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RELATED PAPERS |
1 University of Tübingen, Department of Otorhinolaryngology, Tübingen Hearing Research Centre (THRC), Molecular Neurobiology, Elfriede-Aulhorn-Str. 5, D-72076 Tübingen, Germany
2 Biochemical Institute, Christian-Albrechts-University Kiel, D-24098 Kiel, Germany
3 Anatomical Institute, Christians-Albrecht-University Kiel, D-24098 Kiel, Germany
4 University of Helsinki, Department of Biological and Environmental Sciences, 00014 Helsinki, Finland
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Abstract |
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(Received 11 July 2006;
accepted after revision 8 August 2006;
first published online 10 August 2006)
Corresponding author P. Saftig: Biochemical Institute, Christian-Albrechts-University Kiel, Olshausenstr. 40, D-24098 Kiel, Germany. Email: psaftig{at}biochem.uni-kiel.de
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Introduction |
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We generated LIMP2 knockout mice (Gamp et al. 2003) to address the in vivo function of this lysosomal membrane protein. LIMP2-deficient mice showed an increased postnatal mortality and a uni- or bilateral hydronephrosis caused by an obstruction of the ureteropelvic junction. The obstruction was associated with metaplasia of the ureteric urothelium into simple columnar epithelium and hypertrophy of the smooth muscle layer. In regions with preserved urothelium, disturbed apical expression of uroplakin was observed, suggesting an impairment of membrane transport processes (Gamp et al. 2003). LIMP2-deficient mice also suffered from a peripheral demyelinating neuropathy. Demyelination was found to be associated with a loss of peripheral myelin proteins. Serious hearing impairment in LIMP2-deficient animals has also been observed. This was associated with massive spiral ganglion neuron losses, concomitant with loss of the inner and outer hair cells and a strongly impaired capacity to generate an endocochlear potential (Gamp et al. 2003).
It has long been accepted that marginal cells of stria vascularis are involved in the generation of the endocochlear potential and the secretion of K+ (Wangemann, 1995). K+ is the major cation in the endolymph and the charge carrier for sensory transduction and the generation of the endocochlear potential. The importance of K+ handling in the cochlea is marked by the discovery of several forms of hereditary deafness that are due to mutations of potassium channel genes (Wangemann, 2002). In order to gain more insight into the primary cause of loss of hearing function due to a presumptive loss of the endocochlear potential we examined the hearing deficit of LIMP2-deficient mice in more detail and combined these studies with an analysis of the potassium ion channels involved in the generation of the endocochlear potential. We observed that the developing deafness in LIMP2-deficient mice is characterized by a progressive high-frequency hearing loss which is evident as early as 4 weeks of age. Surprisingly, the hearing loss was temporally linked to a loss of the potassium channel subunits KCNQ1/KCNE1 and the endocytic receptor megalin in the luminal surface membrane of marginal cells long before other overt signs of structural changes or degeneration had occurred, indicating a disturbance of membrane recycling of these protens.
Membrane recycling of apically expressed proteins is a mechanism to regulate the surface expression level of plasma membrane receptor or transport proteins. Once incorporated in endosomes, proteins can recycle back to the trans Golgi network or to the plasma membrane. A subset of membrane proteins can also be targeted to lysosomes for degradation (Gruenberg & Maxfield, 1995; Rodriguez-Boulan et al. 2005).
Our data demonstrate for the first time a significant role of LIMP2 in the correct cell surface expression of functionally relevant ion channels and receptor proteins in the stria vascularis.
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Methods |
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Care and use of the animals, and the experimental protocol were reviewed and approved by the animal welfare commissioners and the regional boards for scientific animal experiments in Tübingen and Kiel.
LIMP2–/– mice were generated as described (Gamp et al. 2003). Between 4 and 18 weeks of age, LIMP2–/– mice were repetitively tested for their hearing thresholds and compared to their wild-type litters. Four to six cochleae of wild-type and LIMP2–/– mice of each age were prepared for histology. As control animals for the experiments with the LIMP2–/– mice we used 12- and 30-week-old mice of a mixed 129svj/C57B6 strain.
Hearing measurements
Hearing thresholds were determined by recordings of auditory evoked brainstem responses (ABR) in animals anaesthetized by intraperitoneal injection of 75 mg kg–1 body weight (b.w.) ketamin hydrochloride (Ketamin 50 Curamed, CuraMED Pharma, Karlsruhe, Germany) and 5 mg kg–1 b.w. xylazin hydrochloride (Rompun 290, Bayer, Leverkusen, Germany), expanded with injection water to give a diluted solution for injection (12.5 ml kg–1 b.w.). Supplemental doses of anaesthetics were administered subcutaneously as needed.
ABRs to click and pure tone auditory stimuli were recorded in a sound attenuating chamber (IAC 400-A, Industrial Acoustics Company, Niederkrüchten, Germany) with a customized electrophysiology set-up using a Multi IO Card (National Instruments E-6052, Austin TX, USA) for stimulus generation and recording of evoked potentials, a free field loudspeaker (Beyerdynamic DT-911, Heilbronn, Germany) at 3 cm distance from the pinna, and a high precision microphone (Brüel & Kjaer 4191, Naerum, Denmark) and measurement amplifier (Brüel & Kjaer 2610) for stimulus control. Click sounds (100 µs) and tone pips (3 ms duration, 1 ms rise and fall time, cosine-shaped) were presented at a rate of 20 s–1 with alternating polarity and alternating phase, respectively. Stimulus intensities ranged from 10 to 100 dB or 20–100 dB sound pressure level (dB SPL), raised in 5 dB steps. Sound pressure was calibrated in-place.
The ABR signals were recorded via subdermal silver wire electrodes (diameter 0.25 mm, Goodfellow, Huntingdon, UK) at the vertex, ventro-lateral to the ear (active) and at the back of the animal (ground). Signals were amplified (100 dB), bandpass filtered (0.2–5 kHz) and averaged for 128 and 256 repetitions for pure tone and click stimuli, respectively. ABR responses were recorded for stimulus frequencies between 2.0 kHz and 45.3 kHz at a resolution of 2 steps per octave. The hearing threshold for a given frequency or click stimulus was calculated from the lowest sound pressure leading to reliable ABR signals.
The cubic 2f1 – f2 distortion product (DP) of the otoacoustic emissions (DPOAE) was measured for f2 = 1.24f1 and L2 = L1 – 10 dB. Sound was directly coupled into the ear canal by means of a metal coupler connected to two loudspeakers (Beyerdynamic DT-911). The emission signals were recorded by means of a microphone connected to the coupler (Brüel & Kjaer 4135; preamplifier Brüel & Kjaer 2670) during sound presentation of 260 ms and averaged 4 times for each sound pressure and frequency presented. First, the 2f1 – f2 distortion product amplitude was measured with L2 = 40 dB SPL and f2 = 4–45.2 kHz. Subsequently, the 2f1 – f2 distortion product amplitude was measured for L1 ranging from 0 to 65 dB SPL at frequencies of f2 between 4.0 and 32.0 kHz. Threshold was determined as the L1 sound pressure that could evoke a 2f1 – f2 DP signal reliably exceeding 5–10 dB above noise level, the latter being typically at –20 dB SPL.
Threshold values of hearing measurements (click-ABR, frequency-specific ABR and DPOAE) were compared for statistical significance by means of Student's two-tailed t test adjusted for multiple testing by the Bonferroni-Holms correction (n.s. P > 0.05, *P < 0.05, **P < 0.01, **P < 0.001).
Immunocytochemistry
Mice were asphyxiated in carbonic acid gas and cochleae were isolated, dissected, fixed in paraformaldehyde, cryosectioned and stained as described (Knipper et al. 1995; Oliver et al. 2003; Schimmang et al. 2003). The following primary antibodies were used: anti-KCNQ4 (Ruttiger et al. 2004); antiprestin (Weber et al. 2002); anti-Kir4.1 (APC-035), anti-Kv1.1 (APC-009), anti-Kv1.2 (APC-010) and anti-Kv3.1 (APC-014) were purchased from Alomone Laboratories (Jerusalem, Israel); anti-megalin (P-20, sc-16478) and anti-KCNQ1 (H-130, sc-20816) came from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA, USA); anti-KCNE1 antibody was a kind gift of J. Barhanin, University of Nice, France. In addition we used the following antibodies: anti-LIMP2 (gift of Y. Tanaka, Osaka University, Japan); anti-LAMP2 (Abl93, Developmental Studies Hybridoma Bank, University of Iowa, USA), anticathepsin-D (SII-9; Pohlmann et al. 1995) for paraffin embedded tissues stained as described (Gamp et al. 2003). Primary antibodies were detected with fluorescently labelled secondary IgG antibodies (Cy3-conjugated antibodies, 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 to stain nuclei (cryosectioned cochlea; Vector Laboratories, Burlingame, CA, USA) and photographed using an Olympus AX70 microscope equipped with epifluorescence illumination or Zeiss Axiovert 200M microscope with the Apotome for generation of optical sections. Immunohistochemical comparisons between control and LIMP2-knockout mice were all performed using the same antibody titre for wild-type and mutant mice and identical exposure times were used when comparative photographs were taken. Immunohistological analyses were performed from chochlea samples taken from three to six LIMP2+/+ and LIMP2–/– mice. Representative images were choosen for presentation.
Microscopical investigations
The cochleae were examined in knockout and age-matched wild-type mice (ages 1, 2, 6, 9, 12–14 months; taken from parallel test groups) by light microscopy (semi-thin sections). Tissues were fixed by transcardial perfusion of deeply anaesthetized (tribromoethanol) mice with 6% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.4). Temporal bones were dissected out and decalcified with EDTA (Coenen et al. 2001). Samples were postfixed with 2% osmium tetroxide and embedded in epon according to routine protocols. Semi-thin sections were stained with toluidine blue.
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Results |
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During the analysis of LIMP2-deficient mice we observed hearing impairment. Our initial observations suggested that the hearing loss correlated with reduced endocochlear potential, most likely starting at an age of 3 months, and probably secondary to degeneration of the stria vascularis (Gamp et al. 2003). To validate this hypothesis we studied the hearing function and phenotype of LIMP2 knockout mice in more detail.
To elucidate the nature of the hearing impairment in LIMP2-deficient mice we first analysed auditory brain stem responses (ABR), an evoked potential response of auditory activity in the auditory nerve and subsequent fibre tracts and nuclei within the auditory brainstem pathways. Thresholds and amplitudes of ABRs provide information on the peripheral hearing status and the integrity of brainstem pathways (Jewett et al. 1970).
Auditory brainstem responses to click (click-ABR) and frequency-specific stimulation (f-ABR) were measured in 1-month- and 2.5-month-old LIMP2 wild-type (Toker & Newton, 2000) and mutant mice. Threshold functions for wild-type animals showed a small but significant improvement in click-ABR thresholds at 2.5 months of age (mean ± S.D., 1 month of age: threshold 10.7 dB SPL ± 2.3, n = 22; 2.5 months of age: 8.3 dB SPL ± 0.9 S.D., n = 12). ABR thresholds documented a profound and significant high-frequency hearing loss in LIMP2-deficient mice at 2.5 months of age (Fig. 1). A deficiency of LIMP2 at this age led to significant threshold loss for click stimuli (Fig. 1A, LIMP2+/+) based on the frequency specific loss of 15–46 dB in the high frequency range > 11.3 kHz (Fig. 1B; 11.3–45 kHz).
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LIMP2-deficient mice exhibited this hearing deficit between 1 and 3 months of age. The earliest age at which hearing loss could be detected was a subset of 1-month-old LIMP2 knockout animals (Fig. 2Ai 1 mth ‘bad’, individual data shown in Fig. 2B, early hearing loss). This group of 1-month-old animals exhibited a hearing loss similar to that observed in all LIMP2-deficient animals at 2.5 months of age (Fig. 2A and B, click-ABR; Fig. 2C, f-ABR; Fig. 2D, DPOAE). Interestingly, some 1-month-old mice of the same litter exhibited normal ABR thresholds in response to click stimuli (Fig. 2A and 1 mth/‘good’) and developed a later deficit appearing as a progressive loss in click-ABR threshold (Fig. 2B, late hearing loss, age 1.7 months: P < 0.05, mean age 2.6 months: P < 0.0001). As a first indication of the hearing deficit in 1-month-old animals ABR thresholds on frequency-specific stimulation and DPOAE thresholds increased at high frequencies (Fig. 2C, f-ABR 1 mth/‘good’: 32–45 kHz; Fig. 2D, DPOAE 1 mth/‘good’: threshold loss 22–32 kHz). By f-ABR and DPOAE hearing threshold, LIMP2-deficient mice aged 1 month with early hearing loss were indistinguishable from knockout animals of mean age 2.5 months (Fig. 2C and D and 1 mth/‘bad’).
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Development of inner ear pathology in LIMP2-deficient mice
To address whether the observed hearing loss in LIMP2-deficient mice was accompanied by morphological alterations we analysed the histology of the stria vascularis in control and LIMP2-deficient mice from 1 month until 14 months of age. In wild-type mice aged between 1 and 12 months (Fig. 3A, C, E and G) 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 was as described in the literature (Schulte & Adams, 1989; Kikuchi et al. 2000). The marginal cells displayed extensive folding which surrounded the endostrial capillaries. At the age of 1 month, the stria vascularis of the LIMP2 knockout mice appeared unaltered (Fig. 3B) as compared with age-matched controls (Fig. 3A). While hearing measurements revealed two groups of 1-month-old LIMP2-deficient mice, hearing impaired and hearing unimpaired (Fig. 2), histological studies using offspring of the same litter revealed no detectable pathological changes. The fibrocytes of the spiral ligament, particularly the intimate relation between the root cells and type II fibrocytes, did not show clear alterations in LIMP2-deficient mice up to the age of 6 months (not shown). At the age of 2 months, the basal folds of the marginal cells showed irregularities and were absent in some places, whereas the capillary profiles were inconspicuous (Fig. 3D). In 6-month-old LIMP2 knockout mice, the strial epithelium was atrophic but still identifiable, although the strial cell types could not clearly be distinguished (Fig. 3F). The marginal cells had completely lost their basal folds. The endostrial capillaries were surrounded by a thick layer of intensely stained extracellular matrix. In LIMP2 knockout mice at the age of 14 months, the cellular organization of the stria vascularis was completely disrupted (Fig. 3H), the remaining cells covering the spiral ligament could not be attributed to any cell type of the normal stria vascularis and endostrial capillary profiles were missing.
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Effect of LIMP2 depletion on ion channel expression of the stria vascularis at 3 months of age
We first investigated the expression pattern of LIMP2 in the stria vascularis. Analysis of 14-day-old wild-type mice revealed expression of LIMP2 in marginal cells, intermediate cells and in basal cells (Fig. 4A). As a first step to validate the hypothesis that progressive hearing loss in LIMP2-deficient mice may have its primary origin in pathological changes of the stria vascularis, we examined the phenotype of strial cells in LIMP2+/+ and LIMP2–/– mice at 3 months of age, when structural alterations were already obvious (see above). LIMP2 was strongly expressed in the stria vascularis of 3-month-old LIMP2+/+ mice (Fig. 4B, red, left). The specificity of the staining is confirmed by an almost complete loss of LIMP2 staining in LIMP2-deficient mice (Fig. 4B, right; only very weak residual background staining is observed).
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In the following we typically illustrate staining in midbasal cochlear turns of both LIMP2+/+ and LIMP2–/– mice. In 3-month-old LIMP2+/+ mice a strong expression of Kir4.1 was noted in presumptive intermediate cells (Fig. 4C, red). Despite considerable atrophy of the stria vascularis in 3-month-old LIMP2–/– mice, Kir4.1 expression was still present (Fig. 4D, red), suggesting deletion of LIMP2 did not effect potassium transport in intermediate cells.
In contrast to Kir4.1, which carries potassium to intermediate cells (Marcus et al. 2002), outward rectifying potassium channels (Kv) have been suggested to primarily carry the potassium from fibrocytes to basal cells and from there to intraluminal cells of the stria vascularis. The outward rectifying potassium channel Kv1.1 was strongly expressed in the stria vascularis, apparently in basal and intermediate cells (Fig. 4E, red). Interestingly, Kv1.1 staining was dramatically reduced in hearing-impaired LIMP2-deficient mice at 3 months of age (Fig. 4F), in comparison with age-matched wild-type animals. Residual amounts of Kv1.1 protein were noted, however, in cells of the most apical cochlear turn in 3-month-old LIMP2-deficient mice (Fig. 4F, inset). This staining was no longer visible in 6 month LIMP2–/– animals (data not shown).
KCNQ1 forms K+ channels by assembly with regulatory subunit KCNE proteins and plays a key role in K+ homeostasis in a variety of tissues. KCNQ1 is coassembled with theKCNE1 to produce a cardiac delayed rectifier K+ current (Jentsch et al. 2000). In the inner ear, the KCNQ1/KNCE1 complex maintains the high concentration of K+ in the endolymph. Accordingly, deletion or disturbance of KCNQ1 and KCNE1 causes deafness by affecting endolymph secretion (Kubisch et al. 1999; Jentsch et al. 2000; Hubner & Jentsch, 2002; Wangemann, 2002). Strong KCNQ1 (Fig. 5A) and KCNE1 (Fig. 5C) expression was localized to the apical surface of the marginal cells, as shown in 3-month-old wild-type mice. In the midbasal and medial cochlear turns of age-matched LIMP2-deficient mice, very small patches of KCNQ1 or KCNE1 protein in marginal cell surfaces were occasionally observed, shown for an example of a midbasal cochlear turn (Fig. 5B and D).
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The results indicate that 3-month-old LIMP2-deficient mice probably suffer from a severe disturbance of potassium transport in basal cells (Kv1.1) and marginal cells (KCNQ1/KCNE1) of the stria vascularis, but less so in intermediate cells (Kir4.1).
Effect of LIMP2 depletion on ion channel expression at 1 month of age
To analyse in more detail the causal relationship between hearing loss and loss of potassium channels, we compared Kv1.1 (Fig. 6A and B), KCNQ1 (Fig. 6C and D) and KCNE1 (data not shown) expression in 1-month-old LIMP2-deficient mice without hearing impairment (Fig. 6A and C (–/–) ‘good’) and in 1-month-old LIMP2-deficient mice which displayed a loss of hearing function (Fig. 6B and D (–/–) ‘bad’; compare also Fig. 2. At this early time point the histological analysis did not reveal any notable differences (Fig. 3). Although Kv1.1 expression had declined significantly at 3 months of age (see Fig. 4F for comparison), no difference in Kv1.1 expression was observed in the stria vascularis of 1-month-old LIMP2-deficient mice with or without a hearing impairment (Fig. 6A and B). In contrast, at this early time point, the expression pattern of KCNQ1 was dramatically reduced in the midbasal and medial cochlear turn in the group of LIMP2-deficient mice with a hearing impairment (Fig. 6D), whereas the age-matched normal-hearing LIMP2–/– animals apparently expressed KCNQ1 protein at qualitatively similar levels to wild-type mice (Fig. 6C, compare also with Fig. 5A). Considering the already beginning hearing loss of 1-month-old LIMP2-deficient mice with good hearing at frequencies below 32 kHz and threshold loss at frequencies above 32 kHz (Fig. 2C), it is worth emphasizing that we are unable to judge KCNQ1 expression in most basal cochlear turns, since we typically maintain the cross-sectioning of the vestibular system at the expense of the most basal cochlear turn. The most basal cochlear turns would correspond to 36–70 kHz (Muller, 1991; Muller et al. 2005) and in the case of a causal relationship of loss of KCNQ1 surface expression and loss of hearing threshold ‘good’ hearing 1-month-old LIMP2–/– mice are expected to have lost KCNQ1 from their marginal cell surfaces.
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LIMP2 depletion also affects apical localization of megalin
The almost complete loss of KCNQ1 from marginal cells in LIMP2 knockout mice may point to a disturbance of directed transport of this ion channel to the apical plasma membrane. Megalin, a member of the LDL receptor family, is a cell surface receptor that transports distinct macromolecules into cells through receptor-mediated endocytosis. This process involves the receptor recognizing a ligand, its internalization through clathrin-coated pits and degradation upon fusion with lysosomes. As with KCNQ1, megalin localizes in the apical membrane of marginal cells, shown for 3-month-old LIMP2+/+ mice (Fig. 7A). Surprisingly, megalin was completely missing in 3-month-old LIMP2-deficient mice (Fig. 7B).
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The retention of KCNQ1/KCNE1 or megalin expression in marginal cell surfaces of the apical turns in 3-month-old LIMP2–/– mice was completely lost in 6-month-old mice (data not shown).
LIMP2 depletion affects expression of KCNQ1 and megalin in dark cells of the vestibular system
Morphological similarities between strial marginal cells and vestibular dark cells exist and it is assumed that both cell types are involved in the secretion of K+ into endolymph and contribute to endolymph homeostasis (Wangemann, 1995; Marcus, 1996; Milhaud et al. 2002). Accordingly, dark cells express LIMP2 (data not shown) and strongly express KCNQ1 (Nicolas et al. 2001), as was also observed in the present study (Fig. 8A). We noted KCNQ1 in the luminal membrane of dark cells of 1-month-old control mice and 1-month-old ‘good-hearing’ LIMP2-deficient mice (Fig. 8A), whereas hearing-impaired 1-month-old LIMP2-deficient mice exhibited no detectable KCNQ1 immunoreactivity (Fig. 8B).
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Effect of LIMP2 deletion on the phenotype of hair cells and spiral ganglion neurons
At the age of 6 months, LIMP2-deficient mice were shown to exhibit a massive decline of spiral ganglion neurons in the cochlea, concomitant with a loss of inner and outer hair cells. Cell death was suggested to occur secondary to the degeneration of the stria vascularis (Gamp et al. 2003). In line with this assumption, hair cells and supporting cells in LIMP2–/– mice appeared structurally normal at the light-microscopy level, even though LIMP2 was found to be expressed in supporting cells. In 3-month-old LIMP2-deficient mice, the expression of the motor-protein prestin and the M-type K+ channel KCNQ4 in outer hair cells as well as the inward rectifying potassium ion channel Kir4.1 in Deiter's cells was normal (data not shown).
Strong expression of LIMP2 was also observed in spiral ganglion neurons. The specificity of the staining was confirmed by the absence of specific staining in 3-month-old LIMP2-deficient mice (Supplemental material, Fig. 1A and B). No altered expression of either KCNJ10/Kir4.1 (Supplemental material, Fig. 1C and D), or any of the outward rectifying potassium channels Kv1.1, Kv1.2 and Kv3.1 (Supplemental material, Fig. 1E–J) could be detected in these cells. This is surprising, since at the same time the expression of Kv1.1 was found to be severely reduced in the stria vascularis (Fig. 4F).
Taken together, these results suggest that hearing loss in LIMP2-deficient mice was associated with a decline of KCNQ1/KCNE1 and of megalin in the marginal cells of the stria vascularis of high-frequency processing cochlear turns. The maintenance of partially intact surface expression of KCNQ1/KCNE1/megalin in apical cochlear turns that process frequencies < 10 kHz together with the retention of normal hearing threshold at frequencies <10 kHz underscores a causal link between loss of hearing and loss of KCNQ1/KCNE1 expression in LIMP2-deficient mice.
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Discussion |
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Role of LIMP2 in the inner ear
LIMP2 is an abundant transmembrane glycoprotein that is located primarily in the limiting membranes of lysosomes and late endosomes. Overexpression of LIMP2 causes an enlargement of early endosomes and late endosomes/lysosomes, probably by impairing the endocytic membrane flow. Impaired membrane/vesicular transport in LIMP2-deficient mice is supported by characteristic alterations of the urothelium in the knockout mice (Gamp et al. 2003). The normally apical localization of uroplakin was disturbed in LIMP2-deficient urothelium. In addition, the lysosomal compartment was increased in size and activity. The apical membrane of the mammalian bladder typically increases as the bladder fills with urine and decreases during emptying. This process involves continuous endocytosis and reinsertion of membrane from a pool of endosomes derived from the apical membrane. The loss of uroplakin from the apical surface of LIMP2-deficient urothelium was suggested to be due to impaired endocytic recycling, possibly leading to the degradation of uroplakin in lysosomes. Similarly, the coincident loss of KCNQ1/KCNE1 and megalin in the stria vascularis and loss of KCNQ1/KCNE1 in dark cells of LIMP2-deficient mice may be due to a failure in the recycling of the protein to the apical membrane after endocytic uptake.
It was recently shown in proximal tubular cells that disturbed endocytosis caused by the deletion of the renal endosome-associated chloride channel, ClC-5, led to an impaired endocytosis of apically expressed membrane proteins including a trafficking defect of megalin (Piwon et al. 2000; Christensen & Birn, 2002; Christensen et al. 2003) similar to our observation in the inner ear of LIMP2-deficient mice. Megalin is known to function as a multiligand receptor involved in endocytic uptake in different absorptive epithelia (Hammes et al. 2005; May et al. 2005; Gonzalez-Villalobos et al. 2006) and has to be recycled to the surface after internalization via clathrin-coated pits (Czekay et al. 1997). While megalin transports various lipoproteins including essential vitamins or steroids in other organs (Hammes et al. 2005; Gonzalez-Villalobos et al. 2006), natural ligands for megalin in the inner ear and stria vascularis are elusive (Moestrup et al. 1995; Mizuta et al. 1999).
Considering that the disturbed megalin expression is a possible cause of deafness in LIMP2-deficient mice, our preliminary data show that megalin knockout mice exhibit normal hearing at least until the first 3 months of age (authors' unpublished results). Thus, the loss of KCNQ1/KCNE1 in marginal cells of the stria vascularis is more likely the primary cause for the early hearing loss observed in LIMP2-deficient mice. These ion channels are responsible for the secretion of K+ in the endolymph (Konishi et al. 1978; Wangemann et al. 1995), generating the endocochlear potential (Wangemann, 2002). As the endocochlear potential directly drives sensory transduction in the hair cells (Jentsch et al. 2004), any loss of KCNQ1/KCNE1 is expected to reduce this driving force and thus directly affect the transduction process and hearing. The spatio/temporal loss of the surface expression of KCNQ1/KCNE1 in LIMP2-deficient mice along the tonotopic axis of the cochlea thus lends weight to a direct causal relationship between the altered protein expression profile and loss of hearing threshold. In 1-month-old bad-hearing and 3-month-old LIMP2-deficient mice, an almost complete loss of KCNQ1/KCNE1 in midbasal and medial cochlear turns was observed, while in apical cochlear turns marginal cell surfaces still exhibited immunoreactivity for these proteins. The apical cochlear half turn in mice processes frequencies below 10 kHz (Muller, 1991; Muller et al. 2005). Since hearing thresholds in LIMP2 mutants were maintained at frequencies <10 kHz, one could hypothesize that a critical level of KCNQ1/KCNE1 surface protein persists, sufficient to act as the driving force for the transduction process in these turns. It is in this context that the selective loss of brainstem responses at frequencies 32 kHz observed in 1-month-old ‘good-hearing’ LIMP2-deficient mice should be regarded. One-month-old ‘good-hearing’ LIMP2–/– mice obviously experienced the first damage in the highest frequency processing basal cochlear turns. It may be suggested from our results that the surface expression of KCNQ1/KCNE1 is lost in this turn. Our data suggest the gradual worsening of hearing loss and surface expression of KCNQ1/KCNE1 towards lower frequency processing cochlear turns until about 3 months of age when the cochlear damage has proceeded towards the 10 kHz region. The hearing loss affecting all frequencies and the entire loss of KCNQ1 surface expression in 6-month-old LIMP2-deficient mice underscores the assumption that the strial defect and the hearing loss progresses from the base to the apex.
Cathepsin-D expression appeared to be enhanced in 3-month-old hearing-impaired LIMP2-deficient mice (data not shown). At this age, however, the severe morphological disturbance of the stria vascularis did not allow any detailed quantification. Due to the overall unchanged expression of cathepsin-D, one of the major lysosomal proteinases, in other inner ear regions, quantification using for example Western blot analysis is not feasible. The loss of KCNQ1/KCNE1 and megalin expression together with the apparent up-regulation of cathepsin-D and LAMP2 in the stria vascularis of LIMP2-deficient mice suggest an increased lysosomal degradation of these proteins. This may be caused by defects in apical protein recycling back to the plasma membrane after endocytosis. In this respect it is intriguing to speculate that under physiological conditions LIMP2 may be one of the intracellular regulators that determine if endocytosed proteins remain in the recycling pathway or if they are targeted towards late endosome/lysosomes for degradation. An endosomal function of LIMP2 is also supported by the endocytic defects observed after LIMP2 overexpression (Kuronita et al. 2002). One could also speculate that a correctly functioning endosomal recycling process is required for those membrane proteins that are localized in megalin expressing surfaces.
Mutations in the gene encoding the K+ channel 
-subunit KCNQ1 have been associated with long QT syndrome and deafness (Yang et al. 1997). In addition to heart and inner ear epithelial cells (see for a review Jentsch et al. 2000), KCNQ1 is expressed in a variety of epithelial cells, including those lining the renal proximal tubule and the gastrointestinal tract (Vallon et al. 2005). In the inner ear KCNQ1 knockout mice exhibit a marked atrophy of the stria vascularis and a degeneration of hair cells and the spiral ganglion (Rivas & Francis, 2005). This phenotype is similar to that observed in LIMP2 knockout mice. It will be interesting to investigate whether a disturbance of KCNQ1/KCNE1 targeting also occurs in the heart and kidney of LIMP2-deficient mice.
In summary, our results show that the lack of LIMP2 causes a selective loss of surface expression of KCNQ1, KCNE1 and megalin in specialized tissues such as the stria vascularis leading to deafness. Further analyses of the roles of LIMP2 and its unidentified interacting partners may provide a better understanding of its function in the endocytic pathway.
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References |
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