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,1
,2
* Audiology and Phoniatry, Charité University Clinic Berlin, Campus Benjamin Franklin, Berlin, Germany;
Molecular Neurobiology and Cell Biology of the Inner Ear and
Regenerative Biology, Department of Otorhinolaryngology, Hearing Research Center Tübingen (THRC), and
Medical and Natural Sciences Research Centre, University of Tübingen, Tübingen, Germany; and
|| Electron Microscopy, Max-Delbrück-Center for Molecular Medicine, Berlin-Buch, Germany
2Correspondence: Universitäts-HNO-Klinik, Elfriede-Aulhorn-Straße 5, D-72076 Tübingen, Germany. E-mail: marlies.knipper{at}uni-tuebingen.de
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: cochlea • stria vascularis • LDL-receptor • sex hormone • presbycusis
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 2)
. It is essential for endocytosis of lipoproteins and low molecular weight proteins in absorptive epithelia (2
3
4)
. Various studies (5
, 6)
have described megalin as an endocytic receptor for lipophilic metabolites and as a carrier for protein bound cholesterol and vitamins A and D. Only recently megalin was shown to maintain the endocrine (hormone) balance in mammals by serving as a carrier for protein bound androgens and estrogens (7)
.
In the inner ear, megalin was detected within the marginal cells of the stria vascularis of the cochlea (8)
. The function of megalin in the inner ear is, however, completely unknown. To study the role of megalin in cochlear function, mutant mice lacking the intact megalin gene (megalin knockout mice) are essential. This is quite difficult since most newborn homozygous knockout pups die of respiratory failures (9)
. Only a few homozygous knockout animals reach adulthood. In this study, we succeeded in analyzing a limited number of homozygous knockout mutants (megalin–/–) at the age of 3 and 6 months. We compared their hearing abilities to those of wild-type (megalin+/+) and heterozygous mutant mice (megalin+/–). Ultrastructural morphological analysis and examination of ion channel expression revealed strial changes reminiscent of presbycusis caused by degeneration of the stria vascularis. Uptake of FITC-labeled 17β-estradiol into the stria vascularis in in vivo and ex vivo studies supports the view that estrogen may be one of the lipophilic metabolites taken up by megalin. The data present evidence of a crucial role of megalin and its ligands in hearing function.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and bathed in a solution of fluorescein isothiocyanate (FITC)-estradiol BSA (22.7 µg/µl; final concentration of 4 µM). FITC-labeled 17β-estradiol was synthesized from 1,3,5(10)-estradien-3–17β-diol-17-hemisuccinate: BSA (Steraloids, London, UK) with FITC. Analysis of the conjugate resulted in a ratio of BSA:17β-estradiol:FITC of 
1:35:2. The protein concentration was 2.5 mg/ml in PBS.
Animals
In this study we used Wistar rats and megalin-deficient mice (megalin+/–, megalin–/–). Megalin C57BL/6 inbred mice were generated as described previously (9)
and bred by mating megalin mice at the animal house of the Institute of the Max-Delbrück-Center for Molecular Medicine (Berlin, Germany).
Care and use of the animals and the experimental protocol were reviewed and approved by the animal welfare commissioner and the regional board for scientific animal experiments in Berlin and Tübingen.
Hearing measurement
Anesthesia of animals was achieved by intraperitoneal injection of a mixture of ketamin hydrochloride (Ketamin 50 Curamed, CuraMED Pharma, Karlsruhe, Germany; 75 mg/kg body weight) and xylazin hydrochloride (Rompun 290, Bayer, Leverkusen, Germany; 5 mg/kg body weight; ref. 11
). Measurements of auditory brain stem responses (ABR) and distortion-product otoacoustic emissions (DPOAE) were performed as described previously (11
12
13)
.
FITC-β-estradiol uptake
Mice were anesthetized as described previously (11)
, and small gel-foam pellets (Gelita Tampon, Braun Aesculap, Germany) were soaked with either FITC-β-estradiol-BSA or artificial perilymph (5–10 µl) placed at the round window (14)
. The animals were allowed to recover from anesthesia and were killed 20 h later by asphyxiation in CO2, and the stria vascularis was dissected from the cochlea. The mice or rat cochleae were prepared ex vivo. A small hole was drilled in the apex of the cochlea and 4 µM GST-RAP (20 µl) in binding buffer (DMEM containing 0.1% ovalbumin) or binding buffer (20 µl; control) was injected through the round window for preincubation (10 min, room temperature), followed by an injection of FITC-β-estradiol-BSA (20 µl) or FITC-BSA (20 µl) for 30 min at 4°C. Since FITC antibodies did not detect the antigen after cochleae decalcification, for detection of FITC-β-estradiol uptake post in vivo or ex vivo application, the stria vascularis was isolated, fixed, and embedded in sagital orientation.
Tissue preparation, immunohistochemistry, and electron microscopy
The cochleae of mice and rats were isolated, dissected, and stained for fluorescence microscopy (12
, 13
, 15)
using the alkaline phosphatase procedure as described in (16)
. For immunohistochemistry the following antibodies were used: anti-megalin (goat polyclonal antibody, Santa Cruz Biotechnology, Santa Cruz, CA, USA, sc-16478), anti-KCNQ1 (rabbit polyclonal antibody, Santa Cruz sc-20816), anti-Kir4.1 (rabbit polyclonal antibody, Alomone Labs, Jerusalem, Israel, APC-035), anti-Kv1.1 (rabbit polyclonal antibody, Alomone Labs APC-009); the rabbit polyclonal antibody against KCNQ4 (17)
and the rabbit polyclonal antibody against prestin (18)
were generated as described previously. Anti-fluorescein-AP Fab fragments (No. 11426338910) were obtained from Roche. Primary antibodies were detected with Cy3-conjugated (Jackson ImmunoResearch Laboratories, Inc., West Grove, PA, USA) or Alexa Fluor 488-conjugated antibodies (Molecular Probes, Eugene, OR, USA). Sections were mounted with Vectashield mounting medium with DAPI (Vector Laboratories, Burlingame, CA, USA) staining cell nuclei in blue. Specimens were photographed with an Olympus AX70 microscope equipped with epifluorescence illumination.
For electron microscopy, 3-month-old megalin+/+ and megalin–/– animals were used. The cochleae were fixed in 8% formaldehyde/0.25% glutaraldehyde in 0.2 M HEPES, decalcified, postfixed with osmium tetroxide, and embedded as described previously (19)
. To detect structural details, the cochleae were cut longitudinally and semithin sections were stained with toluidine blue. Ultrathin sections of the region with the stria marginalis were contrasted with uranyl acetate and lead citrate and examined with a Zeiss 910 electron microscope. Microvilli counts were achieved from 12 ultrathin sections (70 nm thick) cut at variable positions of the cochlea, each section containing 10 marginal cells.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and shown for the midbasal cochlear turn of 3-month-old megalin+/+ mice (Fig. 1
A) strong megalin expression was observed in the marginal cells of the stria vascularis. No megalin immunoreactivity was seen in 3-month-old megalin-deficient mice (Fig. 1B
).
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Hearing function in megalin mutant mice
Three- to eight-month-old megalin+/+, megalin+/–, and megalin–/– mice were analyzed for hearing function. Due to high lethality at and after birth, 1 yr breeding resulted in only one 3-month-old megalin–/– mouse and one 6-month-old megalin–/– mouse available for hearing measurements. For all specimens, hearing function was tested in both ears. Analysis of ABR thresholds showed a profound hearing loss at 3 months of age in megalin–/– mice for click stimuli, which did not change significantly at 6 months of age, leading to an average threshold of 64.9 ± 15.4 dB SPL (mean±SD; Fig. 2
A, –/–) and average threshold loss of 36 dB over the whole frequency range (Fig. 2B
, triangles). In comparison, megalin+/+ mice had an average threshold of 14.5 ± 10.0 dB SPL (Fig. 2A
, +/+). At 3 months of age the hearing function of megalin+/– mice did not differ significantly from controls (Fig. 2A
, +/– 3 months; Fig. 2B
, diamonds). However, megalin+/– mice older than 6 months exhibited a moderate but significant elevation in click-ABR threshold (Fig. 2A
, +/– 6 months: 34.6±13.5 dB SPL). Mean threshold loss over the whole frequency range was 14 dB (Fig. 2B
, squares).
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DPOAE measurements as a test for outer hair cell function (Fig. 2C, D
) could not be determined in 3- and 6-month-old megalin–/– mice (Fig. 2C
), and DPOAE maximum amplitudes were near noise level (Fig. 2D
), while DPOAE thresholds and amplitudes in megalin+/– mice were not significantly reduced (Fig. 2C, D
). This could be an indication that the active cochlear mechanics, i.e., the force generation of the OHCs to amplify basilar membrane movement (20)
may be severely affected in 3-month-old megalin–/– mice.
Effect of megalin deletion on the structure of the stria vascularis
The phenotype of strial cells was examined in more detail in one of the surviving 3-month-old megalin–/– mice. In the cochlea of megalin+/+ and megalin–/– mice, the histological organization of the stria vascularis with three types of cells (basal, intermediate and marginal cells) and the specialized fibrocytes of the spiral ligament were the same as described previously (21)
. The marginal cells displayed extensive folding that surrounded the endostrial capillaries (Fig. 3
A, B). These cells had an unusual phenotype in megalin knockout mice. The basal level of lipofuscin granules (arrows) found in rare numbers in marginal cells (21)
was clearly elevated in 3-month-old megalin–/– mice (Fig. 3B
) in comparison to megalin+/+ mice (Fig. 3A
). In addition, the numbers of microvilli found at the apical membrane of marginal cells (Fig. 3A
, black arrowheads) were reduced in megalin–/– mice (Fig. 3B
). For two randomly chosen series of sections, 3.3 microvilli were counted per cell in megalin+/+ mice (146 cells measured), whereas an average of 1.3 microvilli per cell was detected in megalin–/– mice (91 cells measured). In general, we found that the apical surfaces of marginal cells were more irregular in the mutants (Fig. 3B
).
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Lysosomal membrane glycoproteins such as LIMP2 are major components of the transport processes through the lysosomal membrane. Recent data show that the lysosomal membrane protein LIMP2 plays a critical role in the regulation of the membrane transport processes in the endolymphatic pathway (22)
. In LIMP2-deficient mice the expression of the multiligand endocytotic receptor megalin is reduced in the luminal surfaces of the marginal cells within the stria vascularis (23)
. Data indicate that progressive hearing loss in LIMP2-deficient mice mutants is associated with disturbed expression of the potassium channels KCNQ1, KCNJ10/Kir4.1 and Kv1.1 (23)
. In 3-month-old hearing impaired megalin–/– mice, the expression of all potassium ion channels in the stria vascularis was normal (data not shown). In 6-month-old megalin–/– mice (n=4 ears), but not megalin+/– mice (data not shown), we observed a complete loss of strial KCNQ1 (Supplemental Fig. 1A), Kir4.1 (Supplemental Fig. 1B) and Kv1.1 (Supplemental Fig. 1C) in the basal and midbasal cochlear turns, whereas the expression remained intact in the more apical cochlear turns (Supplemental Fig. 1A-C, inset). In rare cases, very small patches of KCNQ1 could be detected in the marginal cell surfaces within more basal cochlear turns, shown for a midbasal cochlear turn (Supplemental Fig. 1A). Preliminary results done in megalin+/– mice >6 months indicate that also heterozygous animals finally develop a similar disturbance of potassium channels over age. Surface expression of the M-type K+ channel KCNQ4 at the base of outer hair cells (OHCs) was also lost in 6-month-old megalin–/– mice (Supplemental Fig. 2) but not in 3-month-old megalin–/– mice or in 3- or 6-month-old megalin+/– mice (data not shown). The KCNQ4 loss in OHCs was observed solely in basal and midbasal cochlear turns (Supplemental Fig. 2A) but not in apical and medial turns (not shown) or in the hair cells of the vestibular system (Supplemental Fig. 2B). Prestin, the motor protein of OHCs (Supplemental Fig. 2C), and the inward rectifying potassium channel Kir4.1 within the luminal membrane of Deiter’s cells (Supplemental Fig. 1D) exhibited normal expression.
Association of hearing loss and megalin expression in the stria vascularis
For a better understanding of the hearing loss in megalin+/– mice, we compared individual affected megalin+/– animals. Of the megalin+/– mutants older than 6 months, two specimens showed nearly complete loss of megalin protein in the apical surfaces of marginal cells (Fig. 4
B, +/– 001). This was associated with a hearing loss of 40 dB (Fig. 4D
, +/– 001) and significant reductions in DPOAE amplitudes (Fig. 4E
, +/– 001, –4 to –8 dB SPL). In other megalin+/– animals, reduced levels of megalin protein were detected in the apical surface of marginal cells (Fig. 4C
, +/– 002, shown for 6 analyzed cochleae of 3 megalin+/– mice). Hearing thresholds of megalin+/– mutants with maintained megalin expression varied between 24 dB and 39 dB SPL (Fig. 4D
) with DPOAE amplitudes comparable to wild-type mice (Fig. 4E
).
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Estradiol as a ligand of megalin in the stria vascularis
A possible role of estrogen in the development of early presbycusis (for review see ref. 24
) and localization of β-estradiol receptors in the marginal cells of the stria vascularis (25)
motivated us to consider estrogen as a candidate ligand of megalin.
As shown in rats (Fig. 5
A, B) but verified also in mice (data not shown), ex vivo application of FITC-β-estradiol-BSA resulted in FITC-labeling on the same side of the stria vascularis where KCNQ1 (Fig. 5A, B
, left panel) and megalin (Fig. 5A, B
, right panel) are expressed. Preincubation with the specific megalin inhibitor RAP entirely blocked the staining (Fig. 5B
, middle panel). The experiments were repeated twice for rats and mice with similar results.
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As we were unable to breed any more living megalin–/– mice, we examined megalin+/– animals that both lacked megalin protein and had significant hearing loss in order to verify β-estradiol uptake. An overview (Fig. 6
A, B, FITC-17β-estradiol, insets) and a higher magnification (Fig. 6A, B
, FITC-17β-estradiol) show that the application of FITC-17β-estradiol through the round window led to fluorescence signals detectable in megalin+/+ animals (Fig. 6A
, FITC-17β-estradiol) but not in megalin+/– mice (Fig. 6B
, FITC-17β-estradiol). Neighboring cryosections of the stria vascularis of the other ear confirmed strong FITC-labeling in megalin expressing megalin+/+ mice (Fig. 6A
, anti-FITC), while hardly any FITC signals were detected in megalin-negative megalin+/– mice (Fig. 6B
, anti-FITC). In both animals Kir4.1 and KCNQ1 were expressed normally (compare Fig. 6A, B
, Kir4.1, KCNQ1). The data underscore a possible link between the presence of megalin in marginal cells and β-estradiol uptake.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Megalin deficiency-induced hearing loss
Similar to recent findings (8)
, the data in the present study confirm megalin expression in the apical surface of marginal cells but not on the basolateral surface. The function of megalin in the cochlea is elusive, but similar to megalin-expressing proximal tubule cells in kidney uptake (26
27
28
29)
, its internalization in clathrin-coated pits and its return to the apical membrane surface are suggested (8)
.
We report here that loss of megalin within the cochlea leads to progressive hearing loss in 3-month-old homozygous megalin–/– and 6-month-old heterozygous megalin+/– mutants. Hearing loss was accompanied by DPOAE loss indicating a reduction in active cochlear mechanics. A loss of megalin was recently observed in LIMP2 mutant mice, which also suffered from progressive hearing loss and reduced DPOAEs (23)
. In this study, the hearing loss was linked to loss of KCNQ1/KCNE1, known to be responsible for the generation of the endocochlear potential (30
31
32
33)
rather than to loss of megalin. In the present study we demonstrate that the hearing loss in megalin mutants was already profound at a time point when KCNQ1 expression in the stria vascularis and KCNQ4 expression in the organ of Corti was still entirely normal. This strongly indicates that the molecular basis of hearing loss due to megalin deficiency differs from LIMP2 deficiency. Despite normal potassium channel expression at the time of hearing loss, ultrastructural changes were found in megalin mutants reminiscent of early presbycusis. The lysosomal structures found in ultrathin sections of the marginal cells of megalin-deficient mutants are similar to those in rat proximal tubule cells (34)
. Lipofuscin granules often show a characteristic pale smooth component of neutral lipid and a darker globular component (Fig. 3B
, inset) as well as a limiting membrane around some of them, as described for some types of lipofuscinosis (35)
. Lipofuscin granules and lipid droplets are normal cellular compartments in marginal cells of the stria vascularis (21)
. As the end product of lysosomal activity cannot leave the cells by exocytotic pathway, the number of lipofuscin granules within cells increases with age (36)
. An enrichment of lipofuscin deposits in the stria vascularis is therefore a typical feature of strial presbycusis (37
38
39
40)
. The second ultrastructural change observed in hearing impaired megalin mutants was the reduction of microvilli, suggesting an initial pathological change in marginal cells associated with hearing loss. Numerous microvilli are a typical feature of normal secretory activity of marginal cells (41
, 42)
, whereas a reduction in microvilli can be seen when metabolic activities are disturbed by ototoxic drugs such as atoxyl or streptomycin (41
, 43
, 44)
. By the time lipofuscin granule enrichment and microvilli reduction in marginal cells has occurred, hearing loss is already profound (Figs. 2
, 3)
. In conclusion, we predict that the observed ultrastructural changes observed in homozygous megalin mutants precede the observed changes in the expression pattern of potassium channels (Supplemental Figs. 1, 2) and that both changes also gradually occur in heterozygous specimens with a delay of few months. More studies will be required in the future to elucidate the presumed spatiotemporal correlation between hearing loss and phenotype in megalin mutants. However, the lethality of megalin–/– mice is a handicap for a proper analysis. Cell-specific deletion of megalin in conditional mutants may be required to better understand causal relationships between megalin deletion and hearing loss.
Heterozygous megalin+/– animals
Megalin+/– mice have a higher survival rate and display a weaker cochlear phenotype. Additionally, they may even differ from megalin–/– mice at the molecular level. Moreover, neither the analysis of the reproductive organs of megalin mutants (7)
nor the transepithelial transport of vitamins in megalin mutants (5
, 6
, 45)
point to a phenotype in heterozygous megalin+/– mutants. The phenotype observed here in heterozygous megalin+/– mice either indicates a so far undetected semidominant effect through haplo-insufficiency or it mimics an organ-specific feature. For normal functioning, one allele of megalin may be unable to produce enough megalin in the stria vascularis. This may become evident only with analyses of older animals. The data also point to a high variability within heterozygous megalin+/– mutants ranging from those expressing reduced megalin levels, exhibiting moderate hearing loss, to those with severe megalin loss, displaying thresholds very similar to homozygous megalin–/– animals (Fig. 4)
. The entire reduction of megalin protein in the marginal cells of two megalin+/– specimens older than 6 months may be a secondary effect of a reduced megalin level. It is conceivable that incomplete megalin function induces a self-enhancing detoriation of strial pathology leading to megalin depletion from the surface membrane.
Estrogen as a ligand for megalin in the stria vascularis?
Our hypothesis that β-estradiol may be a candidate ligand for strial megalin protein is based on the following observations: 1) recent findings demonstrate that sex hormones bound to plasma carrier sex hormone binding globulin (SHBG) are natural ligands of megalin (7)
; 2) estrogen deficiency correlates with presbycusis (for review see: ref. 24
); and 3) β-estrogen receptors (but not 
-receptors) are expressed in the marginal cells of the stria vascularis (25)
.
Considering these observation, it may be challenging to regard this hypothesis in the context of a role of estrogen in humans in comparison to rodents. Human and rodent gender differences in hearing and the impact of estrogens on hearing ability have been postulated for decades (46
47
48
49
50)
. When 143,843 active workers were tested for occupational hearing loss, a gender gap was found in favor of the female workers (51)
. The females showed a pattern of recordable vs. nonrecordable threshold shifts that occurred 20 yr later than in male workers (51)
. Women with no ovarion estrogen production, like women with Turner’s syndrome, develop early presbycusis (52)
. Estrogen receptors (ER) and β have been found in human and rodent cochleae, but ERβ was found only in the stria vascularis, within the nuclei of the marginal cells (25
, 53)
. Recent observations indicate progressive hearing loss in ERβ knockout mice, which are almost deaf by the time they are 1 yr old (24)
. Furthermore, similar to our present findings, strial presbycusis has been associated with lipofuscin positive particle enrichment (54)
. Despite the fact that we cannot exclude from these correlations that megalin effects the hearing by others than estrogen activities, data nevertheless strongly support the notion that β-estrogen may be transported by megalin. Although previous studies indicate a role of other megalin ligands such as vitamin A and D (55
, 56)
, strial effects were never described in post vitamin deficiency. The role of ERβ within marginal cells of the stria vascularis is quite elusive. The data in the present study support the concept that megalin may be required for β-estrogen transport to target receptors within marginal cell nuclei. The phenotypic analysis of ERβ receptor function in knockouts and discovery of transcriptional target genes in marginal cells will be required in order understand the presumed functional link between megalin and estrogen.
Megalin binding proteins
Megalin acts as a cargo transporter for lipophilic vitamins and steroid hormones bound to carrier proteins. Steroid hormones are assumed to bind to specific carrier proteins within the extracellular space just like sex hormone binding globulin or corticosteroid binding globulin, the transporters for sex steroids and corticosteroids (57
, 58)
. In analogy, complexes of 25-OH vitamin D3 in combination with vitamin D binding protein and vitamin A with the retinol binding protein become internalized by megalin (5
, 6)
but until now have not been reported to be associated with hearing loss.
Although we showed in our experiments that FITC-β-estradiol is taken up when bound to BSA as previously reported (59
, 60)
, the ratio of β-estradiol to BSA (35:1) makes it likely that free β-estradiol is also bound to endogenous SHBG and may be taken up as an FITC-β-estradiol-SHBG complex by means of megalin transport. Investigations are underway to verify the presence of SHBG and estradiol in the endolymphatic fluid.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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Received for publication June 6, 2007. Accepted for publication August 9, 2007.
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REFERENCES |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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-subunit but not the BKβ1-subunit leads to progressive hearing loss. Proc. Natl. Acad. Sci. U. S. A. 101,12922-12927This article has been cited by other articles:
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  R. Simonoska, A. E Stenberg, M. Duan, K. Yakimchuk, A. Fridberger, L. Sahlin, J.-A. Gustafsson, and M. Hultcrantz Inner ear pathology and loss of hearing in estrogen receptor-{beta} deficient mice J. Endocrinol., June 1, 2009; 201(3): 397 - 406. [Abstract] [Full Text] [PDF]
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 C. E. Fleming, F. M. Mar, F. Franquinho, M. J. Saraiva, and M. M. Sousa Transthyretin Internalization by Sensory Neurons Is Megalin Mediated and Necessary for Its Neuritogenic Activity J. Neurosci., March 11, 2009; 29(10): 3220 - 3232. [Abstract] [Full Text] [PDF]
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