Amiel A. Dror

MicroRNAs are essential for development and function of inner ear hair cells in vertebrates

MicroRNAs (miRNAs) inhibit the translation of target mRNAs and affect, directly or indirectly, the expression of a large portion of the protein-coding genes. This study focuses on miRNAs that are expressed in the mouse cochlea and vestibule, the 2 inner ear compartments. A conditional knock-out mouse for Dicer1 demonstrated that miRNAs are crucial for postnatal survival of functional hair cells of the inner ear. We identified miRNAs that have a role in the vertebrate developing inner ear by combining miRNA transcriptome analysis, spatial and temporal expression patterns, and bioinformatics. Microarrays revealed similar miRNA profiles in newborn-mouse whole cochleae and vestibules, but different temporal and spatial expression patterns of six miRNAs (miR-15a, miR-18a, miR-30b, miR-99a, miR-182, and miR-199a) may reflect their roles. Two of these miRNAs, miR-15a-1 and miR-18a, were also shown to be crucial for zebrafish inner ear development and morphogenesis. To suggest putative target mRNAs whose translation may be inhibited by selected miRNAs, we combined bioinformatics-based predictions and mRNA expression data. Finally, we present indirect evidence that Slc12a2, Cldn12, and Bdnf mRNAs may be targets for miR-15a. Our data support the hypothesis that inner ear tissue differentiation and maintenance are regulated and controlled by conserved sets of cell-specific miRNAs in both mouse and zebrafish.

Hearing Loss: Mechanisms Revealed by Genetics and Cell Biology

Hearing loss (HL), or deafness in its most severe form, affects an estimated 28 and 22.5 million Americans and Europeans, respectively. The numbers are higher in regions such as India and the Middle East, where consanguinity contributes to larger numbers of recessively inherited hearing impairment (HI). As a result of work-related difficulties, educational and developmental delays, and social stigmas and exclusion, the economic impact of HL is very high. At the other end of the spectrum, a rich deaf culture, particularly for individuals whose parents and even grandparents were deaf, is a social movement that believes that deafness is a difference in human experience rather than a disability. This review attempts to cover the remarkable progress made in the field of the genetics of HL over the past 20 years. Mutations in a significant number of genes have been discovered over the years that contribute to clinically heterogeneous forms of HL, enabling genetic counseling and prediction of progression of HL. Cell biological assays, protein localization in the inner ear, and detailed analysis of spontaneous and transgenic mouse models have provided an incredibly rich resource for elucidating mechanisms of hereditary hearing loss (HHL). This knowledge is providing answers for the families with HL, who contribute a great deal to the research being performed worldwide.

Hearing Impairment: A Panoply of Genes and Functions

Research in the genetics of hearing and deafness has evolved rapidly over the past years, providing the molecular foundation for different aspects of the mechanism of hearing. Considered to be the most common sensory disorder, hearing impairment is genetically heterogeneous. The multitude of genes affected encode proteins associated with many different functions, encompassing overarching areas of research. These include, but are not limited to, developmental biology, cell biology, physiology, and neurobiology. In this review, we discuss the broad categories of genes involved in hearing and deafness. Particular attention is paid to a subgroup of genes associated with inner ear gene regulation, fluid homeostasis, junctional complex and tight junctions, synaptic transmission, and auditory pathways. Overall, studies in genetics have provided research scientists and clinicians with insight regarding practical implications for the hearing impaired, while heralding hope for future development of therapeutics.

Integration of transcriptomics, proteomics, and microRNA analyses reveals novel microRNA regulation of targets in the mammalian inner ear.

We have employed a novel approach for the identification of functionally important microRNA (miRNA)-target interactions, integrating miRNA, transcriptome and proteome profiles and advanced in silico analysis using the FAME algorithm. Since miRNAs play a crucial role in the inner ear, demonstrated by the discovery of mutations in a miRNA leading to human and mouse deafness, we applied this approach to microdissected auditory and vestibular sensory epithelia. We detected the expression of 157 miRNAs in the inner ear sensory epithelia, with 53 miRNAs differentially expressed between the cochlea and vestibule. Functionally important miRNAs were determined by searching for enriched or depleted targets in the transcript and protein datasets with an expression consistent with the dogma of miRNA regulation. Importantly, quite a few of the targets were detected only in the protein datasets, attributable to regulation by translational suppression. We identified and experimentally validated the regulation of PSIP1-P75, a transcriptional co-activator previously unknown in the inner ear, by miR-135b, in vestibular hair cells. Our findings suggest that miR-135b serves as a cellular effector, involved in regulating some of the differences between the cochlear and vestibular hair cells.

A Myo6 Mutation Destroys Coordination between the Myosin Heads, Revealing New Functions of Myosin VI in the Stereocilia of Mammalian Inner Ear Hair Cells

Myosin VI, found in organisms from Caenorhabditis elegans to humans, is essential for auditory and vestibular function in mammals, since genetic mutations lead to hearing impairment and vestibular dysfunction in both humans and mice. Here, we show that a missense mutation in this molecular motor in an ENU-generated mouse model, Tailchaser, disrupts myosin VI function. Structural changes in the Tailchaser hair bundles include mislocalization of the kinocilia and branching of stereocilia. Transfection of GFP-labeled myosin VI into epithelial cells and delivery of endocytic vesicles to the early endosome revealed that the mutant phenotype displays disrupted motor function. The actin-activated ATPase rates measured for the D179Y mutation are decreased, and indicate loss of coordination of the myosin VI heads or ‘gating’ in the dimer form. Proper coordination is required for walking processively along, or anchoring to, actin filaments, and is apparently destroyed by the proximity of the mutation to the nucleotide-binding pocket. This loss of myosin VI function may not allow myosin VI to transport its cargoes appropriately at the base and within the stereocilia, or to anchor the membrane of stereocilia to actin filaments via its cargos, both of which lead to structural changes in the stereocilia of myosin VI–impaired hair cells, and ultimately leading to deafness.

Calcium Oxalate Stone Formation in the Inner Ear as a Result of an Slc26a4 Mutation

Calcium oxalate stone formation occurs under pathological conditions and accounts for more than 80% of all types of kidney stones. In the current study, we show for the first time that calcium oxalate stones are formed in the mouse inner ear of a genetic model for hearing loss and vestibular dysfunction in humans. The vestibular system within the inner ear is dependent on extracellular tiny calcium carbonate minerals for proper function. Thousands of these biominerals, known as otoconia, are associated with the utricle and saccule sensory maculae and are vital for mechanical stimulation of the sensory hair cells. We show that a missense mutation within the Slc26a4 gene abolishes the transport activity of its encoded protein, pendrin. As a consequence, dramatic changes in mineral composition, size, and shape occur within the utricle and saccule in a differential manner. Although abnormal giant carbonate minerals reside in the utricle at all ages, in the saccule, a gradual change in mineral composition leads to a formation of calcium oxalate in adult mice. By combining imaging and spectroscopy tools, we determined the profile of mineral composition and morphology at different time points. We propose a novel mechanism for the accumulation and aggregation of oxalate crystals in the inner ear.

Lhx3, a LIM domain transcription factor, is regulated by Pou4f3 in the auditory but not in the vestibular system

A dominant mutation of the gene encoding the POU4F3 transcription factor underlies human non‐syndromic progressive hearing loss DFNA15. Using oligonucleotide microarrays to generate expression profiles of inner ears of Pou4f3ddl/ddl mutant and wild‐type mice, we have identified and validated Lhx3, a LIM domain transcription factor, as an in vivo target gene regulated by Pou4f3. Lhx3 is a hair cell‐specific gene expressed in all hair cells of the auditory and vestibular system as early as embryonic day 16. The level of Lhx3 mRNA is greatly reduced in the inner ears of embryonic Pou4f3 mutant mice. Our data also show that the expression of Lhx3 is regulated differently in auditory and vestibular hair cells. This is the first example of a hair cell‐specific gene expressed both in auditory and in vestibular hair cells, with differential regulation of expression in these two closely related systems.

Hearing loss: a common disorder caused by many rare alleles

Perception of sound is a fundamental role of the auditory system. Traveling with the force of their mechanical energy, sound waves are captured by the ear and activate the sensory pathway of this complex organ. The hair cells, specialized sensory cells within the inner ear, transmit the mechanical energy into electrical nerve stimuli that reach the brain. A large number of proteins are responsible for the overarching tasks required to maintain the complex mechanism of sound sensation. Many hearing disorders are due to single gene defects inherited in a Mendelian fashion, thus enabling clinical diagnostics. However, at the same time, hearing impairment is genetically heterogeneous, with both common and rare forms occurring due to mutations in over 100 genes. The crosstalk between human and mouse genetics has enabled comprehensive studies on gene identification and protein function, taking advantage of the tools animal models have to offer. The aim of the following review is to provide background and examples of human deafness genes and the discovery of their function in the auditory system.

CLRN1 Is Nonessential in the Mouse Retina but Is Required for Cochlear Hair Cell Development

Mutations in the CLRN1 gene cause Usher syndrome type 3 (USH3), a human disease characterized by progressive blindness and deafness. Clarin 1, the protein product of CLRN1, is a four-transmembrane protein predicted to be associated with ribbon synapses of photoreceptors and cochlear hair cells, and recently demonstrated to be associated with the cytoskeleton. To study Clrn1, we created a Clrn1 knockout (KO) mouse and characterized the histological and functional consequences of Clrn1 deletion in the retina and cochlea. Clrn1 KO mice do not develop a retinal degeneration phenotype, but exhibit progressive loss of sensory hair cells in the cochlea and deterioration of the organ of Corti by 4 months. Hair cell stereocilia in KO animals were longer and disorganized by 4 months, and some Clrn1 KO mice exhibited circling behavior by 5–6 months of age. Clrn1 mRNA expression was localized in the retina using in situ hybridization (ISH), laser capture microdissection (LCM), and RT–PCR. Retinal Clrn1 transcripts were found throughout development and adulthood by RT–PCR, although expression peaked at P7 and declined to undetectable levels in adult retina by ISH. LCM localized Clrn1 transcripts to the retinas inner nuclear layer, and WT levels of retinal Clrn1 expression were observed in photoreceptor-less retinas. Examination of Clrn1 KO mice suggests that CLRN1 is unnecessary in the murine retina but essential for normal cochlear development and function. This may reflect a redundancy in the mouse retina not present in human retina. In contrast to mouse KO models of USH1 and USH2, our data indicate that Clrn1 expression in the retina is restricted to the Müller glia. This is a novel finding, as most retinal degeneration associated proteins are expressed in photoreceptors, not in glia. If CLRN1 expression in humans is comparable to the expression pattern observed in mice, this is the first report of an inner retinal protein that, when mutated, causes retinal degeneration.

A Novel SLC26A4 (PDS) Deafness Mutation Retained in the Endoplasmic Reticulum

OBJECTIVES To identify mutations in the SLC26A4 gene in individuals with nonsyndromic hearing loss and enlarged vestibular aqueduct, to design a predicted model of the pendrin protein, and to characterize novel mutations by means of localization in mammalian cells and effect of the mutation on the predicted model. DESIGN Validation of the mutation by its exclusion in more than 300 individuals with normal hearing. SETTING A laboratory of genetics of hearing loss research, clinical genetics laboratories, an otolaryngology department at Tel Aviv University, and medical centers in Israel. PATIENTS A patient with nonsyndromic hearing loss and enlarged vestibular aqueduct, 203 deaf probands, and 310 controls with normal hearing. INTERVENTIONS Sequencing the SLC26A4 gene in the patient with nonsyndromic hearing loss and enlarged vestibular aqueduct. Transfection of yellow fluorescent protein (YFP) constructs into mammalian COS7 cells. Designing a computational model of the human SLC26A4 protein. MAIN OUTCOME MEASURE Detection of a novel c.1458_1459insT SLC26A4 mutation. RESULTS A computational model of the human pendrin protein suggests that the novel c.1458_1459insT mutation leads to a prematurely truncated protein, p.Ile487TyrfsX39. Mammalian COS7 cells transfected with the YFP-1458_1459insT construct showed mislocalization of the mutant protein. CONCLUSIONS A novel SLC26A4 mutation was detected in Israel. Because current estimates demonstrate that SLC26A4 mutations are involved in up to 4% of nonsyndromic deafness, our findings emphasize the importance of adding a molecular test for the SLC26A4 gene in the diagnosis of deafness, particularly when bone abnormalities are involved, to the list of genes screened in Israel and elsewhere in the world.