Identification and Characterization of Choline Transporter-Like Protein 2, an Inner Ear Glycoprotein of 68 and 72 kDa That Is the Target of Antibody-Induced Hearing Loss

Nair, Thankam S.; Kozma, Kelley Elizabeth; Hoefling, Nickoleta; Kommareddi, Pavan K.; Ueda, Yo; Gong, Tao; Lomax, Margaret I.; Lansford, Christopher D.; Telian, Steven A.; Satar, Bülent; Arts, H. Alexander; El-Kashlan, Hussam K.; Berryhill, Wayne E.; Raphael, Yehoash; Carey, Thomas E.

doi:10.1523/jneurosci.5063-03.2004

Cited by 107 publications

(139 citation statements)

References 25 publications

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“…The tissue was carefully harvested at the time of labyrinthectomy for Meniere's disease or the labyrinthectomy portion of the translabyrinthine approach for removal of vestibular schwannoma as described previously. 8,12,11 All dissections were performed at room temperature under normal saline. Using an otologic drill, otic capsule bone was removed from the ampullated ends of the semicircular canals.…”

Section: Tissue Harvest and Culturementioning

confidence: 99%

An in vitro model system to study gene therapy in the human inner ear

et al. 2007

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The confined fluid-filled labyrinth of the human inner ear presents an opportunity for introduction of gene therapy reagents designed to treat hearing and balance dysfunction. Here we present a novel model system derived from the sensory epithelia of human vestibular organs and show that the tissue can survive up to 5 days in vitro. We generated organotypic cultures from 26 human sensory epithelia excised at the time of labyrinthectomy for intractable Meniere's disease or vestibular schwannoma. We applied multiply deleted adenoviral vectors at titers between 10 5 and 10 8 viral particles/ml directly to the cultures for 4-24 h and examined the tissue 12-96 h post-transfection. We noted robust expression of the exogenous transgene, green fluorescent protein (GFP), in hair cells and supporting cells suggesting both were targets of adenoviral transfection. We also transfected cultures with a vector that carried the genes for GFP and KCNQ4, a potassium channel subunit that causes dominant-progressive hearing loss when mutated. We noted a positive correlation between GFP fluorescence and KCNQ4 immunolocalization. We conclude that our in vitro model system presents a novel and effective experimental paradigm for evaluation of gene therapy reagents designed to restore cellular function in patients who suffer from inner ear disorders.

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Section: Tissue Harvest and Culturementioning

confidence: 99%

An in vitro model system to study gene therapy in the human inner ear

et al. 2007

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“…In situ hybridization and antisera to cochlin showed cochlin to be expressed in the spiral ligament and limbus , whereas CTL2 is prominently expressed on supporting cells in the organ of Corti (Zajic et al 1991;Ptok et al 1993;Nair et al 2004). Using antibodies raised to unique peptides in each protein, we show in this report that cochlin and CTL2 physically interact and can co-precipitate one another.…”

Section: Introductionmentioning

confidence: 62%

“…The KHRI-3 monoclonal antibody developed from mice immunized with cell suspensions from the organ of Corti (Zajic et al 1991) binds to the CTL2 protein expressed on inner ear supporting cells (Nair et al 2004). Serum-free KHRI-3 antibody was produced in the Cellmax artificial capillary system (Pierce, Rockford, IL, USA; Nair et al 1997).…”

Section: Antibody Production Purification and Couplingmentioning

confidence: 99%

“…Several years ago, we discovered a supporting cell antigen that is a target of antibody-induced hearing loss mediated by the KHRI-3 monoclonal antibody (Nair et al 1999). We identified this antigen as choline transporter-like protein 2 (CTL2), a glycoprotein with 10-11 transmembrane domains (Nair et al 2004) that is a member of the choline transporter-like protein family (CTL1-CTL5; O_Regan et al 2000).…”

Section: Introductionmentioning

confidence: 99%

“…We identified this antigen as choline transporter-like protein 2 (CTL2), a glycoprotein with 10-11 transmembrane domains (Nair et al 2004) that is a member of the choline transporter-like protein family (CTL1-CTL5; O_Regan et al 2000). CTL2 belongs to the solute carrier family of transporters and is also known as solute carrier protein 44A2 (SLC44A2).…”

Section: Introductionmentioning

confidence: 99%

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Cochlin Isoforms and Their Interaction with CTL2 (SLC44A2) in the Inner Ear

Kommareddi

Nair

Raphael

et al. 2007

JARO

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Choline transporter-like protein 2 (CTL2) is a multitransmembrane protein expressed on inner ear supporting cells that was discovered as a target of antibody-induced hearing loss. Its function is unknown. A 64 kDa band that consistently co-precipitates with CTL2 from inner ear extracts was identified by mass spectroscopy as cochlin. Cochlin is an abundant inner ear protein expressed as multiple isoforms. Its function is also unknown, but it is suspected to be an extracellular matrix component. Cochlin is mutated in individuals with DFNA9 hearing loss. To investigate the CTL2-cochlin interaction, antibodies were raised to a cochlin-specific peptide. The antibodies identify several cochlin polypeptides on western blots and are specific for cochlin. We show that the heterogeneity of the cochlin isoforms is caused, in part, by in vivo posttranslational modification by N-glycosylation and, in part, caused by alternative splicing. We verified that antibody to CTL2 co-immunoprecipitates cochlin from the inner ear and antibody to cochlin coimmunoprecipitates CTL2. Using cochlear crosssections, we show that CTL2 is more widely distributed than previously described, and its prominent expression on cells facing the scala media suggests a possible role in homeostasis. A prominent but previously unreported ribbon-like pattern of cochlin in the basilar membrane was demonstrated, suggesting an important role for cochlin in the structure of the basilar membrane. CTL2 and cochlin are expressed in close proximity in the inner sulcus, the spiral prominence, vessels, limbus, and spiral ligament. The possible functional significance of CTL2-cochlin interactions remains unknown.

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Corticosteroid Response and Supporting Cell Antibody in Autoimmune Hearing Loss

Zeitoun¹,

Beckman²,

Arts³

et al. 2005

Arch Otolaryngol Head Neck Surg

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To determine whether antibodies to supporting cells are associated with response to corticosteroids in patients with autoimmune sensorineural hearing loss. Design: Prospective analysis of antibody to inner-ear antigens. Setting: Collaborating otology practices in Pennsylvania, Michigan, and Indiana. Patients: Sixty-three patients with rapidly progressive unilateral or bilateral sensorineural hearing loss of unknown cause suggestive of autoimmune sensorineural hearing loss. Interventions: Pretreatment audiometry, serum analysis by Western blot (WB) and immunofluorescence (IF) tests, corticosteroid therapy, and follow-up audiometry. Main Outcome Measures: Antibody reactivity and audiogram changes were analyzed for association with response to treatment. Results: More than half of the patients (37/63) had antibodies to both a 68-to 72-kDa protein and to innerear supporting cells, 16 patients had positive results on one assay only, and 10 had negative results on both. Twenty-eight patients improved and 35 did not. The WB findings did not correlate with response. Of the WBpositive patients, 49% (21/43) improved, as did 35% (7/ 20) of the WB-negative patients (P=.30). In contrast, 53% (25/47) of IF-positive patients improved, compared with only 19% (3/16) in the IF-negative group (P = .02). Of those who improved, 89% (25/28) were IF positive. Conclusions: Antibody to an inner-ear supporting cell antigen was significantly associated with hearing improvement after corticosteroid therapy (relative rate, 2.8). Patients with IF-positive serum are nearly 3 times more likely to experience improved hearing with corticosteroid treatment than those who are IF negative. Antibodies to inner-ear supporting cell antigen may have value in diagnosis and treatment of patients with autoimmune sensorineural hearing loss.

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Identification and Characterization of Choline Transporter-Like Protein 2, an Inner Ear Glycoprotein of 68 and 72 kDa That Is the Target of Antibody-Induced Hearing Loss

Cited by 107 publications

References 25 publications

An in vitro model system to study gene therapy in the human inner ear

An in vitro model system to study gene therapy in the human inner ear

Cochlin Isoforms and Their Interaction with CTL2 (SLC44A2) in the Inner Ear

Corticosteroid Response and Supporting Cell Antibody in Autoimmune Hearing Loss

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