Mice lacking ganglioside GM3 synthase exhibit complete ... - PNAS

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Jun 9, 2009 - gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. In SAT-I null mice, hearing ability, assessed by brain- ..... Hair cell counts are calculated as mean number of hair cells per 100 μm.
Mice lacking ganglioside GM3 synthase exhibit complete hearing loss due to selective degeneration of the organ of Corti Misato Yoshikawaa,b, Shinji Goa, Kotaro Takasakic, Yasuhiro Kakazud, Mitsuru Ohashid, Masakazu Nagafukua,b, Kazuya Kabayamaa, Junji Sekimotoa, Shun-ichi Suzukia, Kazutaka Takaiwad, Takashi Kimitsukid, Nozomu Matsumotod, Shizuo Komuned, Daisuke Kameie, Masaki Saitof, Michihiro Fujiwarac, Katsunori Iwasakib,c,1, and Jin-ichi Inokuchia,b,1 aDivision

of Glycopathology, Institute of Molecular Biomembranes and Glycobiology, Tohoku Pharmaceutical University, 4-4-1 Aoba-ku, Sendai, Miyagi 981-8558, Japan; bCore Research for Evolutional Science and Technology Program, Science and Technology Agency, 4-1-8, Honcho Kawaguchi, Saitama, 332-0012, Japan; cDepartment of Neuropharmacology, Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan; dDepartment of Otorhinolaryngology, Graduate School of Medical Science, Kyushu University, 3-1-1 Maidashi, Higashi-ku, Fukuoka 812-8582, Japan; eField of Supramolecular Biology, International Graduate School of Arts and Sciences, Yokohama City University, 1-7-29 Suehiro-cho, Tsurumi-ku, Yokohama 230-0045, Japan; and fDepartment of Pharmacodynamics, Meiji Pharmaceutical University, Tokyo 204-8588, Japan

Communicated by Sen-itiroh Hakomori, Pacific Northwest Research Institute, Seattle, WA, March 28, 2009 (received for review November 4, 2008)

The ganglioside GM3 synthase (SAT-I), encoded by a single-copy gene, is a primary glycosyltransferase for the synthesis of complex gangliosides. In SAT-I null mice, hearing ability, assessed by brainstem auditory-evoked potentials (BAEP), was impaired at the onset of hearing and had been completely lost by 17 days after birth (P17), showing a deformity in hair cells in the organ of Corti. By 2 months of age, the organ of Corti had selectively and completely disappeared without effect on balance or motor function or in the histology of vestibule. Interestingly, spatiotemporal changes in localization of individual gangliosides, including GM3 and GT1b, were observed during the postnatal development and maturation of the normal inner ear. GM3 expressed in almost all regions of cochlea at P3, but at the onset of hearing it distinctly localized in stria vascularis, spiral ganglion, and the organ of Corti. In addition, SAT-I null mice maintain the function of stria vascularis, because normal potassium concentration and endocochlear potential of endolymph were observed even when they lost the BAEP completely. Thus, the defect of hearing ability of SAT-I null mice could be attributed to the functional disorganization of the organ of Corti, and the expression of gangliosides, especially GM3, during the early part of the functional maturation of the cochlea could be essential for the acquisition and maintenance of hearing function. auditory system 兩 cochlea 兩 deafness 兩 knockout mice 兩 sialyltransferase

angliosides [i.e., glycosphingolipids (GSLs)] containing sialic acid are abundant in central nervous tissues and are considered to have important roles in controlling development, proliferation, differentiation, and maintenance of neural tissues and cells (1). Gangliosides usually reside in the outer leaflet of the cell membrane and are concentrated in microdomains specialized for cell adhesion and cell signaling (2, 3). The first product in the biosynthetic pathway of the ganglio-series gangliosides is GM3, which serves as a common precursor for the a-series and b-series gangliosides (Fig. 1). The sialyltransferase responsible for GM3 synthesis is the GM3 synthase (EC 2.4.99.9), also known by the names SAT-I (used here), ST3GalV, and Siat 9. The gene encoding this enzyme has been identified by several research laboratories, including ours (4–6). In the study presented here, we generated mice deficient in GM3 synthase (SAT-I⫺/⫺ mice) (7) and examined their general behavior, including their motor function, learning and memory, and sensory function. Interestingly, SAT-I⫺/⫺ mice exhibited no startle reflex in response to various acoustic stimulations, yet they did demonstrate normal startle responses to air puffing, suggesting a hearing impairment. Electrophysiological and histological analyses of the auditory system of the SAT-I⫺/⫺ mice www.pnas.org兾cgi兾doi兾10.1073兾pnas.0903279106

Fig. 1. Biosynthesis of gangliosides and sulfatides. Biosynthetic pathways of ganglio-series gangliosides and sulfated glycosphingolipids are shown. GM3 synthase (SAT-I) is the primary enzyme for the synthesis of a-, b-, and c-series gangliosides. The brain of SAT-I null mice lack a- and b-series gangliosides and contain o-series gangliosides (GM1b and GD1␣), as reported previously (13) (Fig. S1C).

further revealed that the organ of Corti in the inner ear is selectively degenerated in these animals. Results Absence of Acoustic Startle Response in SAT-I Null Mice. GM3 synthase null (SAT-I⫺/⫺) mice were generated as described in

Author contributions: S.K., M.F., K.I., and J.I. designed research; M.Y., S.G., K. Takasaki, Y.K., M.O., M.N., K.K., J.S., S.S., K. Takaiwa, T.K., N.M., D.K., and M.S. performed research; M.Y., S.G., Y.K., M.O., K.I., and J.I. analyzed data; and J.I. wrote the paper. The authors declare no conflict of interest. 1To

whom correspondence may be addressed. E-mail: [email protected] or [email protected].

This article contains supporting information online at www.pnas.org/cgi/content/full/ 0903279106/DCSupplemental.

PNAS 兩 June 9, 2009 兩 vol. 106 兩 no. 23 兩 9483–9488

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Fig. 2. Lack of hearing ability in SAT-I null mice due to the selective degeneration of the organ of Corti. (A) Startle response in 12-week-old SAT-I⫹/⫹ (n ⫽ 10) and SAT-I⫺/⫺ (n ⫽ 9) mice after acoustic stimuli (70, 75, 80, 85, 90, 100, 110, and 120 dBSPL; Left) or air puff (Right). Data represent the means ⫾ SE. *, P ⬍ 0.001; significantly different from the corresponding vehicle-treated group by Student’s t test or the Aspin–Welch test. (B) BAEPs were obtained from 8-week-old mice (SAT-I⫹/⫹ and SAT-I⫺/⫺ littermates) responding to click stimuli up to 85 dBSPL. Patterns from SAT-I⫹/⫹ mice indicate BAEP; the pattern from a SAT-I⫺/⫺ mouse reveals no BAEP. Representative data for each littermate group (n ⫽ 6) are presented. (C) Histology of cochlea. Tissue sections of cochlea in 8-week-old SAT-I⫹/⫹ and SAT-I⫺/⫺ mice were prepared and stained with hematoxylin and eosin as described in Methods. The organ of Corti, with OHCs, IHCs, RM, TM, and SV, appears to be normal in SAT-I⫹/⫹ mice. In contrast, the organ of Corti is completely missing in the cochlea of SAT-I⫺/⫺ mice. (Scale bars: 100 ␮m.) Representative data for each littermate group (n ⫽ 6) are presented.

Methods and Fig. S1. During observations of their general behavior, we found that SAT-I⫺/⫺ mice exhibited no acoustic reflex, indicating a loss of hearing. Three-month-old SAT-I⫹/⫹ mice exhibited acoustic startle responses to stimuli 85 dBSPL and higher; however, the littermate SAT-I⫺/⫺ mice exhibited no startle reflex in response to any acoustic stimulation (Fig. 2A Left). In contrast, all littermate mice demonstrated normal startle responses to air puffs administered to their backs (Fig. 2 A Right), suggesting an impairment in the hearing ability of mice deficient in SAT-I. Lack of Brainstem Auditory-Evoked Potentials (BAEPs) in SAT-I Null Mice Due to Selective Degeneration of the Organ of Corti. The BAEP

represents the average surface field potential recorded activity 9484 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0903279106

of the auditory neural generators of the lower auditory pathway (8). We performed a BAEP study as an objective hearing test on 8-week-old mice by using a click-sound stimulus of up to 85 dBSPL. SAT-I⫹/⫹ mice exhibited reproducible BAEP peaks. The BAEP obtained from SAT-I⫹/⫹ mice (Fig. 2B Left) is not absolutely normal, because the wave 1 derived from cochlea was relatively small, probably because of the congenital genetic defect in C57/BL6 mouse. In SAT-I⫺/⫺ mice, all BAEP peaks were missing, suggesting that the impaired hearing was due to abnormalities in the cochlear nerve and/or inner ear function (Fig. 2B). To further distinguish whether the sensory organs were impaired, we applied electrocochleograms (ECochG), which provide information regarding the cochlear response to sound stimuli. Although the compound action potentials could be obtained from 8-week-old SAT-I⫹/⫹ and SAT-I⫹/⫺ mice, we could not observe any response from SAT-I⫺/⫺ mice by any stimuli (Fig. S2). These results indicate that the cochlea of SAT-I⫺/⫺ has primary lesion of the hearing loss. No structural differences in the middle and the external ear of 6- to 8-week-old SAT-I littermates, SAT-I⫹/⫹, and SAT-I⫺/⫺ mice were observed; however, in SAT-I⫺/⫺ mice, the organ of Corti was completely missing, although the other regions of cochlea, including the Reissner’s membrane (RM), stria vascularis (SV), and the tectorial membrane (TM), appeared to be normal (Figs. 2C and 3B). Probably as a consequence of the loss of hair cells, the spiral ganglion (SG; Fig. 2C) was scattered (9). We considered the possibility that the degeneration of the organ of Corti in the SAT-I⫺/⫺ mice might be secondarily induced by changes in the endolymph, an extracellular fluid produced and maintained by the SV that is critical to mechanoelectric transduction (10). To address this question, we next measured the endocochlear potential (EP) and K⫹ concentrations in the endolymph. There were no significant differences in EPs and the K⫹ concentrations between SAT-I⫹/⫹ and SAT-I⫺/⫺ (Fig. 3A), proving that the profound hearing loss in the SAT-I⫺/⫺ mice at 8 weeks old that resulted from the loss of mechanoelectric transduction was induced by the primary loss of the organ of Corti, not by the secondary consequence from metabolic changes in the endolymph. In addition, histological studies of the vestibule in the SAT-I⫺/⫺ mice revealed no abnormality (Fig. 3C), consistent with the normal balance and motor function of the mice when tested on a rota-rod (Fig. 3D). SAT-I Null Mice Lost Hearing Ability at the Early Phase of Hearing Maturation. To assess the time elapsed for hearing loss due to

SAT-I deficiency, BAEPs were performed on the littermates at days 13, 14, 15, and 17 after birth (P13 to P17). No BAEP was detected in any mouse (SAT-I⫹/⫹, SAT-I⫹/⫺, SAT-I⫺/⫺) at P13, but responses became apparent in SAT-I⫹/⫹ and SAT-I ⫹/⫺ mice between P14 and P15 (Fig. 4A). However, BAEPs in some SAT-I⫺/⫺ mice at P14 could be monitored with decreased responses (Fig. 4B), and ⬇60% had no detectable BAEP (Fig. 4A). By P17, no SAT-I⫺/⫺ mice exhibited a BAEP (Fig. 4A). These results indicate that the functional loss of hearing in SAT-I⫺/⫺ mice begins at the early phase of hearing maturation and becomes complete by P17. We subsequently performed histological examinations of the organ of Corti from SAT-I littermates (Fig. 4C). In SAT-I⫺/⫺ mice, the gross morphology of the organ of Corti indicates that the acute and progressive degeneration of this organ occurred just after the onset of hearing (Fig. 4C). Next, we performed scanning electron microscopy to examine the hair bundles of outer hair cells (OHCs) and inner hair cells (IHCs) at P14 (Fig. 4D). We found a significant deficit of OHCs in SAT-I⫺/⫺ mice at every turn (basal, middle, and apex) of the cochlea. There was no obvious deficit of IHCs in SAT-I⫺/⫺ mice. These results indicate that the degeneration of the organ of Corti already Yoshikawa et al.

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Fig. 3. Function and histology of SV and vestibule in adult SAT-I null mice. (A) Endocochlear potential (EP) and potassium ion (K⫹) concentrations in the endolymph of 6- to 8-week-old SAT-I⫹/⫹ and SAT-I⫺/⫺ mice are illustrated. The vertical axis displays millivolts for EP (Left) and millimolar for K⫹ concentration (Right). Each bar represents the mean ⫾ SE. There were no significant differences in the EP or the K⫹ concentrations between the SAT-I⫹/⫹ and SAT-I⫺/⫺ mice. (Scale bar: 50 ␮m.) (B) Microscopic examination of the SV, stained with hematoxylin and eosin, of 8-week-old SAT-I⫹/⫹ (Left) and SAT-I⫺/⫺ (Right) mice. No gross difference was detected. (C) Histological examination of the vestibule from an 8-week-old SAT-I⫺/⫺ mouse. Despite the disappearance of the organ of Corti (arrowhead), the layer of hair cells at the sacculus (arrow) and macula statica (the structure faintly stained with eosin above the layer of hair cells) is apparent and appears to be normal. (Scale bar: 100 ␮m.) (Inset) A 4-fold magnified view of the hair cell layer is indicated by the arrow, demonstrating stereocilia at the apical side of the hair cells. (D) Motor coordination of SAT-I littermates. SAT-I⫹/⫹ (n ⫽ 10) and SAT-I⫺/⫺ (n ⫽ 11) mice were tested on a rota-rod. The latency to fall was measured at 10 and 15 rpm. Values are expressed as means ⫾ SE.

occurred at the onset of hearing in SAT-I⫺/⫺ mice, consistent with the results of BAEP. GSL Expression in the Inner Ear. GSLs expressed in the inner ear of

SAT-I⫹/⫹, SAT-I⫹/⫺, and SAT-I⫺/⫺ mice were analyzed by high-performance thin-layer chromatography (HPTLC) (Fig. 5A). Sulfatide SM4 (SO3H-Gal-Cer), a major component in mouse inner ear, was apparent in all mice. GM3, GM1, GD1a, GD1b, and GT1b were all detected in SAT-I⫹/⫹ and SAT-I⫹/⫺ mice, with GD1a and GT1b as the major components; trace amounts of several other glycolipids were also detected (Fig. 5A Right). Reports analyzing the ganglioside composition of cochlea in rat (11) and chinchilla (12) identified similar ganglioside expression profiles, although the ratios differed slightly. Gangliosides expressed in the inner ear of SAT-I⫺/⫺ mice included only the o-series, such as GM1b and GD1␣ (Fig. 5A Left). This ganglioside expression pattern is consistent with the absence of GM3 synthase, because o-series gangliosides are produced by bypassing GM3 synthesis (Fig. 1). There have been reports that in SAT-I⫺/⫺ mice, all of the a-series and b-series gangliosides in brain are converted to o-series gangliosides, such as GM1b and GD1␣, because of the lack of GM3 synthesis (13). Consistent Yoshikawa et al.

with this observation, in the brains and inner ear of our SAT-I⫺/⫺ mice, we found 2 major bands comigrating with GM1b and GD1␣ (Fig. 5A and Fig. S1C). Major neutral GSLs in all littermates was glucosylceramide (GlcCer), and lactosylceramide (LacCer) was identified only in SAT-I⫺/⫺ mice, reflecting the blockade of the conversion of LacCer to GM3 (Fig. 5A). Interestingly, we found that in the cochlea of wild-type mice, the distribution of each ganglioside dramatically changes during postnatal development. At P3, GM3 and the other major ganglioside GT1b are expressed in all regions of the cochlea, including the organ of Corti (Fig. 5B). At P14, GM3 is strongly expressed in the SV, SG, and the organ of Corti. GT1b is also expressed in the organ of Corti; however, it is strongly expressed in regions distinct from GM3, such as the spiral ligament and spiral limbus. In adult mice, both GM3 and GT1b exhibit expression patterns similar to that at P14. Thus, spatiotemporal and dynamic localization of individual gangliosides is apparent during the short time that defines the postnatal development and maturation of the inner ear. Discussion Cell surface glycoconjugates, including proteoglycans, glycoproteins, and glycolipids, have been considered to serve important functions in hearing because of their variety and specific expression patterns during the development and maturation of cochlea (14, 15). However, there has been no definitive proof regarding their involvement in auditory functions. We report here deafness in SAT-I null mice with primary selective degeneration of the organ of Corti (sensory organ of hearing in the cochlea). In our histological study, the degeneration of the organ of Corti arises near the onset of hearing in SAT-I null mice, showing the deformities of supporting cells and hair cells (Fig. 4 C and D) and leading to the selective and complete disappearance of this organ by 2 months (Fig. 2C). A role for gangliosides in the development of the central nervous system has been suggested based on the striking changes in ganglioside composition during the differentiation of neurons (for review, see refs. 16 and 17). For example, drastic changes in GT1b distribution in rat cerebellum during early postnatal development has been reported (18). Although similar expression profiles of complex gangliosides, such as GM1, GD1a, GD1b, and GT1b, were observed in both brain and inner ear of adult mice, GM3 expresses a substantial amount in inner ear compared with brain (Fig. 5A and Fig. S1C), suggesting a specific role for GM3 in the auditory system. We observed that SAT-I null mice have a normal life span. Compensatory expression of o-series gangliosides in these mice may have beneficial effects for neurologic function and life span, because mice lacking all ganglio-series gangliosides (after disruption of both the SAT-I and GM2/GD2 synthase genes) develop a severe neurodegenerative disease that results in death soon after weaning (19). In addition, adult mice expressing only GM3 (i.e., double-knockout mice targeting both GM2/GD2 and GD3 synthases) reportedly respond to acoustic stimulation (20, 21), indicating the functional involvement of GM3 in the auditory system. SV, which selectively expresses GM3 in adults (Fig. 5B), has important functions for K⫹ secretion into endolymph and the production of EP (for a review, see ref. 10). However, the lack of GM3 still maintained the normal K⫹ concentration and EP in the endolymph of SAT-I⫺/⫺ (Fig. 3A), despite the fact that the BAEP was abolished completely (Fig. 2B), indicating that deletion of SAT-I and GM3 does not affect the function of SV. Comparing K⫹ concentration and EP results in this paper with other reports in various molecular knockout mice expressing hearing loss, there seems to be no influence of loss of GM3 on the functioning of potassium channel (22), connexin (23, 24), and claudin (25) molecules in the cochlea. However, it remains to be elucidated whether other gangliosides generated in SAT-I⫺/⫺ mice function PNAS 兩 June 9, 2009 兩 vol. 106 兩 no. 23 兩 9485

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Fig. 4. Loss of hearing in SAT-I null mice occurs near the onset of hearing. (A) Rate of positive BAEP as a function of development after the hearing onset. Vertical axis indicates the percentage of mice showing the identical BAEP peaks at 100 dBSPL at all frequencies tested. Data obtained at the sound frequency 8 kHz are presented here. Whereas the positive rate of SAT-I⫹/⫹ (white bars) and SAT-I⫹/⫺ (dotted bars) mice reached 100% at P15, SAT-I⫺/⫺ mice (black bars) showed a low positive rate at onset of hearing (P14, 4 of 10; P15, 3 of 8) and no detectable response at P17 (0 of 6). (B) Thresholds of BAEP at each sound frequency (4, 8, 16, and 32 kHz) were measured from SAT-I littermates at P14. Vertical axis indicates the intensity of sound stimuli (dBSPL) from the speaker. Each point was averaged from the values that induced identical BAEP peak(s). Data represent the means ⫾ SE (vertical bars). There are significant differences between the SAT-I⫹/⫹/SAT-I⫹/⫺ and the SAT-I⫺/⫺ mice (P ⬍ 0.01 by Student’s t test). (C) Progressive deformity of the organ of Corti immediately after hearing onset in SAT-I⫺/⫺ mice. Light micrographs of the cochlea at P14, P15, and P17 in SAT-I⫹/⫹ and SAT-I⫺/⫺ mice. Note the deformities of the hair cells and supporting cells in the SAT-I⫺/⫺ mice at P15. The area of the tunnel of Corti is reduced, and the slits between the hair cells and the pillar cells, which had been obvious at P14, are also abolished in the organ of Corti by P17. (Scale bar: 50 ␮m.) (D) Scanning electron microscopy images of the organ of Corti in SAT-I⫹/⫹ and SAT-I⫺/⫺ mice at P14. These mice show 3 rows of OHCs and 1 row of IHCs in the apex, middle, and base of cochlea. Arrow points to a gap in the regular array of OHCs, suggesting that this cell type is degenerating. (Scale bar: 10 ␮m.) (E) The OHC counts in SAT-I⫹/⫹ (white bar) and SAT-I⫺/⫺ (black bar) mice at P14. The OHCs in SAT-I⫺/⫺ mice were partially lost in every turn at P14. Hair cell counts are calculated as mean number of hair cells per 100 ␮m. Data represent the means ⫾ SE. *, P ⬍ 0.05; by Student’s t test or the Aspin–Welch test. Representative data for each littermate group (⫹/⫹, n ⫽ 5; ⫺/⫺, n ⫽ 10) are presented.

as a substitute for GM3, because of the distinctive staining of GM3 in SV of adult wild-type mice. Our results, summarized in Fig. S3, highlight 2 major findings. First, SAT-I null mice exhibit severe hearing loss near the onset of hearing, with no effect on endolymph EP or K⫹ concentrations and with an elapsed time to complete loss of hearing that parallels the selective degeneration of the organ of Corti. Second, in wild-type mice, GM3 and the other major gangliosides are expressed in nearly all regions of the cochlea, including the organ of Corti, at the early phase of hearing maturation. Considering these points, we propose that the expression of gangliosides, especially GM3, plays a decisive role in the functional organization and prevention of degeneration of the organ of Corti. The exact mechanism of deafness in SAT-I null mice remains to be determined. However, we theorize that the acute deterioration of hearing ability and degeneration of the organ of Corti might originate from multiple (complex) dysfunctions. The loss 9486 兩 www.pnas.org兾cgi兾doi兾10.1073兾pnas.0903279106

of GM3 and complex gangliosides could affect the electrostatic environment of the outer leaflet of plasma membranes, especially in the membrane microdomains (lipid rafts) of hair cells, leading to a loss of function in complexes that act through GM3–protein interactions. GM3 is capable of interacting with and modulating the function of various proteins, including growth factor receptors, Src family kinases, tetraspanins (CD9, CD81, and CD82), and ion channels and cell adhesion molecules in membrane microdomains (for review, see refs. 26 and 27). We also have demonstrated that GM3 dissociates the caveolin-1– insulin receptor functional complex in microdomains during insulin resistance (28). Some proteins known to be important for inner ear functions, such as prestin (29) and the IGF receptor (30), have been suggested to exist in microdomains. Such multiple GM3–protein interactions may be essential for inner ear function. Investigations into the molecular mechanism(s) of the degeneration of the organ of Corti in SAT-I deficient mice are now in progress in our laboratories. Yoshikawa et al.

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Behavioral Studies. Based on a method described previously (8), startle responses were measured by using the SR-LAB Startle Response System (San Diego Instruments). Motor coordination and balance were measured by using the rota-rod test. For detailed methods, see SI Methods.

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Fig. 5. Analysis of gangliosides in mouse inner ear. (A) Thin-layer chromatograms of acidic (Right) and neutral (Left) glycolipids from the inner ear of 6to 8-week-old SAT-I⫹/⫹, SAT-I⫹/⫺, and SAT-I⫺/⫺ mice. In SAT-I⫹/⫹ and SAT-I⫹/⫺ mice, GM3, GM1, GD1a, GD1b, and GT1b are expressed in the inner ear, but these gangliosides are absent in the SAT-I⫺/⫺ mice, and the o-series GM1b and GD1␣ are compensatorily expressed because of blockage of GM3 synthesis (Fig. 1). (B) Localization of GM3 and GT1b in the cochlea of SAT-I⫹/⫹ mice at P3, P14, and P42. At P3, GM3 and GT1b are expressed in almost all regions of the cochlea. At P14, both are expressed in the organ of Corti; however, GM3 is expressed in the SV and SG but, in contrast, GT1b is expressed in regions distinct from GM3, such as the spiral ligament and spiral limbus. At P42, GM3 is selectively expressed in the organ of Corti, SV, and SG. In contrast, GT1b is localized in the organ of Corti, spiral ligament, spiral limbus, and RM. (Scale bars: 200 ␮m.) Lower shows the enlarged pictures of P14 (2-fold magnification).

This previously uncharacterized and progressive work into the functional involvement of gangliosides in the auditory system opens up a new dimension of research and may facilitate its clinical application for sensorineural hearing loss. Methods Mice. GM3 synthase null (SAT-I⫺/⫺) mice were generated as described previously (7). In brief, the second exon of the 7-exon GM3 synthase gene was deleted and replaced with the neomycin resistance gene (Fig. S1 A). Mutant mice were maintained on a C57BL6 –129/SvJ mixed background by heterozygous mating to generate littermate controls. All animal studies were approved by the committees of institutional review boards (IRBs) of Tohoku Pharmaceutical University, Fukuoka University, and Kyushu University. For detailed breeding scheme and primer sequences used for amplification, see SI Methods.

Yoshikawa et al.

BAEPs and ECochG. The head and neck hair of each mice was shaved, and the skin was cleansed with antiseptics. To obtain an adequate anesthesia level, a mixture of ketamine hydrochloride (50 mg/kg) and xylazine hydrochloride (10 mg/kg) was administered intramuscularly while preserving spontaneous respiration; intramuscular injections were repeated as necessary. Each animal was set on a fixed apparatus (Narishige), and body temperature was maintained at 37 °C by using a heating pad. BAEPs were recorded by using 4 stainless steel electrodes s.c. placed at the vertex (positive), behind the bilateral ears (negative), and at the lower back (ground). For 8-week-old mice, click sound stimuli (duration: 0.1 ms; repetitive rate: 10.1 Hz) were delivered from earphones through a hollow ear bar sealed to the external auditory meatus. The maximum intensity of the click sound stimuli was 85 dBSPL, which was calibrated by a microphone (B & K GmbH) and a sound-level meter (B & K GmbH). The ear bar was used both for fixing the mouse head and for access to the external ear canal to administer sound stimuli. For the infant mouse, around the onset of hearing, tone burst stimuli (rise/fall time: 0.2 ms; plateau time: 1 ms; repetitive rate: 10.1 Hz) were applied from the speaker (PT-R100; Pioneer) by using a function generator (DPS-725; Dia Medical) for which the system could provide 100 dBSPL at each frequency. Evoked responses were filtered with a band pass of 100 Hz to 3 kHz, and the average of 500 sweeps was recorded by using a signal processor (Neuropack Micro; Nihon Kohden). Hearing threshold was determined by visual detection using the lowest intensity of sound stimuli yielding a consistent appearance of at least 1 BAEP peak. Subsequent to the BAEP study and never removing the subject from the fixed position, ECochG was performed. For detailed methods, see SI Methods. Measurement of EP and Potassium Concentration. Subsequent to the BAEP study, EP and K⫹ concentrations in 6- to 8-week-old mice were measured as described previously (31). Briefly, the tympanic bulla of mice anesthetized by the mixture of xylazine and ketamine was removed by ventral approach, leaving the tympanic membrane intact. A double-barreled microelectrode (a pair of conventional and K⫹-selective microelectrodes) was inserted into the basal turn scala media after thinning the bone over the stria vascularis and picking a small hole (⬍30 ␮m) under a surgical microscope (OPMI 1; Carl Zeiss). The K⫹-selective, double-barreled microelectrode was fabricated by using a K⫹-selective liquid ion exchanger (IE-190; WPI). EP and potassium potential were recorded with a dual electrometer (FD-223; WPI) against an Ag/AgCl reference inserted under the skin, and they were monitored with a Mac Lab 8s (ADInstruments). The potassium concentration in the endolymph ([K]e) was calculated according to the following equation (25): [K]e ⫽ 150 ⫻ 10(E150–EKe)/␣, where E150 is the voltage from the K⫹-selective microelectrode immersed in a 150 mM KCl solution; EKe is the difference between the EP and the K⫹selective microelectrode, and ␣ is the slope constant of the K⫹-selective microelectrodes. K⫹-selective microelectrode was calibrated immediately after withdrawal from the cochlea. Histological Examination. After the studies described above, animals were kept under deep anesthesia and briefly perfused, via a cannula inserted into the left ventricle, with physiological saline and, subsequently, with 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M phosphate buffer (PB; pH 7.4). Each cochlea was rapidly removed from the temporal bones through microdissection under a stereomicroscope, and the round and oval window membranes were opened. The cochleae were perfused by direct injection of a fixative through the round and oval windows, and then the tissues were immersed in the same fixative overnight at 4 °C. Decalcification was performed by immersing the specimens in 10% ethylenediamine tetraacetic acid in Tris buffer (pH 7.4) for 7–10 days at 4 °C. After a wash in PB, the specimens were cryoprotected in 20% sucrose in PB for 2 h, and subsequently in 40% sucrose in PB overnight at 4 °C. The tissues were infiltrated in a mixture of equal parts 40% sucrose in PB and Tissue-Tek OCT embedding compound (Sakura Finetechnical Co.) overnight at 4 °C (32). The specimens were transferred to an embedding mold (fabricated from household aluminum foil) containing the same infiltration reagent, then rapidly frozen. Sections (5 ␮m thick) were cut by using a Frigocut cryostat (Leica Microsystems) and were mounted on silane-coated microslides (Muto Pure Chemicals). Tissue sections were then exposed to hematoxylin and eosin stain or were immunostained with a series of anti-ganglioside antibodies (GMR6, antibody for GM3; GMR5, antibody for GT1b; Seikagaku Corp.), followed by FITC-conjugated secondary anti-IgM antibody (Vector Laboratories). PNAS 兩 June 9, 2009 兩 vol. 106 兩 no. 23 兩 9487

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A

Scanning Electron Microscopy. Cochleas were dissected to expose the organ of Corti and were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 30 mM Hepes buffer (pH 7.3) for 3 h at 4 °C. Postfixation was performed by alternating tissues in 1% osmium tetroxide in 0.1 M sodium cacodylate buffer for 1 h at 4 °C. Samples were dehydrated through a graded series of ethanol, substituted with t-butyl alcohol, freeze-dried, coated with platinum, and examined in a JEOL JSM-T330A scanning microscope.

(1:1, then 1:2, vol/vol). Preparation of acidic glycolipids and HPTLC were performed as described elsewhere (33, 34). Glycolipids were detected by using an orcinol–sulfuric acid reagent.

GSL Analysis. Inner ears were dissected from mice (SAT-I⫹/⫹, SAT-I⫹/⫺, and SAT-I⫺/⫺) after perfusion with PB, then lyophilized. Dried tissues were crushed, and lipids were extracted from the tissues by using chloroform and methanol

ACKNOWLEDGMENTS. We thank A. Kakigi for instruction for the endocochlear K⫹ measurement, and A. Suzuki, Y. Nomura, Y. Nagai, and E. A. Sweeney for critically reading the manuscript. This work was supported by grants from Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency, and the Ministry of Education, Culture, Sports, Science and Technology to promote multidisciplinary research projects.

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