Mutations of human TMHS cause recessively inherited non-syndromic hearing loss.

BACKGROUND: Approximately half the cases of prelingual hearing loss are caused by genetic factors. Identification of genes causing deafness is a crucial first step in understanding the normal function of these genes in the auditory system. Recently, a mutant allele of Tmhs was reported to be associated with deafness and circling behaviour in the hurry-scurry mouse. Tmhs encodes a predicted tetraspan protein of unknown function, which is expressed in inner ear hair cells. The human homologue of Tmhs is located on chromosome 6p. OBJECTIVE: To determine the cause of deafness in four consanguineous families segregating recessive deafness linked to markers on chromosome 6p21.1-p22.3 defining a novel DFNB locus. RESULTS: A novel locus for non-syndromic deafness DFNB67 was mapped in an interval of approximately 28.51 cM on human chromosome 6p21.1-p22.3. DNA sequence analysis of TMHS revealed a homozygous frameshift mutation (246delC) and a missense mutation (Y127C) in affected individuals of two families segregating non-syndromic deafness, one of which showed significant evidence of linkage to markers in the DFNB67 interval. The localisation of mTMHS in developing mouse inner ear hair cells was refined and found to be expressed briefly from E16.5 to P3. CONCLUSIONS: These findings establish the importance of TMHS for normal sound transduction in humans.

Mutations in Cdh23, encoding a new type of cadherin, cause stereocilia disorganization in waltzer, the mouse model for Usher syndrome type 1D.

Mouse chromosome 10 harbors several loci associated with hearing loss, including waltzer (v), modifier-of deaf waddler (mdfw) and Age-related hearing loss (Ahl). The human region that is orthologous to the mouse 'waltzer' region is located at 10q21-q22 and contains the human deafness loci DFNB12 and USH1D). Numerous mutations at the waltzer locus have been documented causing erratic circling and hearing loss. Here we report the identification of a new gene mutated in v. The 10.5-kb Cdh23 cDNA encodes a very large, single-pass transmembrane protein, that we have called otocadherin. It has an extracellular domain that contains 27 repeats; these show significant homology to the cadherin ectodomain. In v(6J), a GT transversion creates a premature stop codon. In v(Alb), a CT exchange generates an ectopic donor splice site, effecting deletion of 119 nucleotides of exonic sequence. In v(2J), a GA transition abolishes the donor splice site, leading to aberrant splice forms. All three alleles are predicted to cause loss of function. We demonstrate Cdh23 expression in the neurosensory epithelium and show that during early hair-cell differentiation, stereocilia organization is disrupted in v(2J) homozygotes. Our data indicate that otocadherin is a critical component of hair bundle formation. Mutations in human CDH23 cause Usher syndrome type 1D and thus, establish waltzer as the mouse model for USH1D.

Targeted disruption of mouse Pds provides insight about the inner-ear defects encountered in Pendred syndrome.

Following the positional cloning of PDS, the gene mutated in the deafness/goitre disorder Pendred syndrome (PS), numerous studies have focused on defining the role of PDS in deafness and PS as well as elucidating the function of the PDS-encoded protein (pendrin). To facilitate these efforts and to provide a system for more detailed study of the inner-ear defects that occur in the absence of pendrin, we have generated a Pds-knockout mouse. Pds(-/-) mice are completely deaf and also display signs of vestibular dysfunction. The inner ears of these mice appear to develop normally until embryonic day 15, after which time severe endolymphatic dilatation occurs, reminiscent of that seen radiologically in deaf individuals with PDS mutations. Additionally, in the second postnatal week, severe degeneration of sensory cells and malformation of otoconia and otoconial membranes occur, as revealed by scanning electron and fluorescence confocal microscopy. The ultrastructural defects seen in the Pds(-/-) mice provide important clues about the mechanisms responsible for the inner-ear pathology associated with PDS mutations.

Two distinct Ca(2+)-dependent signaling pathways regulate the motor output of cochlear outer hair cells.

The outer hair cells (OHCs) of the cochlea have an electromotility mechanism, based on conformational changes of voltage-sensitive "motor" proteins in the lateral plasma membrane. The translocation of electrical charges across the membrane that accompanies electromotility imparts a voltage dependency to the membrane capacitance. We used capacitance measurements to investigate whether electromotility may be influenced by different manipulations known to affect intracellular Ca(2+) or Ca(2+)-dependent protein phosphorylation. Application of acetylcholine (ACh) to the synaptic pole of isolated OHCs evoked a Ca(2+)-activated apamin-sensitive outward K(+) current. It also enhanced electromotility, probably because of a phosphorylation-dependent decrease of the cell's axial stiffness. However, ACh did not change the voltage-dependent capacitance either in conventional whole-cell experiments or under perforated-patch conditions. The effects produced by the Ca(2+) ionophore ionomycin mimicked those produced by ACh. Hyperpolarizing shifts of the voltage dependence of capacitance and electromotility were induced by okadaic acid, a promoter of protein phosphorylation, whereas trifluoperazine and W-7, antagonists of calmodulin, caused opposite depolarizing shifts. Components of the protein phosphorylation cascade-IP(3) receptors and calmodulin-dependent protein kinase type IV-were immunolocalized to the lateral wall of the OHC. Our results suggest that two different Ca(2+)-dependent pathways may control the OHC motor output. The first pathway modulates cytoskeletal stiffness and can be activated by ACh. The second pathway shifts the voltage sensitivity of the OHC electromotile mechanism and may be activated by the release of Ca(2+) from intracellular stores located in the proximity of the lateral plasma membrane.

The motor and tail regions of myosin XV are critical for normal structure and function of auditory and vestibular hair cells.

Recessive mutations in myosin 15, a class XV unconventional myosin, cause profound congenital deafness in humans and both deafness and vestibular dysfunction in mice homozygous for the shaker 2 and shaker 2(J) alleles. The shaker 2 allele is a previously described missense mutation of a highly conserved residue in the motor domain of myosin XV. The shaker 2(J) lesion, in contrast, is a 14.7 kb deletion that removes the last six exons from the 3"-terminus of the Myo15 transcript. These exons encode a FERM (F, ezrin, radixin and moesin) domain that may interact with integral membrane proteins. Despite the deletion of six exons, Myo15 mRNA transcripts and protein are present in the post-natal day 1 shaker 2(J) inner ear, which suggests that the FERM domain is critical for the development of normal hearing and balance. Myo15 transcripts are first detectable at embryonic day 13.5 in wild-type mice. Myo15 transcripts in the mouse inner ear are restricted to the sensory epithelium of the developing cristae ampularis, macula utriculi and macula sacculi of the vestibular system as well as to the developing organ of Corti. Both the shaker 2 and shaker 2(J) alleles result in abnormally short hair cell stereocilia in the cochlear and vestibular systems. This suggests that Myo15 may be important for both the structure and function of these sensory epithelia.

Expression and localization of prestin and the sugar transporter GLUT-5 during development of electromotility in cochlear outer hair cells.

Electromotility, i.e., the ability of cochlear outer hair cells (OHCs) to contract and elongate at acoustic frequencies, is presumed to depend on the voltage-driven conformational changes of "motor" proteins present in the OHC lateral plasma membrane. Recently, two membrane proteins have been proposed as candidates for the OHC motor. A sugar transporter, GLUT-5, was proposed based on its localization in the OHCs and on the observation that sugar transport alters the voltage sensitivity of the OHC motor mechanism. Another candidate, "prestin," was identified from a subtracted OHC cDNA library and shown to impart voltage-driven shape changes to transfected cultured cells. We used antibodies specific for these two proteins to show that they are highly expressed in the lateral membrane of OHCs. We also compared the postnatal expression patterns of these proteins with the development of electromotility in OHCs of the apical turn of the rat organ of Corti. The patch-clamp recording of transient charge movement associated with electromotility indicates that half of the maximal expression of the motor protein occurs at postnatal day 9. Prestin incorporation in the plasma membrane begins from postnatal day 0 and increases progressively in a time course coinciding with that of electromotility. GLUT-5 is not incorporated into the lateral plasma membrane of apical OHCs until postnatal day 15. Our results suggest that, although GLUT-5 may be involved in the control of electromotility, prestin is likely to be a fundamental component of the OHC membrane motor mechanism.

Water permeability of cochlear outer hair cells: characterization and relationship to electromotility.

The distinguishing feature of the mammalian outer hair cells (OHCs) is to elongate and shorten at acoustic frequencies, when their intracellular potential is changed. This "electromotility" or "electromechanics" depends critically on positive intracellular pressure (turgor), maintained by the inflow of water through yet uncharacterized water pathways. We measured the water volume flow, J(v), across the plasma membrane of isolated guinea pig and rat OHCs after osmotic challenges and estimated the osmotic water permeability coefficient, P(f), to be approximately 10(-2) cm/sec. This value is within the range reported for osmotic flow mediated by the water channel proteins, aquaporins. J(v) was inhibited by HgCl(2), which is known to block aquaporin-mediated water transport. P(f) values that were estimated for OHCs from neonatal rats were of the order of approximately 2 x 10(-3) cm/sec, equivalent to that of membranes lacking water channel proteins. In an immunofluorescence assay we showed that an anti-peptide antibody specific for aquaporins labels the lateral plasma membrane of the OHC in the region in which electromotility is generated. Using patch-clamp recording, we found that water influx into the OHC is regulated by intracellular voltage. We also found that the most pronounced increases of the electromotility-associated charge movement and of the expression of OHC water channels occur between postnatal days 8 and 12, preceding the onset of hearing function in the rat. Our data indicate that electromotility and water transport in OHCs may influence each other structurally and functionally.

Characterization of the human and mouse unconventional myosin XV genes responsible for hereditary deafness DFNB3 and shaker 2.

Mutations in myosin XV are responsible for congenital profound deafness DFNB3 in humans and deafness and vestibular defects in shaker 2 mice. By combining direct cDNA analyses with a comparison of 95.2 kb of genomic DNA sequence from human chromosome 17p11.2 and 88.4 kb from the homologous region on mouse chromosome 11, we have determined the genomic and mRNA structures of the human (MYO15) and mouse (Myo15) myosin XV genes. Our results indicate that full-length myosin XV transcripts contain 66 exons, are >12 kb in length, and encode 365-kDa proteins that are unique among myosins in possessing very long approximately 1200-aa N-terminal extensions preceding their conserved motor domains. The tail regions of the myosin XV proteins contain two MyTH4 domains, two regions with similarity to the membrane attachment FERM domain, and a putative SH3 domain. Northern and dot blot analyses revealed that myosin XV is expressed in the pituitary gland in both humans and mice. Myosin XV transcripts were also observed by in situ hybridization within areas corresponding to the sensory epithelia of the cochlea and vestibular systems in the developing mouse inner ear. Immunostaining of adult mouse organ of Corti revealed that myosin XV protein is concentrated within the cuticular plate and stereocilia of cochlear sensory hair cells. These results indicate a likely role for myosin XV in the formation or maintenance of the unique actin-rich structures of inner ear sensory hair cells.

ATP-Induced Ca(2+) release in cochlear outer hair cells: localization of an inositol triphosphate-gated Ca(2+) store to the base of the sensory hair bundle.

We used a high-performance fluorescence imaging system to visualize rapid changes in intracellular free Ca(2+) concentration ([Ca(2+)](i)) evoked by focal applications of extracellular ATP to the hair bundle of outer hair cells (OHCs): the sensory-motor receptors of the cochlea. Simultaneous recordings of the whole-cell current and Calcium Green-1 fluorescence showed a two-component increase in [Ca(2+)](i). After an initial entry of Ca(2+) through the apical membrane, a second and larger, inositol triphosphate (InsP(3))-gated, [Ca(2+)](i) surge occurred at the base of the hair bundle. Electron microscopy of this intracellular Ca(2+) release site showed that it coincides with the localization of a unique system of endoplasmic reticulum (ER) membranes and mitochondria known as Hensen's body. Using confocal immunofluorescence microscopy, we showed that InsP(3) receptors share this location. Consistent with a Ca(2+)-mobilizing second messenger system linked to ATP-P2 receptors, we also determined that an isoform of G-proteins is present in the stereocilia. Voltage-driven cell shape changes and nonlinear capacitance were monitored before and after ATP application, showing that the ATP-evoked [Ca(2+)](i) rise did not interfere with the OHC electromotility mechanism. This second messenger signaling mechanism bypasses the Ca(2+)-clearance power of the stereocilia and transiently elevates [Ca(2+)](i) at the base of the hair bundle, where it can potentially modulate the action of unconventional myosin isozymes involved in maintaining the hair bundle integrity and potentially influence mechanotransduction.

Stability and plasticity of primary afferent projections following nerve regeneration and central degeneration.

Sensory neurons of the dorsal root ganglia (DRG) regenerate their peripheral axons with relative ease following a nerve lesion. The capacity for central regeneration appears more limited. However, after nerve lesion, some DRG neurons gain a regenerative advantage to sprout centrally. We developed a lesion model in the rat to test whether, after prior lesion of their peripheral axons, subsets of cutaneous afferents benefit differently in their ability to sprout into adjacent spinal segments denervated by dorsal rhizotomy. We found that under identical circumstances, myelinated sensory neurons, small-diameter peptidergic sensory neurons containing calcitonin gene related peptide (CGRP), and small-diameter nonpeptidergic neurons that bind the lectin from the plant Griffonia simplificolia, isolectin B4 (IB4) differ dramatically in their ability to regenerate centrally. Myelinated afferent terminals labelled transganglionically with cholera-toxin beta-subunit gain a small advantage in collaterally sprouting into the adjacent denervated neuropil in lamina III after prior peripheral nerve lesion. This central regenerative response was not mimicked by experimentally induced inflammation of sensory neuron cell bodies. Intact and unlesioned sensory neurons positive for CGRP sprout vigorously into segments denervated by rhizotomy in a nonsomatotopic manner. In contrast, IB4-positive sensory neurons maintain a somatotopic distribution centrally, which is not altered by prior nerve lesion. These data reveal a remarkably heterogeneous response to regeneration-promoting stimuli amongst three different types of cutaneous sensory neurons. In particular, the divergent responses of peptidergic and nonpeptidergic sensory neurons suggests profound functional differences between these neurochemically distinct neurons.

Cochlear outer hair cell electromotility can provide force for both low and high intensity distortion product otoacoustic emissions.

It is generally believed that the force for the otoacoustic emission (OAE) generation is provided by a mechanism of electromotility, observed in isolated cochlear outer hair cells (OHCs). OHC electromotility is resistant to several ototoxic reagents, it does not depend on ATP hydrolysis, but it can be blocked by specific sulfhydryl reagents: p-chloromercuriphenylsulfonic acid (pCMPS) and p-hydroxymercuriphenylsulfonic acid (pHMPS). We have used these reagents to test whether they also affect OAE. Application of pCMPS and pHMPS on the round window membrane of anesthetized guinea pigs produced a dose-dependent inhibition of the cubic (2F1-F2) distortion product OAE (DPOAE). The inhibition developed progressively from high to low frequencies, reflecting the diffusion of the drugs through the cochlear compartment. The effect of pCMPS and pHMPS was different from the effects of furosemide and lethal anoxia, which impair cochlear function but do not block OHC electromotility. pHMPS suppressed DPOAE completely at all sound intensities tested (45-80 dB SPL), whereas furosemide or lethal anoxia caused DPOAE to disappear at low-level stimulation (45-60 dB SPL) only. Our results suggest that the OHC electromotility might provide the force for DPOAE generation not only at low, but also at high stimulus intensities.