Mouse homolog of KIAA0143 protein: hearing deficit induces specific changes of expression in auditory brainstem neurons

Molecular Brain Research 128 (2004) 131 – 140 www.elsevier.com/locate/molbrainres

Research report

Mouse homolog of KIAA0143 protein: hearing deficit induces specific changes of expression in auditory brainstem neurons Yumi Munemotoa,b, Takeshi Houtania, Masahiko Kasea, Satoru Sakumaa, Kazuyasu Babab, Toshio Yamashitab, Tetsuo Sugimotoa,* a

Department of Anatomy and Brain Science, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan b Department of Otolaryngology, Kansai Medical University, Moriguchi, Osaka 570-8506, Japan Accepted 13 June 2004 Available online 21 July 2004

Abstract Hearing deficit induced by mechanical cochlear damage, intense noise or ototoxic drugs produces a variety of structural and functional changes in the inner ear and the auditory brainstem. In the present study, we identified a novel gene that has activity dependent plasticity in the superior olivary complex by using suppression subtractive hybridization. We cloned a gene that encodes mouse homolog of KIAA0143 protein, one derived from a series of unidentified human genes. This gene termed mKIAA0143 shows differential expression of mRNA in the lateral superior olive between mice with hearing deficit and those with normal hearing ability. The mRNA thus obtained encodes a unique membrane-bound protein that consists of 819 amino acids. The gene locus was mapped using genomic DNA databases to the mouse chromosome 15D1. Green fluorescent protein-tagged mKIAA0143 was expressed in COS-1 cells. It was amply seen in the cellular plasma membrane. D 2004 Elsevier B.V. All rights reserved. Theme: Sensory systems Topic: Auditory systems: central anatomy Keywords: Suppression subtractive hybridization; Differential expression; Superior olivary complex; Olivocochlear neuron; COS-1 cell; Hearing ability

1. Introduction Hearing ability is considered to reflect integral soundresponsive activities of cells in the inner ear as well as in central auditory trajectories. In the auditory brainstem stations, plastic changes may take place in response to sound stimulation or reduction of sound input into the inner ear (reviewed in Refs. [15,19]). Major changes include altered electrical properties with or without changing morphologies of cell and processes. Phenotypical changes detectable at cellular levels have been reported; most of these are related to transmitters in the cochlea and central auditory trajectories [1,22–24]. Transneuronal changes indicating reduced fibers and synapses of centrally placed remote neurons are clearly * Corresponding author. Tel.: +81 6 6993 9420; fax: +81 6 6995 2708. E-mail address: [email protected] (T. Sugimoto). 0169-328X/$ - see front matter D 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbrainres.2004.06.022

shown to occur following loss of sound input [2,10,14, 15,19]. Several attempts have been made to discover expression of novel genes underlying some of the disease processes in the auditory pathway including recently developed technology using cDNA microarray [4,17]. In the present study, we identified a novel gene that has activity dependent plasticity in the auditory brainstem of mice with hearing deficit by using suppression subtractive hybridization, alternative sensitive method for isolating specific genes based on suppression polymerase chain reaction (PCR) [7,8]. Combined with high-fidelity expression analysis, this method contributes largely to accurate identification of differentially expressing genes in particular cases (see Ref. [12] for further references). In our present study, specific changes of gene expression were confirmed in brainstem neurons. Some additional properties of the expressed protein were shown at the molecular and cellular levels.

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2. Materials and methods 2.1. Animal and measurement of auditory brainstem response All experiments were performed in compliance with the NIH guidelines for the care and use of laboratory animals. Ten-week-old male ICR mice were used in the present study. Hearing ability was measured with auditory brainstem response (ABR) before surgical manipulation [18,21]. Mice were anesthetized with a combination of 5% ketamine and 2% xyladine (2:1, 0.1 ml/animal, i.p.). Hearing thresholds were measured in bilateral ears after applying tone bursts (rise and decay, 1 ms; rate, 10 Hz; duration, 500 times) with sound frequencies of 4, 12 and 20 kHz. Thresholds of ABR correspond to the lowest intensities of the sound below which significant ABR waves disappear. The mice whose bilateral auditory thresholds were below 40 dB sound pressure level (SPL) at all sound frequencies were determined to have normal hearing ability and they were used for the following studies. 2.2. Surgical procedure 2.2.1. Removal of malleus The malleus was removed bilaterally through ear canals under general anesthesia. After the operation, the mice showed a threshold shift of 20–30 dB SPL by ABR measurement. They were assumed to have conductive hearing loss. 2.2.2. Ablation of cochlea Under deep anesthesia, postauricular skin was incised and the tympanic membrane was visualized. After removal of the malleus and the incus, the cochlea was ablated mechanically. The tympanic cavity was filled with the gelfoam and the wound was closed with cyanoacrylate glue. The postoperative hearing ability was measured by ABR. All the mice showed no response to sound stimulation at 110 dB SPL. 2.3. RNA extraction and purification Total RNA was extracted from mouse brainstem (n=3) by the guanidium thiocyanate method with the yield of 500– 600 Ag total RNA. The quality of RNA was checked on a denaturing agarose gel. Messenger RNA was prepared by using Oligotex-dT30 Super (Takara Bio, Otsu, Japan), which yields about 20 Ag poly(A)+ mRNA. 2.4. Suppression subtractive hybridization Total RNA was extracted from the brainstem of mice with hearing deficit at 5 days after bilateral malleus removal (XX) and age-matched mice with normal hearing ability (CC). Reverse transcription was performed on poly(A)+

mRNA purified from XX and CC total RNAs. Doublestrand cDNA was then synthesized. The cDNA was digested with RsaI and ligated to the adapter 1 and adapter 2 supplied in the PCR-select cDNA subtraction kit (Clontech Laboratories, Palo Alto, CA, USA) [8]. Two-directional (XX–CC and CC–XX) subtractive hybridization was performed between XX and CC. The PCR products were cloned into pGEM-T Easy vector (Promega, Madison, WI, USA), and transfected with E. coli. 2.5. Screening of the subtracted cDNA A total of 180 clones were randomly picked and placed in colony arrays (XX–CC, 100 clones; CC–XX, 80 clones). These plasmid DNAs were purified and screened with dot hybridization to identify differentially expressing clones. In brief, the DNAs were diluted to 10 pg/Al with a dilution buffer and 1 Al of the aliquot was placed on nylon membranes (180 spots/sheet). Hybridization was carried out with four kinds of digoxigenin (Dig)-labeled DNA probes (CC–XX, XX–CC, CC unsubtracted and XX unsubtracted DNA probes). Hybridized membranes were reacted with alkaline phosphatase-labeled anti-Dig antibody. The membranes were then incubated with the enzyme reaction mixture containing nitroblue tetrazolium and 5bromo-4-chloro-3-indolyl-phosphate. Each spot on dot hybridization membranes was captured as image files on Photoshop 6.0. Spot image was then analyzed on NIH Image 1.62. Spot density was converted to gray-scale values by adjusting white balance on the membrane. Black sample spots served as the maximal limit of the signal intensity for the calibration of gray-scale values. For the purposes of in situ hybridization screening of eight XX–CC clones and nine CC–XX clones, we used Diglabeled riboprobe and carried out the hybridization according to the protocol described in Section 2.8. 2.6. RT-PCR For the purposes of cDNA cloning, total RNA was extracted from 10-week-old male C57BL6J mouse brain by the guanidium thiocyanate method. Messenger RNA was prepared by using Oligotex-dT30 Super (Takara Bio). A 0.1-Ag aliquot of mRNA obtained from mouse brain was reverse-transcribed with oligo(dT)30 as a primer and 100 units of ReverTra Ace (Toyobo, Osaka, Japan). A 20 Al of reaction mix was incubated for 1 h at 42 8C. One microliter of the RT product was amplified by PCR. For PCR primers, F1 (forward) 5V-gtcatgctgcggtcggcgcggctcgcgctt-3V (30mer) and R1 (reverse) 5V-catgtataagcattagtgagacaaatgagtcccgt-3V (35-mer) were synthesized. F1 and R1, respectively, correspond to the nucleotide number 123– 152 and 5094–5128 of mKIAA0143 cDNA. PCR mix (10 Al) contained 1 Al RT product, 1 AM forward and reverse primers, 0.25 unit of Ex Taq Hot Start Version (Takara Bio). PCR parameters used here are as follows: preheat, 94 8C for

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2 min; cycles, first, 94 8C for 0.5 min; second, 68 8C for 0.5 min; third, 72 8C for 5 min; a total of 30 cycles. PCR product was electrophoresed and analyzed on 1% agarose gel. Amplified fragments were ligated to pGEM-T Easy vector (Promega) and sequenced with ABI PRISM BigDye Terminator Cycle using Sequencing Ready Reaction Kits (Applied Biosystems, Tokyo, Japan). Subcloning yielded the plasmid (pmKIAA0143) harboring a 751-base pair (bp) SacI-digested fragment of mKIAA0143 that shows particularly low similarity to known sequences. This fragment was used for riboprobe synthesis and the resulting sense and antisense riboprobes were subjected to Northern blot and in situ hybridization analyses. 2.7. Northern blotting Total RNA was obtained as described in RT-PCR. After gel electrophoresis, the RNA on the agarose gel was capillary-blotted to GeneScreen nylon membrane (NEN Research Products, Boston, MA, USA) in 10 standard saline citrate (SSC). The membrane was hybridized with Dig-labeled antisense riboprobes (1 Ag/ml) in hybridization buffer overnight at 55 8C. After hybridization, the membrane was washed with 2, 0.5 SSC at room temperature and 0.1 SSC at 55 8C for 30 min. Then, the membrane was blocked with 1% blocking reagent in Dig buffer 1 (0.1 M Tris–HCl pH 7.5/0.15 M NaCl) for 30 min. It was incubated with alkaline phosphatase-labeled anti-Dig antibody (Roche Diagnostics, Mannheim, Germany; 1:500 dilution) for 30 min at room temperature and subsequently incubated with the reaction mixture containing nitroblue tetrazolium and 5bromo-4-chloro-3-indolyl-phosphate for 30 min at room temperature. 2.8. In situ hybridization In situ hybridization with Dig-labeled riboprobe was performed with the procedure described in detail previously

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Fig. 2. Signal intensity of spots in dot hybridization. Spots with high signal intensity are measured on the hybridization membranes for screening of differentially expressing clones. Blast search results are as follows. XX–CC clones (solid bar) (15) KIAA0143 homolog; (18) adenosine deaminase, RNA-specific, B1; (20) latent transforming growth factor beta binding protein 4; (33) eukaryotic translation initiation factor 2, subunit 2 (beta); (40) mouse mRNA for neurobeachin; (52) mouse mRNA for mKIAA0567 protein; (73) AT-rich interactive domain 4B (Rbp1-like) (Arid4b), rat retinoblastoma-binding protein 1-related protein homolog; (28) mouse 28S ribosomal RNA. CC–XX clones (open bar) (25) hypothetical protein UniGene Cluster Mm. 237095; (33) UniGene Cluster Mm. 207496 protein kinase C, beta; (38) NADH dehydrogenase (ubiquinone) 1 alpha subcomplex 10; (39) CLIP-170-related protein homolog; (47) ATPase, aminophospholipid transporter (APLT), class I, type 8A, member 1; (52) UniGene Cluster Mm. 326825 transcribed sequences; (59) rat pannexin 2; (68) mouse ADP-ribosylation factor related protein 1; (76) protein phosphatase 4 regulatory subunit 2 homolog.

[13]. After linearization of the plasmid harboring 751-bp SacI-digested fragment of mKIAA0143, it was subjected as a template to riboprobe synthesis in which in vitro transcription was done with SP6 or T7 RNA polymerase in the presence of Dig-11-UTP. Mice were perfused with a fixative containing 4% paraformaldehyde and 2% glutaraldehyde in 0.12 M sodium phosphate buffer (pH 7.4). The brain was saturated with sucrose and frozen coronal sections of 40-Am thickness were cut on a cryotome. Free-floating sections were treated with hydrochloric acid, Triton X-100

Fig. 1. ABR wave patterns before (a) and 2 days after malleus removal (b). Sound intensity is shown in dB SPL at the left side of the wave. Thresholds of ABR shift from 25 dB SPL (a) to 55 dB SPL (b).

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and proteinase K, and they were incubated with a prehybridization solution. Hybridization was carried out overnight at 55 8C. The hybridization mixture contained a Dig-labeled RNA probe. Hybridized sections were rinsed with 4, 0.5, 0.25 and 0.125 SSC at room temperature. The hybridized sections were processed for Dig immunostaining. Briefly, they were conjugated with alkaline phosphatase-labeled anti-Dig antibody for 2 h at room temperature and subsequently incubated with the enzyme reaction mixture containing nitroblue tetrazolium and 5bromo-4-chloro-3-indolyl-phosphate for 2 h at room temperature. Adjacent sections were stained with 0.1% cresyl violet and utilized for cell count and cytoarchitectonic studies. The hybridized sections were viewed under Nikon Eclipse microscope and the areas including hybridized neurons were captured as image files on Photoshop 6.0. Using gray-scale conversion mode, densitometric values for unit area were obtained. Statistical analysis of these values was made on GraphPad Prism version 3.0c. Comparisons were analyzed by ANOVA followed by post-hoc Tukey’s MCT and Bonferroni’s MCT.

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was then reacted with horseradish peroxidase-labeled goat anti-mouse IgG and finally colored with diaminobenzidine tetrahydrochloride and hydrogen peroxide. 2.11. Information processing The sequence of cDNAs derived from subtractive hybridization was analyzed with computer program BLAST search (http://www.ncbi.nlm.nih.gov/BLAST/) of NCBI. Multiple alignment and Kyte and Doolittle hydrophobicity plot for constituting amino acids were performed with CLUSTALW (http://hypernig.nig.ac.jp/homology/clustalw.shtml) and GENETYX (SDC, Tokyo, Japan), respectively. Chromosomal localization of the gene was examined with NCBI Map Viewer (http://www.ncbi.nlm.nih.gov/mapview/). Potential residues for transmembrane stretches were analyzed with reference to human KIAA0143 (NiceProt View of SwissProt: accession Q14156). Prosite MotifFinder (http://au. expasy.org/prosite/) served for the characterization of protein motifs.

3. Results 2.9. GFP fusion protein 3.1. Measurement of ABR Using the pEGFP expression system (Clontech), the mKIAA0143 cDNA (number of amino acid and calculated molecular weight of the protein; 819 amino acid, 92.6 kDa) was fused with EGFP cDNA (239 amino acid, 27 kDa) utilizing a linker (17 amino acid, 1.9 kDa). The cDNA construct was transfected into COS-1 cells by PolyFect transfection reagent (QIAGEN, Tokyo, Japan). The expressed protein (1075 amino acid, 121 kDa) was expected to contain EGFP on the carboxyterminal side. Green fluorescence derived from this fusion protein was observed with brightness in COS-1 cells after 1–3 days. Fluorescent cells were photographed with a Nikon Optiphot fluorescence microscope using an FITC excitation filter. 2.10. Western blotting The mKIAA0143-EGFP fusion protein described above was purified from COS-1 cells and subjected to Western blot following processes of SDS-PAGE and immunostaining of the membrane (Clear Blot Membrane-p; ATTO, Tokyo, Japan) with anti-GFP antibody (GFP, B-2, sc-9996; Santa Cruz Biotechnology, Santa Cruz, CA, USA). The membrane

All untreated mice showed thresholds of ABR below 40 dB SPL. On the other hand, following ear operation, thresholds of ABR shifted in all the operated mice. After removal of malleus, ABR waveform appeared only with intense sound (Fig. 1). After cochlear ablation, thresholds of ABR became elevated to greater than 100 dB SPL. 3.2. Suppression subtractive hybridization and BLAST search A total of 100 XX–CC subtracted clones were obtained from hearing deficit cDNAs subtracted by normal hearing ability cDNAs, and 80 CC–XX subtracted clones were also obtained. These 180 clones were screened with dot hybridization to identify differentially expressing clones. A total of 27 clones were identified as differentially expressing and all of them were sequenced. Of these, six clones belong to the same gene and four clones encode chimeric proteins. Finally, we could identify 17 cDNA fragments by the BLAST search (eight XX–CC clones and nine CC–XX clones, Fig. 2). These clones were screened with in situ

Fig. 3. Diagram illustrating nucleotide and amino acid sequences of mKIAA0143 (deposited in GenBank databases under accession number AB158474). Sites of F1 PCR primer (dextral arrow) and in-frame stop codon (double underline) are shown in 5V-noncoding region. Underlined in the coding region are four membrane-spanning stretches (TM1–4), each consisting of 21 amino acids and corresponding to amino acid positions 312–332 (TM1), 399–419 (TM2), 550– 570 (TM3) and 595–615 (TM4). Potential N-glycosylation sites are marked with squares. Potential phosphorylation sites are shown with numbered circles around serine/threonine/tyrosine residues designating cAMP phosphorylation site (1), protein kinase C phosphorylation site (2), casein kinase II phosphorylation site (3) and tyrosine kinase phosphorylation site (4). Underlined in the 3V-noncoding region is the fragment corresponding to the subtractive hybridization product XX–CC 15. Sites of R1 PCR primer (sinistral arrow) and two polyadenylation signals (broken line) are also marked at the 3V-terminal region.

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hybridization. One cDNA (XX–CC 15) was selected based on in situ hybridization that visualized differential mRNA expression within the auditory brainstem. 3.3. Isolation of full-length cDNA The 628-bp fragment (XX–CC 15) was found to carry cDNA highly homologous to human KIAA0143 protein (5292-bp; GenBank accession, XM_035825.3) in nucleotide sequence. Mouse cDNAs related in part to human KIAA0143 protein include 1688-bp spinal cord cDNA (AK039483.1) and 3980-bp RIKEN C0006C10 cDNA

(BC007482.1). On the base of such knowledge, a mouse homolog of human KIAA0143 protein was cloned (Fig. 3). We used reverse transcription–polymerase chain reaction (RT-PCR; see Section 2.6 for detail) with poly(A)+ mRNA from C57BL6 mouse brain and with a forward and a reverse primers to obtain an RT-PCR product (5006-bp, Fig. 4c). This fragment subserved the construction of a full-length cDNA (5225-bp). Information on additional nucleotide sequence flanking 5006-bp fragment was obtained by computer search for mouse cDNA and EST databases. The cDNA clone has an open-reading frame for translating 819 amino acids (Fig. 3). Northern blot analysis showed that

Fig. 4. (a) Alignment of amino acid sequence of mKIAA0143 and four related proteins. Analysis with CLUSTALW (http://hypernig.nig.ac.jp/homology/ clustalw.shtml). Residues cited here, species and GenBank accession are shown in parenthesis: mKIAA0143 (16–206, mouse, AB158474), DMcmp44E (15– 204, Drosophila melanogaster, NP_724711), YQD3_CAEEL (14–207, Caenorhabditis elegans, Q09263), NP_198037 (33–229, Arabidopsis thaliana, NP_198037), YM62_YEAST (13–217, Saccharomyces cerevisiae, Q03653). Boxes indicate three or more identical residues. The alignment indicates a considerable number of amino acids located in the aminoterminal region of each protein are conserved. (b) Kyte and Doolittle hydrophobicity profile of mKIAA0143 protein. Analysis was performed by GENETYX program with a window size of 10 amino acids. A series of hydrophobic stretches (bar) provide this protein with four membrane-spanning regions. (c) RT-PCR analysis of C57BL6J mouse brain poly(A)+ RNA with primers derived from the mKIAA0143 sequence (Fig. 3). A 5.0-kbp fragment (arrow) was amplified. Marker (right lane) indicates E/HindIII fragments comprising (size in bp) 23130, 9416, 6557, 4361, 2322, 2027, 564 and 125. (d) Northern analysis of total RNA from mouse brain by using a riboprobe. A major transcript with maximal length of 5.9 knt (arrow) was successfully hybridized. Arrowheads indicate positions of 28S and 18S ribosomal RNA. Hybridization with Dig-labeled riboprobe generated from 751-bp SacI-digested fragment of mKIAA0143 cDNA.

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a riboprobe derived from the 751-bp SacI-digested fragment successfully hybridized with major RNA species of 5.9 knt in length (Fig. 4d). The novel cDNA (mKIAA0143; deposited in GenBank databases under accession number AB158474) is attributed to a mouse homolog of human KIAA0143 protein. Nested deletions of mKIAA0143 were made and DNA sequencing was carried out on both strands. 3.4. Structure of mouse homolog of human KIAA0143 protein As shown in Fig. 3, the translation initiation site was assigned to the first ATG triplet that is located downstream of a stop codon found in-frame. In the coding region, potential residues for transmembrane stretches were analyzed with reference to human KIAA0143. As shown in the hydrophobicity plot (Fig. 4b), this protein contains four membrane-spanning regions, which was enforced by sequence homologies with some membrane-bound proteins in other species (Fig. 4a). In the coding region, membranespanning stretches (TM1–4) appear to divide the remaining hydrophilic regions into five fragments. The first fragment is the longest of all. Protein motif analysis shed more light on membrane topology. Potential phosphorylation sites are populated in the first and the fifth fragments. An Nglycosylation site is seen in the second and fourth fragments possibly assuming them on the extracellular side. A translation termination codon appeared in the frame following the codon specifying tyrosine. The sequence corresponding to the subtractive hybridization product (XX–CC 15) was found in the 3V-untranslated region. Polyadenylation signals were seen immediately upstream of poly(dA) tract. As stated above, RT-PCR analysis indicated that mRNA comparable in length to the major amplified product is at least contained in mouse brain (Fig. 4c) and the size of

Fig. 6. Expression of mKIAA0143 mRNA in the superior olivary complex of the control mouse. Hybridization signal is primarily seen in the shell region of the LSO consisting of large neurons. In contrast, the LSO core that consists of small neurons shows insignificant amount of the signal. Hybridization signal was also seen in the MNTB, LNTB, VNTB, SPON and MSO. In situ hybridization displayed in Figs. 6–8 was done with Diglabeled antisense riboprobe generated from 751-bp SacI-digested fragment of mKIAA0143 cDNA. Scale bar=100 Am.

mKIAA0143 mRNA has been estimated about 5.9 knt by Northern blot (Fig. 4d), indicating that the nucleotide sequence of the cDNA (Fig. 3) corresponds to that of almost full-length mRNA. Computer analysis revealed that the gene locus for mKIAA0143 is mapped to mouse chromosome 15D1. 3.5. Expression of GFP fusion protein in COS-1 cells The cDNA fragments encoding mKIAA0143 and EGFP were fused in tandem into a mammalian expression vector. The resulting fusion protein was transiently expressed in COS-1 cells and could be observed for 1–3 days. Strong green fluorescence was noted at the cell membrane as well as in some intracellular elements (Fig. 5a). 3.6. Western blot of GFP-tagged mKIAA0143 protein The fusion protein which comprises 1075 amino acid (121 kDa) was purified from COS-1 cells. Bands specifying the fusion protein were confirmed in the immunoblot (Fig. 5b). Major band (about 120 kDa) may be derived from GFPtagged mKIAA0143 protein. A low-molecular-weight mass (about 27 kDa) may represent GFP judging from the presence of Kozak consensus translation initiation site just upstream of GFP-coding region.

Fig. 5. Expression of mKIAA0143-GFP fusion protein in COS-1 cells at the second post-transfection day. (a) Strong green fluorescence. Note that cellular plasma membrane along with some intracellular elements has enhanced green fluorescence. Scale bar=10 Am. (b) Western blot of the COS-1 cells analyzed with anti-GFP antibody. Bands indicating the fusion protein (121 kDa) and unfused GFP (about 27 kDa) are seen.

3.7. In situ hybridization of mKIAA0143 mRNA in the auditory brainstem For in situ hybridization, brainstem sections from mice with normal hearing ability were first examined as controls. They were compared with those from mice operated for

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Fig. 7. Expression of mKIAA0143 mRNA in the cochlear nuclear complex (a), lateral lemniscal nuclei (b) and inferior colliculus (c) of the control mouse. Hybridization-positive neurons are sparsely distributed in these nuclei. (d) Control section through the LSO that was hybridized with sense riboprobe showing no appreciable staining. Scale bar=200 Am.

malleus removal and cochlear ablation. In all groups, hybridization signal derived from antisense riboprobe was populated in neurons in the superior olivary complex (Fig. 6). It was seen less numerously in the cochlear nucleus (Fig. 7a), the lateral lemniscal nuclei (Fig. 7b) and the inferior colliculus (Fig. 7c). In all the other brainstem structures including the reticular formation, hybridization-positive neurons were scarce and dispersed among unlabeled neurons. Hybridization signal was not detected when sense riboprobe was hybridized (Fig. 7d). In the mice with normal hearing ability, the hybridization-positive neurons were regionally distributed in the superior olivary complex (Fig. 6). They were predominant in the periolivary region dorsal and lateral to the lateral superior olive (LSO), and also seen in the medial nucleus of the trapezoid body (MNTB), the lateral nucleus of the

trapezoid body (LNTB), the ventral nucleus of the trapezoid body (VNTB) and the medial superior olive (MSO). The hybridized neurons found in the LNTB, VNTB, MNTB and MSO are mainly composed of medium-sized neurons, and those in the periolivary region encircling the LSO dorsally and laterally were large, suggesting regional expression associated with heterogeneous cell groups. Some hybridized neurons were also present in and around the superior paraolivary nucleus (SPON). They have medium-sized to large cell bodies. The small neurons in the LSO core lacked distinct labeling and displayed only trace amount of the hybridization signal. Expression of mKIAA0143 mRNA in the LSO in the operated animals differs from normal controls. In the mice that underwent malleus removal and allowed to survive for 1 day, mKIAA0143 mRNA was still expressed in the LSO

Fig. 8. Changes of mKIAA0143 mRNA expression in the LSO 1 day (a) and 5 days (b) after malleus removal and 5 days after cochlear ablation (c). In the mice that underwent malleus removal (a, b), mKIAA0143 mRNA expressed in the shell neurons (arrowhead in a) surrounding the LSO core disappeared gradually over the observation periods (1 and 5 days). In the mice with cochlear ablation (c), such decrease of expression in the shell neurons was reproduced. On the other hand, the small-celled LSO core increased neuronal expression of mKIAA0143 mRNA (arrow) to the maximum at 5 days following surgery (b, c). These changes of expression were similarly seen on both sides of the LSO. Scale bar=100 Am.

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Fig. 9. Levels of expression of mKIAA0143 mRNA in the LSO core and shell regions. In control animals and those operated for malleus removal (1 and 5 days) and cochlear ablation, levels of expression are quantified using gray-scale images of areas including hybridized neurons (n=9–11). ANOVAs for each group are all significant. Post-hoc comparisons between operated groups and control are shown to be significant by asterisk ( pb0.01).

shell region (arrowhead, Fig. 8a). At postoperative 5 days of malleus removal and cochlear ablation, its expression reduced much further (Fig. 8b,c). On the other hand, the small neurons in the LSO core (arrow, Fig. 8b,c) showed distinct hybridization signal. We confirmed these findings in the LSO bilaterally. 3.8. Changes of mKIAA0143 mRNA in the LSO core and shell regions Levels of expression of mKIAA0143 mRNA were quantified in the LSO core and shell regions (Fig. 9) by measuring intensity of hybridization signal per unit area in the control, 1 and 5 days of malleus removal and cochlear ablation. Compared with control, a notable increase of the expression was observed in the core region in both types of hearing deficit. Conversely, expression levels in the hearing deficit were significantly lowered in the shell region. Nissl-stained adjacent sections showed that neither core nor shell neurons showed any difference on the cell count between hearing deficit and control. Notable changes of expression could not be detected in other structures of the brainstem.

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elements. The intracellular localization may relate to an association with the cellular secretion transport pathway. Alignment study showed that this mouse protein shares some features with four related proteins (Fig. 4). One of these proteins isolated and studied in detail in Drosophila (cmp44E) has been shown that it is required for cell viability [9]. In situ hybridization analysis in mice with hearing deficit revealed significant changes of mKIAA0143 mRNA expression in the superior olivary complex. Although we used malleus-removal animals for subtraction hybridization, mice with two different kinds of ear surgery have shown similar results on in situ data, suggesting that such changes in gene expression may have arisen from hearing deficit and little affected by the difference in the mode of deafness. Large neurons surrounding the LSO were called bshellQ neurons by Vetter and Mugnaini [25]. Shell neurons are one of the populations that constitute lateral olivocochlear (LOC) neurons. The enrichment of mRNA in large cells of the mice with normal hearing ability and its depletion in those with hearing deficit suggest a role of mKIAA0143 protein in close association with the normal function of LOC system. Since the LOC as well as medial olivocochlear neurons send axons far toward hair cells of the cochlea [27], the changes of this mRNA may represent a marker for monitoring the damage to the axon terminal portions. Hearing deficit-induced increase in mKIAA0143 mRNA was recognized in the small LSO core neurons. This region contains not only efferent neurons but also centrally projecting neurons [11,26]. Among the efferent neurons are those bearing the principal site of the origin of efferent fibers and further roles in directly influencing both the central auditory nuclei and the periphery [3,26]. Stimulation or surgical impairment of the olivocochlear bundle has been shown to produce changes in cochlear mechanics, afferent fiber activities and efficiencies of signal detection [5,6,16,20]. Further characterization of the small neurons must be needed to disclose the significance of the increased expression of mKIAA0143 and its functions in hearing deficit.

4. Discussion 5. Conclusion By subtracting genes expressed in the brainstem between mice with malleus removal and normal controls, we could obtain mouse homolog of KIAA0143 with the aid of human databases for unidentified protein. The mRNA obtained encodes a unique membrane-bound protein that consists of 819 amino acids (Fig. 3). By computer analysis, the gene locus was mapped to the mouse chromosome 15D1. Green fluorescent proteintagged mKIAA0143 was expressed in COS-1 cells (Fig. 5). It was amply seen in association with the cellular plasma membrane and also with some intracellular

Suppression subtractive hybridization enabled us to detect a novel gene in the auditory brainstem in the mouse. By BLAST search, the novel gene obtained here proved to code for a unique membrane protein called mKIAA0143 protein. The gene for this protein revealed significant alterations of mRNA expression in the superior olivary complex of the mice with hearing deficit. The present results emphasize the association of mKIAA0143 protein with altered conditions of hearing ability.

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Acknowledgements The authors thank Fumio Yamashita and Tetsuji Yamamoto for technical assistance, and Yuki Okada for expert secretarial work. Supported in part by grants from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and the Science Research Promotion Fund of the Japan Private School Promotion Foundation.

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