,25-(OH)2vitamin D3 (VitD) significantly up-regulated transcripts of TRPV5 in SCCD, calbindin-D9K in SCCD and LW, NCX2 in LW, while PMCA4 in SCCD and PMCA3 in LW were down-regulated. The expression of TRPV5 relative to TRPV6 was in the sequence SV > Neonatal SCCD > Adult SCCD > LW > primary culture SCCD. Expression of TRPV5 protein from primary culture of SCCD did not increase significantly when cells were incubated with VitD (1.2 times control; P > 0.05). Immunolocalization showed the distribution of TRPV5 and TRPV6. TRPV5 was found near the apical membrane of strial marginal cells and both TRPV5 and TRPV6 in outer and inner sulcus cells of the cochlea and in the SCCD of the vestibular system.
Conclusions
These findings demonstrate for the first time the expression of a complete Ca2+ absorptive system in native cochlear and vestibular tissues. Regulation by vitamin D remains equivocal since the results support the regulation of this system at the transcript level but evidence for control of the TRPV5 channel protein was lacking.
doi:10.1186/1472-6793-10-1
PMCID: PMC2825184  PMID: 20113508
17.  A Claudin-9–Based Ion Permeability Barrier Is Essential for Hearing 
PLoS Genetics  2009;5(8):e1000610.
Hereditary hearing loss is one of the most common birth defects, yet the majority of genes required for audition is thought to remain unidentified. Ethylnitrosourea (ENU)–mutagenesis has been a valuable approach for generating new animal models of deafness and discovering previously unrecognized gene functions. Here we report on the characterization of a new ENU–induced mouse mutant (nmf329) that exhibits recessively inherited deafness. We found a widespread loss of sensory hair cells in the hearing organs of nmf329 mice after the second week of life. Positional cloning revealed that the nmf329 strain carries a missense mutation in the claudin-9 gene, which encodes a tight junction protein with unknown biological function. In an epithelial cell line, heterologous expression of wild-type claudin-9 reduced the paracellular permeability to Na+ and K+, and the nmf329 mutation eliminated this ion barrier function without affecting the plasma membrane localization of claudin-9. In the nmf329 mouse line, the perilymphatic K+ concentration was found to be elevated, suggesting that the cochlear tight junctions were dysfunctional. Furthermore, the hair-cell loss in the claudin-9–defective cochlea was rescued in vitro when the explanted hearing organs were cultured in a low-K+ milieu and in vivo when the endocochlear K+-driving force was diminished by deletion of the pou3f4 gene. Overall, our data indicate that claudin-9 is required for the preservation of sensory cells in the hearing organ because claudin-9–defective tight junctions fail to shield the basolateral side of hair cells from the K+-rich endolymph. In the tight-junction complexes of hair cells, claudin-9 is localized specifically to a subdomain that is underneath more apical tight-junction strands formed by other claudins. Thus, the analysis of claudin-9 mutant mice suggests that even the deeper (subapical) tight-junction strands have biologically important ion barrier function.
Author Summary
Hereditary deafness is a common birth defect in the human population, yet the majority of genes required for audition is thought to be unidentified. Genetic approaches in the mouse have greatly contributed to our understanding of the molecular mechanisms that underlie hearing. Random mutagenesis of mice, identification of deaf mutants, and subsequent analysis of the deafness-causing gene defects has led to the discovery of several previously unrecognized gene functions. Here, we report on the characterization of a new mutant mouse line (nmf329) that exhibits profound hearing loss and loss of sensory cells in the auditory organ. Genetic analysis reveals that these animals carry a mutation in the claudin-9 gene, which encodes a protein with hitherto unknown biological function. We have found that normal claudin-9—but not a mutant form—inhibits the paracellular movement of certain ions. The lack of the claudin-9 ion barrier in the inner ear leads to changes in ionic conditions that can account for the loss of sensory cells in the mutant mice. Within the cell–cell junctions, the claudin-9 layer is located basal to those of other claudins. Thus, our analysis of claudin-9–deficient animals suggests that even the deeper layers of claudins have important ion barrier function.
doi:10.1371/journal.pgen.1000610
PMCID: PMC2720454  PMID: 19696885
18.  Lack of pendrin HCO3- transport elevates vestibular endolymphatic [Ca2+] by inhibition of acid-sensitive TRPV5 and TRPV6 channels 
The low Ca2+ concentration of mammalian endolymph in the inner ear is required for normal hearing and balance. We reported [Yamauchi et al. Biochem Biophys Res Commun, 2005] that the epithelial Ca2+ channels TRPV5 and TRPV6 are expressed in the vestibular system and that TRPV5 expression is stimulated by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3), as also reported in kidney. TRPV5/6 channels are known to be inhibited by extracellular acidic pH. Endolymphatic pH, [Ca2+] and transepithelial potential of the utricle (UP) were measured in Cl-/ HCO3− exchanger pendrin (SLC26A4) knockout mice in vivo. Slc26a4-/- mice exhibit reduced pH and UP and increased [Ca2+]. Monolayers of primary cultures of rat semicircular canal duct (SCCD) cells were grown on permeable supports and cellular uptake of 45Ca2+ was measured individually from the apical and basolateral sides. Net uptake of 45Ca2+ was greater after incubation with 1,25(OH)2D3. Net 45Ca2+ absorption was dramatically inhibited by low apical pH and was stimulated by apical alkaline pH. Gadolinium, lanthanum and ruthenium red reduced apical uptake. These observations support the notion that one aspect of vestibular dysfunction in Pendred syndrome is a pathological elevation of endolymphatic [Ca2+] due to luminal acidification and consequent inhibition of TRPV5/6-mediated Ca2+ absorption.
doi:10.1152/ajprenal.00432.2006
PMCID: PMC2515270  PMID: 17200157
Epithelial Ca Channel; vitamin D; SLC26A4; HCO3- secretion; TRPV5; TRPV6
19.  Loss of cochlear HCO3− secretion causes deafness via endolymphatic acidification and inhibition of Ca2+ reabsorption in a Pendred syndrome mouse model 
Pendred syndrome, characterized by childhood deafness and postpuberty goiter, is caused by mutations of SLC26A4, which codes for the anion exchanger pendrin. The goal of the present study was to determine how loss of pendrin leads to hair cell degeneration and deafness. We evaluated pendrin function by ratiometric microfluorometry, hearing by auditory brain stem recordings, and expression of K+ and Ca2+ channels by confocal immunohistochemistry. Cochlear pH and Ca2+ concentrations and endocochlear potential (EP) were measured with double-barreled ion-selective microelectrodes. Pendrin in the cochlea was characterized as a formate-permeable and DIDS-sensitive anion exchanger that is likely to mediate HCO3− secretion into endolymph. Hence endolymph in Slc26a4+/− mice was more alkaline than perilymph, and the loss of pendrin in Slc26a4−/− mice led to an acidification of endolymph. The stria vascularis of Slc26a4−/− mice expressed the K+ channel Kcnj10 and generated a small endocochlear potential before the normal onset of hearing at postnatal day 12. This small potential and the expression of Kcnj10 were lost during further development, and Slc26a4−/− mice did not acquire hearing. Endolymphatic acidification may be responsible for inhibition of Ca2+ reabsorption from endolymph via the acid-sensitive epithelial Ca2+ channels Trpv5 and Trpv6. Hence the endolymphatic Ca2+ concentration was found elevated in Slc26a4−/− mice. This elevation may inhibit sensory transduction necessary for hearing and promote the degeneration of the sensory hair cells. Degeneration of the hair cells closes a window of opportunity to restore the normal development of hearing in Slc26a4−/− mice and possibly human patients suffering from Pendred syndrome.
doi:10.1152/ajprenal.00487.2006
PMCID: PMC2020516  PMID: 17299139
pendrin; stria vascularis; Slc26a4; Kcnj10; Trpv5
20.  Microarray-based comparison of three amplification methods for nanogram amounts of total RNA 
Gene expression profiling using microarrays requires microgram amounts of RNA, which limits its direct application for the study of nanogram RNA samples obtained using microdissection, laser capture microscopy, or needle biopsy. A novel system based on Ribo-SPIA technology (RS, Ovation-Biotin amplification and labeling system) was recently introduced. The utility of the RS system, an optimized prototype system for picogram RNA samples (pRS), and two T7-based systems involving one or two rounds of amplification (OneRA, Standard Protocol, or TwoRA, Small Sample Prototcol, version II) were evaluated in the present study. Mouse kidney (MK) and mouse universal reference (MUR) RNA samples, 0.3 ng to 10 μg, were analyzed using high-density Affymetrix Mouse Genome 430 2.0 GeneChip arrays. Call concordance between replicates, correlations of signal intensity, signal intensity ratios, and minimal fold increase necessary for significance were determined. All systems amplified partially overlapping sets of genes with similar signal intensity correlations. pRS amplified the highest number of genes from 10-ng RNA samples. We detected 24 of 26 genes verified by RT-PCR in samples prepared using pRS. TwoRA yielded somewhat higher call concordances than did RS and pRS (91.8% vs. 89.3% and 88.1%, respectively). Although all target preparation methods were suitable, pRS amplified the highest number of targets and was found to be suitable for amplification of as little as 0.3 ng of total RNA. In addition, RS and pRS were faster and simpler to use than the T7-based methods and resulted in the generation of cDNA, which is more stable than cRNA.
doi:10.1152/ajpcell.00258.2004
PMCID: PMC2020524  PMID: 15613496
gene expression microarray analysis; microdissection; nucleic acid amplification techniques
21.  Macrophage invasion contributes to degeneration of stria vascularis in Pendred syndrome mouse model 
BMC Medicine  2006;4:37.
Background
Pendred syndrome, an autosomal-recessive disorder characterized by deafness and goiter, is caused by a mutation of SLC26A4, which codes for the anion exchanger pendrin. We investigated the relationship between pendrin expression and deafness using mice that have (Slc26a4+/+ or Slc26a4+/-) or lack (Slc26a4-/-) a complete Slc26a4 gene. Previously, we reported that stria vascularis of adult Slc26a4-/- mice is hyperpigmented and that marginal cells appear disorganized. Here we determine the time course of hyperpigmentation and marginal cell disorganization, and test the hypothesis that inflammation contributes to this tissue degeneration.
Methods
Slc26a4-/- and age-matched control (Slc26a4+/+ or Slc26a4+/-) mice were studied at four postnatal (P) developmental stages: before and after the age that marks the onset of hearing (P10 and P15, respectively), after weaning (P28-41) and adult (P74-170). Degeneration and hyperpigmentation stria vascularis was evaluated by confocal microscopy. Gene expression in stria vascularis was analyzed by microarray and quantitative RT-PCR. In addition, the expression of a select group of genes was quantified in spiral ligament, spleen and liver to evaluate whether expression changes seen in stria vascularis are specific for stria vascularis or systemic in nature.
Results
Degeneration of stria vascularis defined as hyperpigmentation and marginal cells disorganization was not seen at P10 or P15, but occurred after weaning and was associated with staining for CD68, a marker for macrophages. Marginal cells in Slc26a4-/-, however, had a larger apical surface area at P10 and P15. No difference in the expression of Lyzs, C3 and Cd45 was found in stria vascularis of P15 Slc26a4+/- and Slc26a4-/- mice. However, differences in expression were found after weaning and in adult mice. No difference in the expression of markers for acute inflammation, including Il1a, Il6, Il12a, Nos2 and Nos3 were found at P15, after weaning or in adults. The expression of macrophage markers including Ptprc (= Cd45), Cd68, Cd83, Lyzs, Lgals3 (= Mac2 antigen), Msr2, Cathepsins B, S, and K (Ctsb, Ctss, Ctsk) and complement components C1r, C3 and C4 was significantly increased in stria vascularis of adult Slc26a4-/- mice compared to Slc26a4+/+ mice. Expression of macrophage markers Cd45 and Cd84 and complement components C1r and C3 was increased in stria vascularis but not in spiral ligament, liver or spleen of Slc26a4-/- compared to Slc26a4+/- mice. The expression of Lyzs was increased in stria vascularis and spiral ligament but not in liver or spleen.
Conclusion
The data demonstrate that hyperpigmentation of stria vascularis and marginal cell reorganization in Slc26a4-/- mice occur after weaning, coinciding with an invasion of macrophages. The data suggest that macrophage invasion contributes to tissue degeneration in stria vascularis, and that macrophage invasion is restricted to stria vascularis and is not systemic in nature. The delayed onset of degeneration of stria vascularis suggests that a window of opportunity exists to restore/preserve hearing in mice and therefore possibly in humans suffering from Pendred syndrome.
doi:10.1186/1741-7015-4-37
PMCID: PMC1796619  PMID: 17187680
22.  Pharmacological reversal of endothelin-1 mediated constriction of the spiral modiolar artery: a potential new treatment for sudden sensorineural hearing loss 
Background
Vasospasm of the spiral modiolar artery (SMA) may cause ischemic stroke of the inner ear. Endothelin-1 (ET-1) induces a strong, long-lasting constriction of the SMA by increasing contractile apparatus Ca2+ sensitivity via Rho-kinase. We therefore tested several Rho-kinase inhibitors and a cell-permeable analogue of cAMP (dbcAMP) for their ability to reverse ET-1-induced constriction and Ca2+-sensitization.
Methods
The present study employed SMA isolated from gerbil temporal bones. Ca2+sensitivity was evaluated by correlating vascular diameter and smooth muscle cell [Ca2+]i, measured by fluo-4-microfluorometry and videomicroscopy.
Results
The Rho-kinase inhibitors Y-27632, fasudil, and hydroxy-fasudil reversed ET-1-induced vasoconstriction with an IC50 of 3, 15, and 111 μmol/L, respectively. DbcAMP stimulated a dose-dependent vasodilation (Ec50 = 1 mmol/L) and a reduction of [Ca2+]i (EC50 = 0.3 μmol/L) of ET-1-preconstricted vessels (1 nmol/L). Fasudil and dbcAMP both reversed the ET-1-induced increase in Ca2+ sensitivity.
Conclusion
Rho-kinase inhibition and dbcAMP reversed ET-1-induced vasoconstriction and Ca2+-sensitization. Therefore, Rho-kinase inhibitors or cAMP modulators could possess promise as pharmacological tools for the treatment of ET-1-induced constriction, ischemic stroke and sudden hearing loss.
doi:10.1186/1472-6815-5-10
PMCID: PMC1315339  PMID: 16316469
23.  Apical membrane P2Y4 purinergic receptor controls K+ secretion by strial marginal cell epithelium 
Background
It was previously shown that K+ secretion by strial marginal cell epithelium is under the control of G-protein coupled receptors of the P2Y family in the apical membrane. Receptor activation by uracil nucleotides (P2Y2, P2Y4 or P2Y6) leads to a decrease in the electrogenic K+ secretion. The present study was conducted to determine the subtype of the functional purinergic receptor in gerbil stria vascularis, to test if receptor activation leads to elevation of intracellular [Ca2+] and to test if the response to these receptors undergoes desensitization.
Results
The transepithelial short circuit current (Isc) represents electrogenic K+ secretion and was found to be decreased by uridine 5'-triphosphate (UTP), adenosine 5'-triphosphate (ATP) and diadenosine tetraphosphate (Ap4A) but not uridine 5'-diphosphate (UDP) at the apical membrane of marginal cells of the gerbil stria vascularis. The potencies of these agonists were consistent with rodent P2Y4 and P2Y2 but not P2Y6 receptors. Activation caused a biphasic increase in intracellular [Ca2+] that could be partially blocked by 2-aminoethoxy-diphenyl borate (2-APB), an inhibitor of the IP3 receptor and store-operated channels. Suramin (100 μM) did not inhibit the effect of UTP (1 μM). The ineffectiveness of suramin at the concentration used was consistent with P2Y4 but not P2Y2. Transcripts for both P2Y2 and P2Y4 were found in the stria vascularis. Sustained exposure to ATP or UTP for 15 min caused a depression of Isc that appeared to have two components but with apparently no chronic desensitization.
Conclusion
The results support the conclusion that regulation of K+ secretion across strial marginal cell epithelium occurs by P2Y4 receptors at the apical membrane. The apparent lack of desensitization of the response is consistent with two processes: a rapid-onset phosphorylation of KCNE1 channel subunit and a slower-onset of regulation by depletion of plasma membrane PIP2.
doi:10.1186/1478-811X-3-13
PMCID: PMC1298316  PMID: 16266433
24.  Loss of KCNJ10 protein expression abolishes endocochlear potential and causes deafness in Pendred syndrome mouse model 
BMC Medicine  2004;2:30.
Background
Pendred syndrome, a common autosomal-recessive disorder characterized by congenital deafness and goiter, is caused by mutations of SLC26A4, which codes for pendrin. We investigated the relationship between pendrin and deafness using mice that have (Slc26a4+/+) or lack a complete Slc26a4 gene (Slc26a4-/-).
Methods
Expression of pendrin and other proteins was determined by confocal immunocytochemistry. Expression of mRNA was determined by quantitative RT-PCR. The endocochlear potential and the endolymphatic K+ concentration were measured with double-barreled microelectrodes. Currents generated by the stria marginal cells were recorded with a vibrating probe. Tissue masses were evaluated by morphometric distance measurements and pigmentation was quantified by densitometry.
Results
Pendrin was found in the cochlea in apical membranes of spiral prominence cells and spindle-shaped cells of stria vascularis, in outer sulcus and root cells. Endolymph volume in Slc26a4-/- mice was increased and tissue masses in areas normally occupied by type I and II fibrocytes were reduced. Slc26a4-/- mice lacked the endocochlear potential, which is generated across the basal cell barrier by the K+ channel KCNJ10 localized in intermediate cells. Stria vascularis was hyperpigmented, suggesting unalleviated free radical damage. The basal cell barrier appeared intact; intermediate cells and KCNJ10 mRNA were present but KCNJ10 protein was absent. Endolymphatic K+ concentrations were normal and membrane proteins necessary for K+ secretion were present, including the K+ channel KCNQ1 and KCNE1, Na+/2Cl-/K+ cotransporter SLC12A2 and the gap junction GJB2.
Conclusions
These observations demonstrate that pendrin dysfunction leads to a loss of KCNJ10 protein expression and a loss of the endocochlear potential, which may be the direct cause of deafness in Pendred syndrome.
doi:10.1186/1741-7015-2-30
PMCID: PMC516044  PMID: 15320950

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