A Novel Antigen Sensitive to Calcium Chelation That is Associated with the Tip Links and Kinocilial Links of Sensory Hair Bundles

Tip links are extracellular, cell-surface-associated filaments of unknown molecular composition that are thought to gate the mechanotransducer channel of the sensory hair cell. They disappear from the hair bundle in response to calcium chelation and lanthanum treatment and resist degradation by the protease subtilisin. A monoclonal antibody derived from a hybridoma screen identified a novel antigen associated with tip links, the tip-link antigen. The tip-link antigen is also associated with kinocilial links, subtilisin-resistant filaments that are sensitive to calcium chelation and connect the kinocilium to the tallest stereocilia of the hair bundle. Furthermore, the tip-link antigen is expressed in the retina, where it is associated with the ciliary calyx, a ring of microvilli that surrounds the outer segment of the photoreceptor. The tip-link antigen rapidly disappears from the surface of the hair bundle in response to calcium chelation. It is also subtilisin resistant, relative to the ankle-link antigen, an antigen associated with another type of hair bundle link. The tip-link antigen is lanthanum sensitive and, like tip links, reappears on the surface of the hair bundle after calcium chelation. The monoclonal antibody to the tip-link antigen immunoprecipitates two concanavalin A-reactive polypeptides with apparent molecular masses of 200 and 250 kDa from detergent extracts of the retina. These results provide the first identification of a cell surface antigen associated with tip links, indicate that tip links share properties in common with kinocilial links, and reveal a second epitope that, along with the ankle-link antigen, is common to both sensory hair bundles and the ciliary calyx of photoreceptors

Aminoglycoside-Induced Phosphatidylserine Externalization in Sensory Hair Cells Is Regionally Restricted, Rapid, and Reversible

The aminophospholipid phosphatidylserine (PS) is normally restricted to the inner leaflet of the plasma membrane. During certain cellular processes, including apoptosis, PS translocates to the outer leaflet and can be labeled with externally applied annexin V, a calcium-dependent PS-binding protein. In mouse cochlear cultures, annexin V labeling reveals that the aminoglycoside antibiotic neomycin induces rapid PS externalization, specifically on the apical surface of hair cells. PS externalization is observed within ~75 s of neomycin perfusion, first on the hair bundle and then on membrane blebs forming around the apical surface. Whole-cell capacitance also increases significantly within minutes of neomycin application, indicating that blebbing is accompanied by membrane addition to the hair cell surface. PS externalization and membrane blebbing can, nonetheless, occur independently. Pretreating hair cells with calcium chelators, a procedure that blocks mechanotransduction, or overexpressing a phosphatidylinositol 4,5-biphosphate (PIP2)-binding pleckstrin homology domain, can reduce neomycin-induced PS externalization, suggesting that neomycin enters hair cells via transduction channels, clusters PIP2, and thereby activates lipid scrambling. The effects of short-term neomycin treatment are reversible. After neomycin washout, PS is no longer detected on the apical surface, apical membrane blebs disappear, and surface-bound annexin V is internalized, distributing throughout the supranuclear cytoplasm of the hair cell. Hair cells can therefore repair, and recover from, neomycin-induced surface damage. Hair cells lacking myosin VI, a minus-end directed actin-based motor implicated in endocytosis, can also recover from brief neomycin treatment. Internalized annexin V, however, remains below the apical surface, thereby pinpointing a critical role for myosin VI in the transport of endocytosed material away from the periphery of the hair cell

Hearing Loss and Retarded Cochlear Development in Mice Lacking Type 2 Iodothyronine Deiodinase

The later stages of cochlear differentiation and the developmental onset of hearing require thyroid hormone. Although thyroid hormone receptors (TRs) are a prerequisite for this process, it is likely that other factors modify TR activity during cochlear development. The mouse cochlea expresses type 2 deiodinase (D2), an enzyme that converts thyroxine, the main form of thyroid hormone in the circulation, into 3,5,3\u27-triiodothyronine (T3) the major ligand for TRs. Here, we show that D2-deficient mice have circulating thyroid hormone levels that would normally be adequate to allow hearing to develop but they exhibit an auditory phenotype similar to that caused by systemic hypothyroidism or TR deletions. D2-deficient mice have defective auditory function, retarded differentiation of the cochlear inner sulcus and sensory epithelium, and deformity of the tectorial membrane. The similarity of this phenotype to that caused by TR deletions suggests that D2 controls the T3 signal that activates TRs in the cochlea. Thus, D2 is essential for hearing, and the results suggest that this hormone-activating enzyme confers on the cochlea the ability to stimulate its own T3 response at a critical developmental period

The supporting-cell antigen: a receptor-like protein tyrosine phosphatase expressed in the sensory epithelia of the inner ear

After noise- or drug-induced hair-cell loss, the sensory epithelia of the avian inner ear can regenerate new hair cells. Few molecular markers are available for the supporting-cell precursors of the hair cells that regenerate, and little is known about the signaling mechanisms underlying this regenerative response. Hybridoma methodology was used to obtain a monoclonal antibody (mAb) that stains the apical surface of supporting cells in the sensory epithelia of the inner ear. The mAb recognizes the supporting-cell antigen (SCA), a protein that is also found on the apical surfaces of retinal Müller cells, renal tubule cells, and intestinal brush border cells. Expression screening and molecular cloning reveal that the SCA is a novel receptor-like protein tyrosine phosphatase (RPTP), sharing similarity with human density-enhanced phosphatase, an RPTP thought to have a role in the density-dependent arrest of cell growth. In response to hair-cell damage induced by noise in vivo or hair-cell loss caused by ototoxic drug treatment in vitro, some supporting cells show a dramatic decrease in SCA expression levels on their apical surface. This decrease occurs before supporting cells are known to first enter S-phase after trauma, indicating that it may be a primary rather than a secondary response to injury. These results indicate that the SCA is a signaling molecule that may influence the potential of nonsensory supporting cells to either proliferate or differentiate into hair cell

Spontaneous otoacoustic emissions in TectaY1870C/+ mice reflect changes in cochlear amplification and how it is controlled by the tectorial membrane

Spontaneous otoacoustic emissions (SOAEs) recorded from the ear canal in the absence of sound reflect cochlear amplification, an outer-hair-cell (OHC) process required for the extraordinary sensitivity and frequency selectivity of mammalian hearing. Although wild-type mice rarely emit, those with mutations that influence the tectorial membrane (TM) show an incidence of SOAEs similar to that in humans. In this report, we characterized mice with a missense mutation in Tecta, a gene required for the formation of the striated-sheet matrix within the core of the TM. Mice heterozygous for the Y1870C mutation (TectaY1870C/+) are prolific emitters, despite a moderate hearing loss. Additionally, Kimura’s membrane, into which the OHC stereocilia insert, separates from the main body of the TM, except at apical cochlear locations. Multimodal SOAEs are also observed in TectaY1870C/+ mice where energy is present at frequencies that are integer multiples of a lower-frequency SOAE (the primary). Second-harmonic SOAEs, at twice the frequency of a lower-frequency primary, are the most frequently observed. These secondary SOAEs are found in spatial regions where stimulus-evoked OAEs are small or in the noise floor. Introduction of high-level suppressors just above the primary SOAE frequency reduce or eliminate both primary and second-harmonic SOAEs. In contrast, second-harmonic SOAEs are not affected by suppressors, either above or below the second-harmonic SOAE frequency, even when they are much larger in amplitude. Hence, second-harmonic SOAEs do not appear to be spatially separated from their primaries, a finding that has implications for cochlear mechanics and the consequences of changes to TM structure

The very large G-protein coupled receptor VLGR1: a component of the ankle link complex required for the normal development of auditory hair bundles

Sensory hair bundles in the inner ear are composed of stereocilia that can be interconnected by a variety of different link types, including tip links, horizontal top connectors, shaft connectors, and ankle links. The ankle link antigen is an epitope specifically associated with ankle links and the calycal processes of photoreceptors in chicks. Mass spectrometry and immunoblotting were used to identify this antigen as the avian ortholog of the very large G-protein-coupled receptor VLGR1, the product of the Usher syndrome USH2C (Mass1) locus. Like ankle links, Vlgr1 is expressed transiently around the base of developing hair bundles in mice. Ankle links fail to form in the cochleae of mice carrying a targeted mutation in Vlgr1 (Vlgr1/del7TM), and the bundles become disorganized just after birth. FM1-43 [N-(3-triethylammonium)propyl)-4-(4-(dibutylamino)styryl) pyridinium dibromide] dye loading and whole-cell recordings indicate mechanotransduction is impaired in cochlear, but not vestibular, hair cells of early postnatal Vlgr1/del7TM mutant mice. Auditory brainstem recordings and distortion product measurements indicate that these mice are severely deaf by the third week of life. Hair cells from the basal half of the cochlea are lost in 2-month-old Vlgr1/del7TM mice, and retinal function is mildly abnormal in aged mutants. Our results indicate that Vlgr1 is required for formation of the ankle link complex and the normal development of cochlear hair bundles

Accelerated age-related degradation of the tectorial membrane in the Ceacam16 βgal/βgal null mutant mouse, a model for late-onset human hereditary deafness DFNB113

CEACAM16 is a non-collagenous protein of the tectorial membrane, an extracellular structure of the cochlea essential for normal hearing. Dominant and recessive mutations in CEACAM16 have been reported to cause postlingual and progressive forms of deafness in humans. In a previous study of young Ceacam16 βgal/βgal null mutant mice on a C57Bl/6J background, the incidence of spontaneous otoacoustic emissions (SOAEs) was greatly increased relative to Ceacam16+/+ and Ceacam16+/βgal mice, but auditory brain-stem responses (ABRs) and distortion product otoacoustic emissions (DPOAEs) were near normal, indicating auditory thresholds were not significantly affected. To determine if the loss of CEACAM16 leads to hearing loss at later ages in this mouse line, cochlear structure and auditory function were examined in Ceacam16+/+, Ceacam16+/βgal and Ceacam16βgal/βgal mice at 6 and 12 months of age and compared to that previously described at 1 month. Analysis of older Ceacam16βgal/βgal mice reveals a progressive loss of matrix from the core of the tectorial membrane that is more extensive in the apical, low-frequency regions of the cochlea. In Ceacam16βgal/βgal mice at 6-7 months, the DPOAE magnitude at 2f1-f2 and the incidence of SOAEs both decrease relative to young animals. By ~12 months, SOAEs and DPOAEs are not detected in Ceacam16βgal/βgal mice and ABR thresholds are increased by up to ~40 dB across frequency, despite a complement of hair cells similar to that present in Ceacam16+/+ mice. Although SOAE incidence decreases with age in Ceacam16βgal/βgal mice, it increases in ageing heterozygous Ceacam16+/βgal mice and is accompanied by a reduction in the accumulation of CEACAM16 in the tectorial membrane relative to controls. An apically-biased loss of matrix from the core of the tectorial membrane, similar to that observed in young Ceacam16βgal/βgal mice, is also seen in Ceacam16+/+ and Ceacam16+/βgal mice, and other strains of wild-type mice, but at much later ages. The loss of Ceacam16 therefore accelerates age-related degeneration of the tectorial membrane leading, as in humans with mutations in CEACAM16, to a late-onset progressive form of hearing loss

The acquisition of mechano-electrical transducer current adaptation in auditory hair cells requires myosin VI

Mutations in Myo6, the gene encoding the (F-actin) minus end-directed unconventional myosin, myosin VI, cause hereditary deafness in mice (Snell's waltzer) and humans. In the sensory hair cells of the cochlea, myosin VI is expressed in the cell bodies and along the stereocilia that project from the cells’ apical surface. It is required for maintaining the structural integrity of the mechanosensitive hair bundles formed by the stereocilia. In this study we investigate whether myosin VI contributes to mechano-electrical transduction. We report that Ca²+-dependent adaptation of the mechano-electrical transducer (MET) current, which serves to keep the transduction apparatus operating within its most sensitive range, is absent in outer and inner hair cells from homozygous Snell's waltzer mutant mice, which fail to express myosin VI. The operating range of the MET channels is also abnormal in the mutants, resulting in the absence of a resting MET current. We found that cadherin 23, a component of the hair bundle's transient lateral links, fails to be downregulated along the length of the stereocilia in maturing Myo6 mutant mice. MET currents of heterozygous littermates appear normal. We propose that myosin VI, by removing key molecules from developing hair bundles, is required for the development of the MET apparatus and its Ca²+-dependent adaptation

The tip-link antigen, a protein associated with the transduction complex of sensory hair cells, is protocadherin-15

Sound and acceleration are detected by hair bundles, mechanosensory structures located at the apical pole of hair cells in the inner ear. The different elements of the hair bundle, the stereocilia and a kinocilium, are interconnected by a variety of link types. One of these links, the tip link, connects the top of a shorter stereocilium with the lateral membrane of an adjacent taller stereocilium and may gate the mechanotransducer channel of the hair cell. Mass spectrometric and Western blot analyses identify the tip-link antigen, a hitherto unidentified antigen specifically associated with the tip and kinocilial links of sensory hair bundles in the inner ear and the ciliary calyx of photoreceptors in the eye, as an avian ortholog of human protocadherin-15, a product of the gene for the deaf/blindness Usher syndrome type 1F/DFNB23 locus. Multiple protocadherin-15 transcripts are shown to be expressed in the mouse inner ear, and these define four major isoform classes, two with entirely novel, previously unidentified cytoplasmic domains. Antibodies to the three cytoplasmic domain-containing isoform classes reveal that each has a different spatiotemporal expression pattern in the developing and mature inner ear. Two isoforms are distributed in a manner compatible for association with the tip-link complex. An isoform located at the tips of stereocilia is sensitive to calcium chelation and proteolysis with subtilisin and reappears at the tips of stereocilia as transduction recovers after the removal of calcium chelators. Protocadherin-15 is therefore associated with the tip-link complex and may be an integral component of this structure and/or required for its formatio

Combined bezafibrate and medroxyprogesterone acetate: potential novel therapy for acute myeloid leukaemia

Background: The majority of acute myeloid leukaemia (AML) patients are over sixty years of age. With current treatment regimens, survival rates amongst these, and also those younger patients who relapse, remain dismal and novel therapies are urgently required. In particular, therapies that have anti-leukaemic activity but that, unlike conventional chemotherapy, do not impair normal haemopoiesis. Principal Findings: Here we demonstrate the potent anti-leukaemic activity of the combination of the lipid-regulating drug bezafibrate (BEZ) and the sex hormone medroxyprogesterone acetate (MPA) against AML cell lines and primary AML cells. The combined activity of BEZ and MPA (B/M) converged upon the increased synthesis and reduced metabolism of prostaglandin D2 (PGD2) resulting in elevated levels of the downstream highly bioactive, anti-neoplastic prostaglandin 15-deoxy Δ12,14 PGJ2 (15d-PGJ2). BEZ increased PGD2 synthesis via the generation of reactive oxygen species (ROS) and activation of the lipid peroxidation pathway. MPA directed prostaglandin synthesis towards 15d-PGJ2 by inhibiting the PGD2 11β -ketoreductase activity of the aldo-keto reductase AKR1C3, which metabolises PGD2 to 9α11β-PGF2α. B/M treatment resulted in growth arrest, apoptosis and cell differentiation in both AML cell lines and primary AML cells and these actions were recapitulated by treatment with 15d-PGJ2. Importantly, the actions of B/M had little effect on the survival of normal adult myeloid progenitors. Significance: Collectively our data demonstrate that B/M treatment of AML cells elevated ROS and delivered the anti-neoplastic actions of 15d-PGJ2. These observations provide the mechanistic rationale for the redeployment of B/M in elderly and relapsed AML