表观遗传学——耳聋研究的新视野

耳聋是一种常见的人类感觉系统缺陷,新生儿发病率可达1/1000～3/1000。耳蜗感觉神经上皮毛细胞的结构或功能异常可导致耳聋,遗传因素在其中起重要作用。虽然一些与遗传性耳聋相关的基因及染色体位点已经被定位或克隆,仍有很多耳聋的病因尚不清楚。人们发现,除了常见的热点基因突变(GJB2、SLC26A4、线粒体DNA C1494T和A1555G等)外,一些表观遗传学的改变也在耳聋的发生中起重要作用。例如,miR-96突变会导致人和小鼠的渐进性失聪,异常的CpG岛甲基化与一些耳聋综合征的发生有关等。文章着重对表观遗传学在耳聋领域的研究现状和进展进行了综述。     

表观遗传学——耳聋研究的新视野

耳聋是一种常见的人类感觉系统缺陷, 新生儿发病率可达1/1000～3/1000。耳蜗感觉神经上皮毛细胞的结构或功能异常可导致耳聋,遗传因素在其中起重要作用。虽然一些与遗传性耳聋相关的基因及染色体位点已经被定位或克隆, 仍有很多耳聋的病因尚不清楚。人们发现, 除了常见的热点基因突变(GJB2、SLC26A4、线粒体DNA C1494T和A1555G等)外, 一些表观遗传学的改变也在耳聋的发生中起重要作用。例如, miR-96 突变会导致人和小鼠的渐进性失聪, 异常的CpG岛甲基化与一些耳聋综合征的发生有关等。文章着重对表观遗传学在耳聋领域的研究现状和进展进行了综述。     

Pluripotent stem cells from the adult mouse inner ear

In mammals, the permanence of acquired hearing loss is mostly due to the incapacity of the cochlea to replace lost mechanoreceptor cells, or hair cells. In contrast, damaged vestibular organs can generate new hair cells, albeit in limited numbers. Here we show that the adult utricular sensory epithelium contains cells that display the characteristic features of stem cells. These inner ear stem cells have the capacity for self-renewal, and form spheres that express marker genes of the developing inner ear and the nervous system. Inner ear stem cells are pluripotent and can give rise to a variety of cell types in vitro and in vivo, including cells representative of ectodermal, endodermal and mesodermal lineages. Our observation that these stem cells are capable of differentiating into hair cell-like cells implies a possible use of such cells for the replacement of lost inner-ear sensory cells.     

Hair cell regeneration

The mammalian inner ear largely lacks the capacity to regenerate hair cells, the sensory cells required for hearing and balance. Recent studies in both lower vertebrates and mammals have uncovered genes and pathways important in hair cell development and have suggested ways that the sensory epithelia could be manipulated to achieve hair cell regeneration. These approaches include the use of inner ear stem cells, transdifferentiation of nonsensory cells, and induction of a proliferative response in the cells that can become hair cells.     

A novel locus for autosomal dominant nonsyndromic hearing loss, DFNA13, maps to chromosome 6p.

Nonsyndromic hearing loss (NSHL) is the most common type of hearing impairment in the elderly. Environmental and hereditary factors play an etiologic role, although the relative contribution of each is unknown. To date, 39 NSHL genes have been localized. Twelve produce autosomal dominant hearing loss, most frequently postlingual in onset and progressive in nature. We have ascertained a large, multigenerational family in which a gene for autosomal dominant NSHL is segregating. Affected individuals experience progressive hearing loss beginning in the 2d-4th decades, eventually making the use of amplification mandatory. A novel locus, DFNA13, was identified on chromosome 6p; the disease gene maps to a 4-cM interval flanked by D6S1663 and D6S1691, with a maximum two-point LOD score of 6.409 at D6S299.     

Cell cycle regulation in hair cell development and regeneration in the mouse cochlea

Cell cycle inhibitors play important roles in the development of mammalian cochleae. Loss of function of those factors in mice at various developmental stages results in distinct phenotypes characterized by overproduction or loss of cochlear sensory cells. Our recent study showed that acute deletion of the retinoblastoma protein (Rb) induces rapid cell cycle reentry and subsequent loss of postnatal cochlear hair cells in mice. Clearly, these regulators play multiple roles in cell cycle exit and differentiation of hair cell and supporting cell progenitors. They are also crucial in maintenance of postmitotic states and survival of differentiated hair cells and supporting cells. In mammals, lost hair cells cannot be spontaneously replaced, leading to permanent deafness. However, lower vertebrates such as birds and fish can naturally regenerate damaged hair cells from the underlying supporting cells through proliferation and transdifferentiation. Thus, manipulating cell cycle inhibitors in mammalian cochleae could provide a new avenue to restore hearing in deaf people caused by a variety of genetic mutations and environmental insults.     

Residual microRNA expression dictates the extent of inner ear development in conditional Dicer knockout mice

Inner ear development requires coordinated transformation of a uniform sheet of cells into a labyrinth with multiple cell types. While numerous regulatory proteins have been shown to play critical roles in this process, the regulatory functions of microRNAs (miRNAs) have not been explored. To demonstrate the importance of miRNAs in inner ear development, we generated conditional Dicer knockout mice by the expression of Cre recombinase in the otic placode at E8.5. Otocyst-derived ganglia exhibit rapid neuron-specific miR-124 depletion by E11.5, degeneration by E12.5, and profound defects in subsequent sensory epithelial innervations by E17.5. However, the small and malformed inner ear at E17.5 exhibits residual and graded hair cell-specific miR-183 expression in the three remaining sensory epithelia (posterior crista, utricle, and cochlea) that closely corresponds to the degree of hair cell and sensory epithelium differentiation, and Fgf10 expression required for morphohistogenesis. The highest miR-183 expression is observed in near-normal hair cells of the posterior crista, whereas the reduced utricular macula demonstrates weak miR-183 expression and develops presumptive hair cells with numerous disorganized microvilli instead of ordered stereocilia. The correlation of differential and delayed depletion of mature miRNAs with the derailment of inner ear development demonstrates that miRNAs are crucial for inner ear neurosensory development and neurosensory-dependent morphogenesis.     

Molecular mechanisms that regulate auditory hair-cell differentiation in the mammalian cochlea

Mechanosensory hair cells of the vertebrate cochlea offer an excellent developmental system to study cell-fate specification, and to gain insight into the many human neurological deficits which result in a hearing loss, by affecting primarily the hair cells. Therefore, there is great interest in studying the molecular mechanisms that regulate their specification and differentiation. Recent studies, based mostly on loss-of-function experiments that target the role of Notch signaling and basic helix-loop-helix genes in inner-ear development have indicated that they can regulate mechanosensory hair cell-fate specification and their initial differentiation.     

Mutations in LOXHD1, an Evolutionarily Conserved Stereociliary Protein, Disrupt Hair Cell Function in Mice and Cause Progressive Hearing Loss in Humans

Hearing loss is the most common form of sensory impairment in humans and is frequently progressive in nature. Here we link a previously uncharacterized gene to hearing impairment in mice and humans. We show that hearing loss in the ethylnitrosourea (ENU)-induced samba mouse line is caused by a mutation in Loxhd1. LOXHD1 consists entirely of PLAT (polycystin/lipoxygenase/α-toxin) domains and is expressed along the membrane of mature hair cell stereocilia. Stereociliary development is unaffected in samba mice, but hair cell function is perturbed and hair cells eventually degenerate. Based on the studies in mice, we screened DNA from human families segregating deafness and identified a mutation in LOXHD1, which causes DFNB77, a progressive form of autosomal-recessive nonsyndromic hearing loss (ARNSHL). LOXHD1, MYO3a, and PJVK are the only human genes to date linked to progressive ARNSHL. These three genes are required for hair cell function, suggesting that age-dependent hair cell failure is a common mechanism for progressive ARNSHL.     

Homozygosity mapping identifies a novel GIPC3 mutation causing congenital nonsyndromic hearing loss in a Saudi family

Hearing loss is one of the most common sensory disorders in humans and has a genetic cause in 50% of the cases. Our recent studies indicate that nonsyndromic hearing loss (NSHL) in the Saudi Arabian population is genetically heterogeneous and is not caused by mutations in GJB2 and GJB6, the most common genes for deafness in various populations worldwide. Identification of the causative gene/mutation in affected families is difficult due to extreme genetic heterogeneity and lack of phenotypic variability. We utilized an SNP array-based whole-genome homozygosity mapping approach in search of the causative gene, for the phenotype in a consanguineous Saudi family, with five affected individuals presenting severe to profound congenital NSHL. A single shared block of homozygosity was identified on chromosome 19p13.3 encompassing GIPC3, a recently identified hearing loss gene. Subsequently, a novel mutation c.122 C>A (p.T41K) in GIPC3 was found. This is the first report of GIPC3 mutation in a Saudi family. The presence of the GIPC3 mutations in only one of 100 Saudi families with congenital NSHL suggests that it appears to be a rare cause of familial or sporadic deafness in this population.     

Nonsense mutations in SMPX, encoding a protein responsive to physical force, result in X-chromosomal hearing loss

The fact that hereditary hearing loss is the most common sensory disorder in humans is reflected by, among other things, an extraordinary allelic and nonallelic genetic heterogeneity. X-chromosomal hearing impairment represents only a minor fraction of all cases. In a study of a Spanish family the locus for one of the X-chromosomal forms was assigned to Xp22 (DFNX4). We mapped the disease locus in the same chromosomal region in a large German pedigree with X-chromosomal nonsyndromic hearing impairment by using genome-wide linkage analysis. Males presented with postlingual hearing loss and onset at ages 3-7, whereas onset in female carriers was in the second to third decades. Targeted DNA capture with high-throughput sequencing detected a nonsense mutation in the small muscle protein, X-linked (SMPX) of affected individuals. We identified another nonsense mutation in SMPX in patients from the Spanish family who were previously analyzed to map DFNX4. SMPX encodes an 88 amino acid, cytoskeleton-associated protein that is responsive to mechanical stress. The presence of Smpx in hair cells and supporting cells of the murine cochlea indicates its role in the inner ear. The nonsense mutations detected in the two families suggest a loss-of-function mechanism underlying this form of hearing impairment. Results obtained after heterologous overexpression of SMPX proteins were compatible with this assumption. Because responsivity to physical force is a characteristic feature of the protein, we propose that long-term maintenance of mechanically stressed inner-ear cells critically depends on SMPX function.     

Cell Cycle,Differentiation and Regeneration: Where to Begin?

Hair cells, the sensory cells of inner ear, perform essential functions in hearing and balance. However, mammalian hair cells, like most of the CNS neurons, lack the capacity to regenerate. This is in sharp contrast to lower vertebrates in which hair cell regeneration occurs spontaneously through cell division of supporting cells, which leads to hearing restoration. It is believed that the lack of regeneration in mammals is, to a large degree, due to the block of cell cycle re-entry imposed by negative cell growth genes in the inner ear. Recent studies have identified retinoblastoma gene, a well-known tumor suppressor, as the key gene involved in cell cycle exit of inner ear sensory cells. In the inner ear of pRb conditional knockout mice, hair cells undergo continuous cell division, and at the same time differentiate and become functional. Cell division continues in early postnatal cochlea and adult vestibule. Remarkably, the vestibular hair cells without pRb survive, and function at both the cellular and system levels. The time course and effects of pRb inhibition shows that there is a separation between the roles of pRb in cell cycle exit, and subsequent maturation and apoptosis. Those studies reveal distinctly different roles of pRb in the cochlear and vestibular sensory epithelia. The review discusses additional areas to be studied for regeneration of mature hair cells, and highlights the importance of transient and reversible block of pRb function as one of the routes to be explored for regeneration.     

Mutations in Grxcr1 Are The Basis for Inner Ear Dysfunction in the Pirouette Mouse

Recessive mutations at the mouse pirouette (pi) locus result in hearing loss and vestibular dysfunction due to neuroepithelial defects in the inner ear. Using a positional cloning strategy, we have identified mutations in the gene Grxcr1 (glutaredoxin cysteine-rich 1) in five independent allelic strains of pirouette mice. We also provide sequence data of GRXCR1 from humans with profound hearing loss suggesting that pirouette is a model for studying the mechanism of nonsyndromic deafness DFNB25. Grxcr1 encodes a 290 amino acid protein that contains a region of similarity to glutaredoxin proteins and a cysteine-rich region at its C terminus. Grxcr1 is expressed in sensory epithelia of the inner ear, and its encoded protein is localized along the length of stereocilia, the actin-filament-rich mechanosensory structures at the apical surface of auditory and vestibular hair cells. The precise architecture of hair cell stereocilia is essential for normal hearing. Loss of function of Grxcr1 in homozygous pirouette mice results in abnormally thin and slightly shortened stereocilia. When overexpressed in transfected cells, GRXCR1 localizes along the length of actin-filament-rich structures at the dorsal-apical surface and induces structures with greater actin filament content and/or increased lengths in a subset of cells. Our results suggest that deafness in pirouette mutants is associated with loss of GRXCR1 function in modulating actin cytoskeletal architecture in the developing stereocilia of sensory hair cells.     

Growth factor regulation of the cell Cycle in developing and mature inner ear sensory epithelia

Growth factors and other extracellular signals regulate cell division in many tissues. Consequently, growth factors may have therapeutic uses to stimulate the production of replacement sensory hair cells in damaged human inner ears, thereby assisting in alleviating hearing loss and vestibular dysfunction. Assessment of the ability of growth factors to stimulate cell proliferation in inner ear sensory epithelia is at an early stage. This paper provides a brief account of what we know regarding growth factor regulation of cell proliferation in developing and mature inner ear sensory epithelia.     

Mouse models to study inner ear development and hereditary hearing loss

Hereditary sensorineural hearing loss, derived from inner ear defects, is the most common hereditary disability with a prevalence of 1 in 1,000 children, although it can be present in up to 15% of births in isolated communities. The mouse serves as an ideal animal model to identify new deafness-related genes and to study their roles in vivo. This review describes mouse models for genes that have been linked with hearing impairment (HI) in humans. Mutations in several groups of genes have been linked with HI in both mice and humans. Mutant mice have been instrumental in elucidating the function and mechanisms of the inner ear. For example, the roles of collagens and tectorins in the tectorial membrane, as well as the necessity of intact links between the hair cell projections, stereocilia and kinocilia, have been discovered in mice. Accurate endolymph composition and the proteins which participate in its production were found to be crucial for inner ear function, as well as several motor proteins such as prestin and myosins. Two systematic projects, KOMP and EUCOMM, which are currently being carried out to create knock-out and conditional mutants for every gene in the mouse genome, promise that many additional deafness-related genes will be identified in the next years, providing models for all forms of human deafness.     

Multiple quantitative trait loci modify cochlear hair cell degeneration in the Beethoven (Tmc1Bth) mouse model of progressive hearing loss DFNA36

Dominant mutations of transmembrane channel-like gene 1 (TMC1) cause progressive sensorineural hearing loss in humans and Beethoven (Tmc1Bth/+) mice. Here we show that Tmc1Bth/+ mice on a C3HeB/FeJ strain background have selective degeneration of inner hair cells while outer hair cells remain structurally and functionally intact. Inner hair cells primarily function as afferent sensory cells, whereas outer hair cells are electromotile amplifiers of auditory stimuli that can be functionally assessed by distortion product otoacoustic emission (DPOAE) analysis. When C3H-Tmc1Bth/Bth is crossed with either C57BL/6J or DBA/2J wild-type mice, F1 hybrid Tmc1Bth/+ progeny have increased hearing loss associated with increased degeneration of outer hair cells and diminution of DPOAE amplitudes but no difference in degeneration of inner hair cells. We mapped at least one quantitative trait locus (QTL), Tmc1m1, for DPOAE amplitude on chromosome 2 in [(C/B)F1xC]N2-Tmc1Bth/+ backcross progeny, and three other QTL on chromosomes 11 (Tmc1m2), 12 (Tmc1m3), and 5 (Tmc1m4) in [(C/D)F1xC]N2-Tmc1Bth/+ progeny. The polygenic basis of outer hair cell degeneration in Beethoven mice provides a model system for the dissection of common, complex hearing loss phenotypes, such as presbycusis, that involve outer hair cell degeneration in humans.     

Transgenic and gene targeting studies of hair cell function in mouse inner ear

Despite the rapid discovery of a large number of genes in sensory hair cells of the inner ear, the functional roles of these genes in hair cells remain largely undetermined. Recent advances in transgenic and gene targeting technologies in mice have offered unprecedented opportunities to genetically manipulate the expression of these genes and to study their functional roles in hair cells in vivo. Transgenic analyses have revealed the presence of hair-cell-specific promoters in the genes encoding Math1, myosin VIIa, Pou4f3, and the alpha9 subunit of the acetylcholine receptor (alpha9 AChR). Targeted inactivation using embryonic stem cell technology and transgenic expression studies have revealed the roles of several genes involved in hair cell lineage (Math1), differentiation (Pou4f3), mechanotransduction (Myo1c, and Myo7a), electromotility (Prestin), and efferent modulation (Chrna9, encoding alpha9 AChR). Although many of these genes also play roles in other tissues, inactivation of these genes in hair cells alone will soon be possible by using the Cre-loxP system. Also imminent is the development of genetic methods to inactivate genes specifically in mouse hair cells at a desired time, by using inducible systems established in other types of neurons. Combining these types of manipulation of gene expression will enable hearing researchers to elucidate some of the fundamental and unique features of hair cell function such as mechanotransduction, frequency tuning, active mechanical amplification, and efferent modulation.     

Drosophila auditory organ genes and genetic hearing defects

The Drosophila auditory organ shares equivalent transduction mechanisms with vertebrate hair cells, and both are specified by atonal family genes. Using a whole-organ knockout strategy based on atonal, we have identified 274 Drosophila auditory organ genes. Only four of these genes had previously been associated with fly hearing, yet one in five of the genes that we identified has a human cognate that is implicated in hearing disorders. Mutant analysis of 42 genes shows that more than half of them contribute to auditory organ function, with phenotypes including hearing loss, auditory hypersusceptibility, and ringing ears. We not only discover ion channels and motors important for hearing, but also show that auditory stimulus processing involves chemoreceptor proteins as well as phototransducer components. Our findings demonstrate mechanosensory roles for ionotropic receptors and visual rhodopsins and indicate that different sensory modalities utilize common signaling cascades.