Human Molecular Genetics, 2003, Vol. 12, No. 9 995-1004
DOI: 10.1093/hmg/ddg116
© 2003 Oxford University Press
Transgenic expression of a dominant-negative connexin26 causes degeneration of the organ of Corti and non-syndromic deafness
1Department of Medical Genetics and 2Department of Otorhinolaryngology—Head and Neck Surgery, Tohoku University Graduate School of Medicine, Sendai, Japan
Received December 18, 2002; Accepted March 2, 2003
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Hereditary deafness affects about 1 in 2000 children and mutations in the GJB2 gene are the major cause in various ethnic groups. GJB2 encodes connexin26, a putative channel component in cochlear gap junction. However, the pathogenesis of hearing loss caused by the GJB2 mutations remains obscure. The generation of a mouse model to study the function of connexin26 during hearing has been hampered by the fact that Gjb2 knockout mice are embryonic lethal. To establish viable model mice we generated transgenic mice expressing a mutant connexin26 with R75W mutation that was identified in a deaf family with autosomal-dominant inheritance. The previous expression analysis revealed that the R75W connexin26 inhibited the gap channel function of the co-expressed normal connexin26 in a dominant-negative fashion. We established two lines of transgenic mice that showed severe to profound hearing loss, deformity of supporting cells, failure in the formation of the tunnel of Corti and degeneration of sensory hair cells. Despite robust expression of the transgene, no obvious structural change was observed in the stria vascularis or spiral ligament that is rich in connexin26 and generates the endolymph. The high resting potential in cochlear endolymph essential for hair cell excitation was normally sustained. These results suggest that the GJB2 mutation disturbs homeostasis of cortilymph, an extracellular space surrounding the sensory hair cells, due to impaired K+ transport by supporting cells, resulting in degradation of the organ of Corti, rather than affecting endolymph homeostasis in mice and probably in humans.
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Hereditary deafness affects about 1 in 2000 children and mutations in the GJB2 gene, which encodes gap junction protein connexin26, are the major cause in various ethnic groups (1–4). It has been hypothesized that gap junctions in cochlea, especially connexin26, provide an intercellular passage by which potassium ions are transported to maintain high levels of the endocochlear potential (EP), which is essential for sensory hair cell excitation (5). However, the pathogenesis of deafness due to GJB2 mutations remains obscure partly because histological and electrophysiological examination that can be carried out in human is limited and partly because Gjb2-deficiency mice is embryonic lethal (6).
To elucidate the pathological role of connexin26 in the inner ear, we generated transgenic mice carrying human conne-xin26 cDNA with a dominant-negative mutation. Most connexin26 mutations show autosomal-recessive inheritance while several missense mutations, including R75W, have been found to segregate with deafness in an autosomal-dominant fashion. In a Xenopus oocyte expression system, the mutant connexin26 with R75W not only failed to show channel-forming activity but also blocked the activity of co-expressed wild-type connexin26, indicating the dominant-negative effect of the mutant connexin26 (7). To inhibit the function of mouse connexin26 we expressed the dominant negative connexin26 with R75W under control of the CAG promoter that has potent promoter activities in various types of cells including germ cells (8,9). We chose to use Cre-loxP system to suppress the expression of the dominant-negative connexin26 in founder mice (10), considering that the null mutant of GJB2 is embryonic lethal (6). The expression of the dominant-negative connexin26 was then induced by Cre recombinase in the offspring for examination of the phenotype.
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RESULTS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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We constructed a transgenic vector that expressed human connexin26 cDNA with R75W mutation under control of the CAG promoter (Fig. 1A). A fluorescent marker gene flanked by a pair of loxP sequences was inserted between the promoter and the mutant connexin26 cDNA (10). The transgenic vector was introduced into 253 fertilized eggs and nine lines of transgenic mice were obtained. The dominant negative connexin26 was not expressed in the founder mice. To induce the mutant protein expression we mated them with transgenic mice harboring Cre recombinase gene under the control of CAG promoter (CAG-Cre mice). Previous study revealed that a DNA fragment flanked by a pair of loxP sequences in embryonic cells was efficiently excised out from mouse genome when mated with CAG-Cre mice (11). Two lines of mice that had both mutant GJB2 and Cre (R75W+ mice) were obtained (line nos 9-10 and 10-1) and used for further analysis. The R75W+ mice were viable and fertile and born at the expected mendelian ratio. No obvious anomaly was found and no growth retardation was observed. Relative expression levels of the transgene mRNA against mouse connexin26 mRNA was examined by a reverse transcribed-polymerase chain reaction (RT–PCR) method. We introduced a four-base deletion in 3' untranslated region of the transgene to distinguish the mRNA of the transgene from that of the mouse connexin26 gene (Fig. 1B). The transgene mRNA produces a 157 bp cDNA fragment while mouse connexin26 mRNA generates a 161 bp cDNA fragment. The relative ratios of the transgene mRNA against normal mice connexin26 mRNA in inner ear were 4.1±1.6 and 2.9±0.8 in 10-1 and 9-10 strains, respectively (Fig. 1C). Mutant GJB2 mRNA was also detected in brain, liver, kidney, skin and placenta at various levels.
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The R75W+ mice exhibited no apparent phenotypic abnormalities except deafness. The auditory brainstem response (ABR) revealed that the mice at 7 weeks old (line nos 9-10 and 10-1) showed severe to profound hearing loss as compared with littermate controls (R75W- mice) that carried the transgene allele but not the Cre allele (Fig. 2A and B). Both lines of R75W+ mice at 2 weeks old also showed severe to profound hearing impairment. The thresholds of 2-week-old 9-10 R75W+ mice (n=3), 2-week-old 10-1 R75W+ mice (n=6), 7-week-old 9-10 R75W+ mice (n=8), and 7-week-old 10-1 R75W+ mice (n=4) were 103.3±2.9, 103.3±2.6, 100±3.8 and 103±2.7 dB SPL, respectively. In control mice, the thresholds of 2-week-old 9-10 R75W- mice (n=5), 2-week-old 10-1 R75W- mice (n=3), 7-week-old 9-10 R75W- mice (n=8), and 7-week-old 10-1 R75W- mice (n=4) were 37±2.7, 36.7±2.9, 27.2±4.4 and 33.8±2.5 dB SPL, respectively. We then measured the EP values in cochlea. The average EP value in non-transgenic mice was 97.4±7.1 mV and that in R75W+ mice was 87.7±2.5 mV, both of which remained within a normal range (80–100 mV), as shown in Figure 2C.
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Because connexin26 is also distributed in balance-related organs (12), we examined the vestibular function of the mice using a rotarod apparatus with a gradual increase of rotation speed (13). A total of 20 mice (R75W+ mice from line nos 9-10 and 10-1 and non-transgenic mice from line nos 9-10 and 10-1) were assessed. Both lines of R75W+ mice could stay on an accelerating rotarod normally, indicating that their vestibular function was not impaired (Fig. 3).
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Histological examinations of the inner ear revealed significant changes in the mutant mice. Hematoxylin and eosin (H–E) staining of the midmodiolar section (Fig. 4) showed a collapse of the tunnel of Corti in 2-week-old mice (Fig. 4C) and a degeneration of the organ of Corti and spiral ganglia at 7 weeks (Fig. 4E). There was no other gross anomaly or hydrops of endolymph. Toluidin blue staining of Epon embedded samples showed deformity of supporting cells in 2-week-old mice (Fig. 4G) and degeneration of hair cells at 7 weeks (Fig. 4I). There was no change in Reissner's membrane, tectorial membrane, spiral ligament or stria vascularis. To elucidate whether these changes were directly caused by the transgene expression, we performed immunostaining using specific antibody against enhanced green fluorescent protein (EGFP), which was co-expressed with the mutant connexin26 allele. Expression was observed in the organ of Corti, the spiral ligament, stria vascularis and spiral limbus (Fig. 5B). The connexin26 immunoreactivity in the non-transgenic mice was observed in the spiral ligament, the organ of Corti and spiral limbus (Fig. 5C), as shown in the previous study (12). Transgenic mice showed obscure staining in the organ of Corti while distinct immunostaining was detected in the lateral wall and spiral limbus, suggesting degenerative changes in the organ of Corti (Fig. 5D). The connexin30, another gap junction protein, was expressed in the cochlea similar to connexin26 (Fig. 5E and F). Sensory macula and fibrocytes in balance organs appeared normal (Fig. 6A and B), although EGFP was apparently expressed throughout the vestibular tissue (Fig. 6C and D).
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An analysis by transmission electron microscopy demonstrated further details of histological alterations in the organ of Corti (Fig. 7). In 2-week-old transgenic mice, sensory hair cells were present in the cochlea (Fig. 7C). However, deformity of the cell shapes together with presumed increase of the cytoplasm in the supporting cells appeared to diminish the extracellular fluid space surrounding the outer hair cells and the tunnel of Corti space. In 7-week-old mutant mice, the outer hair cells had degenerated and disappeared (Fig. 7E). Synapse formation was clearly observed in inner hair cells, suggesting that extension of neuronal fibers from spiral ganglion to sensory hair cells was not interrupted. Fibrocytes of the spiral ligament and stria vascularis, which are responsible for the generation of the EP, showed no alteration (Fig. 7F and G). There was no evidence of apoptotic cell death in the sensory epithelium studied by transmission electron microscopy or TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling) method using 2- and 7-week-old 9-10 mice (data not shown).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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We generated two lines of transgenic mice that expressed the dominant-negative connexin26 in various organs including inner ear, and found profound hearing loss, degeneration of outer and inner hair cells, and deformity of organ of Corti in both strains as early as 2 weeks after birth. The observation is in line with congenital hearing loss in human, because auditory neurons and sensory hair cells in mice develop not only during embryogenesis but also postnatally and mature at around 2 weeks after birth (14), while the auditory system has already matured at birth in humans. A previous histological study of temporal bone specimen of a patient with GJB2 mutations revealed that the hair cells in the organ of Corti were near-totally degenerated (15), which resembles the histological findings of our model mice. No abnormality was observed in balance functions in patients with GJB2 mutations, which was also consistent with the normal vestibular structure and function in our model mice.
K+ ions in endolymph exit from outer hair cells and are removed, partially by uptake into Deiters' cells (Fig. 8). In the recent hypothesis of the potassium recycling model of the cochlea (5), K+ diffuses through an epithelial gap junction system connecting Deiters' cells to adjacent supporting cells. Another network of a connective tissue fibrocyte gap junction of the lateral wall provides K+ ions to the stria vascularis, which contributes to the secretion of K+ ions into endolymph and the generation of a high resting potential in scala media. Inactivation of transport proteins of the marginal cells of the stria vascularis impairs K+ secretion (16–18), resulting in collapse of the Reissner's membrane separating the scala media from the scala vestibuli. Targeted disruption of the Pou3f4 gene in mice (19), which resulted in profound deafness, showed morphological changes in the fibrocytes of spiral ligament and it was associated with reduced EP. In the present study, however, there was no evidence for morphological changes of the stria vascularis or fibrocytes or collapse of the Reissner's membrane, despite the fact that the transgene was clearly expressed in the cochlear lateral wall. Lack of morphological changes in the stria vascularis or fibrocytes appears to be consistent with the normal EP, although a relatively small number of animals were used to measure the EP. These observations imply the preservation of ionic and electric environment in endolymph in transgenic mice. Different gap junction proteins from connexin26 may compensate the K+ pathway across the fibrocytes and stria vascularis.
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A recent work on mice lacking the K-Cl cotransporter Kcc4 revealed that removal of K+ ions by the supporting Deiters' cells would change the ionic composition of small extracellular space, cortilymph, surrounding the basolateral membrane of outer hair cells, eventually leading to their degeneration (20). Connexin26 is present in the cochlear gap junctions between supporting cells and supposed to play a role as a K+ channel (Fig. 8A). Dominant-negative inhibition of connexin26 function in the supporting cells may cause deafness by blocking K+ removal further downstream from Deiters' cells at the epithelial gap junction network. Outer hair cells of Kcc4-/- mice degenerated before Deiters' cells were lost, which is interestingly similar to the observations in our transgenic mouse. Another key to resolve the underlying mechanism of deafness is the difference of pathology between the cochlear and vestibular organs. Although the dominant-negative effect of the transgene is also expected in the vestibular supporting cells, no significant change was recognized in the vestibular organ using functional and morphological analyses. This may be explained by the fact that the vestibule do not possesses the cortilymph generated specifically by the cochlear supporting cells. Thus, severe hearing loss observed as early as 2 weeks after birth, in spite of the presence of both the hair cells and the spiral ganglion cells, can be brought about by a possible physiological dysfunction of the supporting cells that disrupts proper ionic environment around hair cells, leading to disturbances in the membrane integrity, the membrane excitation, and the transmitter release of the hair cells. Especially, an extreme disturbance of extracellular homeostasis surrounding outer hair cells may lead to death of these cells by osmotic stress or membrane depolarization.
There are supposed to be two gap junction systems (21): one is the epithelial cell system composed of Deiters, Hensen, Claudius and outer sulcus cells, and the other is the spiral ligament system composed of fibrocytes and strial basal and intermediate cells. There is the consequent discontinuity in direct communication between these two gap junction systems (22). The epithelial cell system is required to maintain homeostasis of both outer hair cells and cortilymph by uptake of K+ through supporting cells. The connective tissue spiral ligament system contributes to the function of the stria vascularis where K+ is pumped out into the endolymph and the high resting potential in the endolymph is generated (Fig. 8B). Our results suggest connexin26 play a major role in the epithelial cell system rather than in the spiral ligament system although connexin26 is expressed in both systems.
In preparation of this paper another model mouse for connexin26 deficiency was reported (23). They performed targeted ablation of connexin26 specifically in epithelial gap junction network by using Cre recombinase under control of otogelin promoter that was expressed only in supporting cells. Their model mice developed moderate to profound deafness and degradation of the organ of Corti, which were similar to our model mice. However, EP was reduced in their model mice. Taking into account their findings together with ours, we would like to suggest that impaired K+ homeostasis of endolymph and reduced EP are not essential for development of deafness. Instead, deficiency of connexin26 disturbs homeostasis of cortilymph due to impaired K+ transport by supporting cells, resulting in degradation of the organ of Corti and hearing loss.
GJB2 mutations are responsible for a large proportion of congenital deafness. The mouse model generated in this study would provide a valuable tool for investigating therapeutic means to prevent the degeneration of supporting cells in the organ of Corti at an early stage of development.
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METHODS |
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TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMETHODSREFERENCES |
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Generation of R75W+ mice
Human connexin26 cDNA (681 bp) with the R75W mutation was subcloned into a pIRES-EGFP vector (BD Biosciences Clontech, Palo Alto, CA, USA), and digested with Ecl136II and SspI to generate a fragment that contained mutant connexin26 cDNA (CX26-R75W), internal ribosomal entry site (IRES), and EGFP (Fig. 1A). The entire coding region of the red fluorescent protein (DsRed) cDNA (Clontech) was subcloned into the SwaI site between the two loxP sites in the pCALwL vector (9), which was then designated as CAG-DR. The pCALwL vector was obtained from RIKEN gene bank (www.rtc.riken.go.jp/). The R75W-IRES-EGFP fragment was then subcloned into the SmaI site that locates downstream of the two loxP sequences of the CAG-DR vector. The plasmid was digested with Sal I and SfiI and injected into fertilized eggs of C57BL/6 to generate the transgenic mice (Fig. 1A). To identify transgenic mice, genomic DNA of the offspring were extracted from tail samples using DNesay Tissue kit (Qiagen, GmbH, Hilden, Germany). Then the 3' untranslated regions of both the transgene and mouse Gjb2 gene were amplified with CX26TG5 and Cy5-labeled CX26TG6C primers that amplified (Fig. 1B). The sequences of primers CX26TG5 and CX26TG6C were 5'-GGCCCAC(A/G)GA(G/A)AAGACTGTCTTCAC-3' and 5'-CCATCTTTGCTCTAGCAGCCAGGCA-3', respectively. Mixed nucleotides were used for the underlined positions. To distinguish the PCR product of the transgene from that of the mouse Gjb2 gene, we introduced a four-base deletion in the transgene. The transgene produces a 157 bp DNA fragment while mouse genomic Gjb2 generates a 161 bp fragment. The PCR products were size-separated by an 8% polyacrylamide urea gel electrophoresis in A.L.F. Express II DNA sequencer (Amersham Bioscience Corp., Piscataway, NJ, USA). The size and amount of DNA fragments were measured by software, Fragment analysis (Amersham Bioscience Corp.). A plasmid vector pxCACre, which carried Cre recombinase cDNA under control of the CAG promoter (24), was used for generation of mice with Cre recombinase. We introduced it into C57BL/6 strain mice and obtained a transgenic mouse line (CAG-Cre mice). We then mated 9-10 or 10-1 mice with CAG-Cre mice to obtain mice that had both mutant connecin26 cDNA and Cre recombinase gene (R75+ mice) for further study.
RT–PCR analysis
Under deep anesthesia with sodium pentobarbital we sacrificed 7-week-old mice and collected the inner ear, brain, liver, kidney, heart, skin and placenta. Total RNA extraction and the digestion with RNase-free DNase were performed with the RNeasy kit (Qiagen GmbH). Complementary DNA was synthesized with oligo-dT8-12 primer and Superscript II reverse transcriptase (Invitrogen Corp, Carlsbad, CA, USA) followed by amplification with CX26TG5 and CX26TG6C primers. The size and amount of the PCR products were measured as described above. The area ratio of the 157 and 161 bp peaks was calculated for estimation of the relative expression level (Fig. 1C).
Measurement of ABR and EP
Mice were anesthetized with sodium pentobarbital (70 mg/kg) delivered intraperitoneally and maintained in a headholder within an acoustically and electrically insulated and grounded test room. Stainless-steel needle electrodes were placed on the tympanic bulla (positive lead) and scalp vertex (negative lead). The ABRs were measured using an evoked potential recording system (NEC Corp, Tokyo, Japan). Acoustic stimuli evoked by a click were delivered to the mice through a loudspeaker. The peak amplitude was measured as the peak-to-trough and the threshold was defined as 1 µV. For EP measurement, each mouse was artificially ventilated with a respirator through a tracheal cannula after deep anesthesia and muscular relaxation. The rectal temperature was kept at 37°C and an electrocardiometer was monitored. A glass microelectrode filled with 150 mM of KCl was inserted into the scala media of the basal turn through the lateral wall of the cochlea and the output was recorded by high-impedance dual electrometer (19).
Rotarod analysis
The balance abilities of 10–14-week-old mice from the two transgenic line nos 9-10 and 10-1, were measured using an accelerating revolving rod 3.0 cm in diameter (Rota-rod treadmill for mice MK-600, Muromachi Co. Ltd, Tokyo, Japan). For each test, a mouse was placed on the rod that was rotated at a speed of six rotations per minute (rpm). The speed was then increased from 6 to 28 rpm over 2 min and the time until the mouse fell from the rod (latency to fall) was measured. Each mouse was tested for 7 consecutive days. For each group of mice, mean±SD of the latency to fall was calculated on each day.
Histological analysis
Mice of line nos 9-10 and 10-1 (2 and 7 weeks old) were used for histological analysis. Perfusion, decalcification, dehydration and the preparation of paraffin blocks were performed as described (19). Sections were cut at 6 µm and mounted on slides. For immunohistochemistry, rabbit antiserum against connexin26 (Zymed Laboratory, USA), rabbit antiserum against connexin30 (Zymed Laboratory) or mouse monoclonal antibody against EGFP (Clontech) was employed. Primary antibodies were detected with Vectastain ABC reagent (Vector Laboratories, USA) and 3,3'-diaminobentidine-H2O2 as previously reported. The examination of the ultrastructure by transmission electron microscopy was performed as described (25). The samples were sectioned at 0.1 µm thickness and stained with uracyl acetate and lead.
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ACKNOWLEDGEMENT |
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We are grateful to Ms Kato for her excellent technical assistance. This work was supported by grants from the Ministry of Education, Culture, Sports, Science and Technology, and the Ministry of Health, Labor and Public Welfare, Japan.
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FOOTNOTES |
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* To whom correspondence should be addressed at: Department of Medical Genetics, Tohoku University Graduate School of Medicine, 1-1 Seiryomachi, Aobaku, Sendai 980-8574, Japan. Tel: +81 227178140; Fax: +81 227178142; Email: skure{at}mail.cc.tohoku.ac.jp
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H. Hibino and Y. Kurachi Molecular and physiological bases of the k+ circulation in the Mammalian inner ear. Physiology, October 1, 2006; 21: 336 - 345. [Abstract] [Full Text] [PDF]
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 D. J. Jagger and A. Forge Compartmentalized and Signal-Selective Gap Junctional Coupling in the Hearing Cochlea J. Neurosci., January 25, 2006; 26(4): 1260 - 1268. [Abstract] [Full Text] [PDF]
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 Y. Maeda, K. Fukushima, K. Nishizaki, and R. J.H. Smith In vitro and in vivo suppression of GJB2 expression by RNA interference Hum. Mol. Genet., June 15, 2005; 14(12): 1641 - 1650. [Abstract] [Full Text] [PDF]
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 W.-L. Di, Y. Gu, J. E. A. Common, T. Aasen, E. A. O'Toole, D. P. Kelsell, and D. Zicha Connexin interaction patterns in keratinocytes revealed morphologically and by FRET analysis J. Cell Sci., April 1, 2005; 118(7): 1505 - 1514. [Abstract] [Full Text] [PDF]
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R. D. Hawkins and M. Lovett The developmental genetics of auditory hair cells Hum. Mol. Genet., October 1, 2004; 13(suppl_2): R289 - R296. [Abstract] [Full Text] [PDF]
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