Piotr Kazmierczak

Cadherin 23 and protocadherin 15 interact to form tip-link filaments in sensory hair cells.

Hair cells of the inner ear are mechanosensors that transduce mechanical forces arising from sound waves and head movement into electrochemical signals to provide our sense of hearing and balance. Each hair cell contains at the apical surface a bundle of stereocilia. Mechanoelectrical transduction takes place close to the tips of stereocilia in proximity to extracellular tip-link filaments that connect the stereocilia and are thought to gate the mechanoelectrical transduction channel. Recent reports on the composition, properties and function of tip links are conflicting. Here we demonstrate that two cadherins that are linked to inherited forms of deafness in humans interact to form tip links. Immunohistochemical studies using rodent hair cells show that cadherin 23 (CDH23) and protocadherin 15 (PCDH15) localize to the upper and lower part of tip links, respectively. The amino termini of the two cadherins co-localize on tip-link filaments. Biochemical experiments show that CDH23 homodimers interact in trans with PCDH15 homodimers to form a filament with structural similarity to tip links. Ions that affect tip-link integrity and a mutation in PCDH15 that causes a recessive form of deafness disrupt interactions between CDH23 and PCDH15. Our studies define the molecular composition of tip links and provide a conceptual base for exploring the mechanisms of sensory impairment associated with mutations in CDH23 and PCDH15.

The Usher syndrome proteins cadherin 23 and harmonin form a complex by means of PDZ-domain interactions

Usher syndrome type 1 (USH1) patients suffer from sensorineuronal deafness, vestibular dysfunction, and visual impairment. Several genetic loci have been linked to USH1, and four of the relevant genes have been identified. They encode the unconventional myosin VIIa, the PDZ-domain protein harmonin, and the putative adhesion receptors cadherin 23 (CDH23) and protocadherin 15 (PCDH15). We show here that CDH23 and harmonin form a protein complex. Two PDZ domains in harmonin interact with two complementary binding surfaces in the CDH23 cytoplasmic domain. One of the binding surfaces is disrupted by sequences encoded by an alternatively spliced CDH23 exon that is expressed in the ear, but not the retina. In the ear, CDH23 and harmonin are expressed in the stereocilia of hair cells, and in the retina within the photoreceptor cell layer. Because CDH23-deficient mice have splayed stereocilia, our data suggest that CDH23 and harmonin are part of a transmembrane complex that connects stereocilia into a bundle. Defects in the formation of this complex are predicted to disrupt stereocilia bundles and cause deafness in USH1 patients.

Physical and Functional Interaction between Protocadherin 15 and Myosin VIIa in Mechanosensory Hair Cells

Hair cells of the mammalian inner ear are the mechanoreceptors that convert sound-induced vibrations into electrical signals. The molecular mechanisms that regulate the development and function of the mechanically sensitive organelle of hair cells, the hair bundle, are poorly defined. We link here two gene products that have been associated with deafness and hair bundle defects, protocadherin 15 (PCDH15) and myosin VIIa (MYO7A), into a common pathway. We show that PCDH15 binds to MYO7A and that both proteins are expressed in an overlapping pattern in hair bundles. PCDH15 localization is perturbed in MYO7A-deficient mice, whereas MYO7A localization is perturbed in PCDH15-deficient mice. Like MYO7A, PCDH15 is critical for the development of hair bundles in cochlear and vestibular hair cells, controlling hair bundle morphogenesis and polarity. Cochlear and vestibular hair cells from PCDH15-deficient mice also show defects in mechanotransduction. Together, our findings suggest that PCDH15 and MYO7A cooperate to regulate the development and function of the mechanically sensitive hair bundle.

Harmonin Mutations Cause Mechanotransduction Defects in Cochlear Hair Cells

In hair cells, mechanotransduction channels are gated by tip links, the extracellular filaments that consist of cadherin 23 (CDH23) and protocadherin 15 (PCDH15) and connect the stereocilia of each hair cell. However, which molecules mediate cadherin function at tip links is not known. Here we show that the PDZ-domain protein harmonin is a component of the upper tip-link density (UTLD), where CDH23 inserts into the stereociliary membrane. Harmonin domains that mediate interactions with CDH23 and F-actin control harmonin localization in stereocilia and are necessary for normal hearing. In mice expressing a mutant harmonin protein that prevents UTLD formation, the sensitivity of hair bundles to mechanical stimulation is reduced. We conclude that harmonin is a UTLD component and contributes to establishing the sensitivity of mechanotransduction channels to displacement.

Inhibition of Akt kinase signalling and activation of Forkhead are indispensable for upregulation of FasL expression in apoptosis of glioma cells

Activation of Akt signalling pathway is frequently found in glioma cells and may contribute to their resistance to undergo apoptosis in response to conventional therapies. We found that cyclosporin A (CsA) induces apoptosis of C6 glioma cells, which is associated with transcriptional activation of fasL. In the present paper, we investigated an involvement of Akt signalling in the regulation of FasL expression in CsA-induced apoptosis. We demonstrated that the level of active Akt decreases significantly after CsA treatment, which results in the decrease of Forkhead phosphorylation and its translocation to the nucleus. It correlated with an increase of binding to the Forkhead-responsive element FHRE from the FasL promoter, as demonstrated by gel-shift assays. Although treatment with LY294002, a specific inhibitor of PI3 K, decreased the phosphorylation of Akt and increased Fkhr translocation to the nucleus, these events were not sufficient to induce FasL expression and apoptosis of C6 glioma cells. Interference with Akt/Forkhead signalling by membrane-targeted Akt or removal of the FKHR-binding sites from the FasL promoter significantly abolished its activation. These results indicate that downregulation of Akt signalling and activation of Forkhead is a prerequisite for the induction of FasL promoter. It may be clinically important for pharmacological intervention in gliomas.

Sensing sound: molecules that orchestrate mechanotransduction by hair cells

Animals use acoustic signals to communicate and to obtain information about their environment. The processing of acoustic signals is initiated at auditory sense organs, where mechanosensory hair cells convert sound-induced vibrations into electrical signals. Although the biophysical principles underlying the mechanotransduction process in hair cells have been characterized in much detail over the past 30 years, the molecular building-blocks of the mechanotransduction machinery have proved to be difficult to determine. We review here recent studies that have both identified some of these molecules and established the mechanisms by which they regulate the activity of the still-elusive mechanotransduction channel.

Structure of the N terminus of cadherin 23 reveals a new adhesion mechanism for a subset of cadherin superfamily members.

The cadherin superfamily encodes more than 100 receptors with diverse functions in tissue development and homeostasis. Classical cadherins mediate adhesion by binding interactions that depend on their N-terminal extracellular cadherin (EC) domains, which swap N-terminal β-strands. Sequence alignments suggest that the strand-swap binding mode is not commonly used by functionally divergent cadherins. Here, we have determined the structure of the EC1–EC2 domains of cadherin 23 (CDH23), which binds to protocadherin 15 (PCDH15) to form tip links of mechanosensory hair cells. Unlike classical cadherins, the CDH23 N terminus contains polar amino acids that bind Ca2+. The N terminus of PCDH15 also contains polar amino acids. Mutations in polar amino acids within EC1 of CDH23 and PCDH15 abolish interaction between the two cadherins. PCDH21 and PCDH24 contain similarly charged N termini, suggesting that a subset of cadherins share a common interaction mechanism that differs from the strand-swap binding mode of classical cadherins.

Development and Regeneration of Sensory Transduction in Auditory Hair Cells Requires Functional Interaction Between Cadherin-23 and Protocadherin-15

Tip links are extracellular filaments that connect pairs of hair cell stereocilia and convey tension to mechanosensitive channels. Recent evidence suggests that tip links are formed by calcium-dependent interactions between the N-terminal domains of cadherin-23 (CDH23) and protocadherin-15 (PCDH15). Mutations in either CDH23 or PCDH15 cause deafness in mice and humans, indicating the molecules are required for normal inner ear function. However, there is little physiological evidence to support a direct role for CDH23 and PCDH15 in hair cell mechanotransduction. To investigate the contributions of CDH23 and PCDH15 to mechanotransduction and tip-link formation, we examined outer hair cells of mouse cochleas during development and after chemical disruption of tip links. We found that tip links and mechanotransduction with all the qualitative properties of mature transduction recovered within 24 h after disruption. To probe tip-link formation, we measured transduction currents after extracellular application of recombinant CDH23 and PCDH15 fragments, which included putative interaction domains (EC1). Both fragments inhibited development and regeneration of transduction but did not disrupt transduction in mature cells. PCDH15 fragments that carried a mutation in EC1 that causes deafness in humans did not inhibit transduction development or regeneration. Immunolocalization revealed wild-type fragments bound near the tips of hair cell stereocilia. Scanning electron micrographs revealed that hair bundles exposed to fragments had a reduced number of linkages aligned along the morphological axis of sensitivity of the bundle. Together, the data provide direct evidence implicating CDH23 and PCDH15 proteins in the formation of tip links during development and regeneration of mechanotransduction.

The Mechanotransduction Machinery of Hair Cells

Studying genes linked to deafness identifies components of the ears mechanotransduction apparatus. Mechanotransduction, the conversion of mechanical force into an electrochemical signal, allows living organisms to detect touch, hear, register movement and gravity, and sense changes in cell volume and shape. Hair cells in the vertebrate inner ear are mechanoreceptor cells specialized for the detection of sound and head movement. Each hair cell contains, at the apical surface, rows of stereocilia that are connected by extracellular filaments to form an exquisitely organized bundle. Mechanotransduction channels, localized near the tips of the stereocilia, are gated by the gating spring, an elastic element that is stretched upon stereocilia deflection and mediates rapid channel opening. Components of the mechanotransduction machinery in hair cells have been identified and several are encoded by genes linked to deafness in humans, which indicates that defects in the mechanotransduction machinery are the underlying cause of some forms of hearing impairment.

Progressive Hearing Loss in Mice Carrying a Mutation in Usp53

Disordered protein ubiquitination has been linked to neurodegenerative disease, yet its role in inner ear homeostasis and hearing loss is essentially unknown. Here we show that progressive hearing loss in the ethylnitrosourea-generated mambo mouse line is caused by a mutation in Usp53, a member of the deubiquitinating enzyme family. USP53 contains a catalytically inactive ubiquitin-specific protease domain and is expressed in cochlear hair cells and a subset of supporting cells. Although hair cell differentiation is unaffected in mambo mice, outer hair cells degenerate rapidly after the first postnatal week. USP53 colocalizes and interacts with the tight junction scaffolding proteins TJP1 and TJP2 in polarized epithelial cells, suggesting that USP53 is part of the tight junction complex. The barrier properties of tight junctions of the stria vascularis appeared intact in a biotin tracer assay, but the endocochlear potential is reduced in adult mambo mice. Hair cell degeneration in mambo mice precedes endocochlear potential decline and is rescued in cochlear organotypic cultures in low potassium milieu, indicating that hair cell loss is triggered by extracellular factors. Remarkably, heterozygous mambo mice show increased susceptibility to noise injury at high frequencies. We conclude that USP53 is a novel tight junction-associated protein that is essential for the survival of auditory hair cells and normal hearing in mice, possibly by modulating the barrier properties and mechanical stability of tight junctions. SIGNIFICANCE STATEMENT Hereditary hearing loss is extremely prevalent in the human population, but many genes linked to hearing loss remain to be discovered. Forward genetics screens in mice have facilitated the identification of genes involved in sensory perception and provided valuable animal models for hearing loss in humans. This involves introducing random mutations in mice, screening the mice for hearing defects, and mapping the causative mutation. Here, we have identified a mutation in the Usp53 gene that causes progressive hearing loss in the mambo mouse line. We demonstrate that USP53 is a catalytically inactive deubiquitinating enzyme and a novel component of tight junctions that is necessary for sensory hair cell survival and inner ear homeostasis.