New treatment options for hearing loss.

Nature Reviews Drug Discovery | AOP, published online 20 March 2015; doi:10.1038/nrd4533

REVIEWS New treatment options for hearing loss Ulrich Müller1 and Peter G. Barr-Gillespie2

Abstract | Hearing loss is the most common form of sensory impairment in humans and affects more than 40 million people in the United States alone. No drug-based therapy has been approved by the Food and Drug Administration, and treatment mostly relies on devices such as hearing aids and cochlear implants. Over recent years, more than 100 genetic loci have been linked to hearing loss and many of the affected genes have been identified. This understanding of the genetic pathways that regulate auditory function has revealed new targets for pharmacological treatment of the disease. Moreover, approaches that are based on stem cells and gene therapy, which may have the potential to restore or maintain auditory function, are beginning to emerge.

Nonsyndromic deafness A genetic form of hearing loss that affects hearing function without any other symptoms in different tissues and organs.

Syndromic deafness A genetic form of hearing loss that affects not only hearing but also the function of other tissues and organs.

Ototoxic A substance that is toxic to the ear, such as certain antibiotics and chemotherapy agents.

Tinnitus Perception of a ringing tone within the ear without an external sound trigger.

Department of Molecular and Cellular Neuroscience, Dorris Neuroscience Center, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, San Diego, California 92037, USA. 2 Oregon Hearing Research Center, Vollum Institute, Oregon Health & Science University, 3181 South West Sam Jackson Park Road, Portland, Oregon 97239, USA. e-mails: [email protected]; [email protected] doi:10.1038/nrd4533 Published online 20 March 2015 1

Hearing loss is the most common form of sensory impairment in humans, affecting people of any age and manifesting in various forms that range from mild auditory impairment to complete deafness. In nonsyndromic deafness, only hearing function is noticeably altered, whereas syndromic deafness is accompanied by other defects, such as visual impairment or kidney dysfunction. Disease onset and progression are tremendously diverse. Although approximately 1 in 500 children are born with impaired hearing1, sudden or progressive forms of hearing loss can manifest at any age. Hearing loss that is associated with ageing (that is, presbycusis) is remarkably common, affecting nearly two-thirds of individuals over 70 years of age2. Hearing loss can be caused by environmental factors, such as exposure to noise, viruses or ototoxic chemicals. At least one in ten working-age American individuals suffers from a loss of hearing that is associated with exposure to loud sound, either from a work environment or through recreational activities3. Traumatic injury, such as injury caused by exposure to an explosion or to the firing of a gun, can lead to sudden hearing loss; sometimes this hearing loss is accompanied by the perception of a constant ringing noise (a condition known as tinnitus). It has become increasingly clear that genetic factors also have a central role in disease aetiology; mutations in more than 100 genetic loci have already been linked to nonsyndromic deafness4,5,6 (see the Hereditary Hearing Loss website). It is estimated that mutations in hundreds more genes cause or predispose people to congenital, progressive, noise-induced and age-related forms of hearing loss. There are many syndromic forms of the disease (see the OMIM database), although they account for only approximately one-third of the patients with hearing loss7.

Despite the immense scale of these problems, the treatment options for hearing loss are mostly based on medical devices, such as hearing aids and cochlear implants. There are no pharmacological therapeutics currently in widespread use. Identification of the genetic defects that cause hearing impairment has been instrumental in the discovery of molecular pathways that are involved in the regulation of auditory perception. Knowledge of these pathways provides a starting point for the development of therapeutic options for individuals that are affected by hearing impairment. Exciting advances in regenerative medicine and gene therapy offer potential alternative routes for restoring lost auditory function or for slowing down its progression. Here, we discuss the basic features of the auditory system, the molecular pathways that control its development and function, and the promises and challenges of regenerative approaches and pharmacological interventions for the treatment of hearing loss.

Signal processing in the auditory system Within the mammalian auditory system, the organ of Corti, which is located in the snail-shaped cochlea of the inner ear, is responsible for the detection of sound (FIG. 1a,b). The organ of Corti harbours the auditory sensory epithelium, which, in humans, contains approximately 16,000 hair cells (the principal sensory cells for sound) that are patterned into three rows of outer hair cells (OHCs) and one row of inner hair cells (IHCs)8. Each hair cell contains, at its apical surface, a mechanically sensitive organelle — the hair bundle — that consists of rows of actin-filled stereocilia that increase in height (FIG. 1c,d). An extracellular matrix, the tectorial membrane,

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REVIEWS a

b Scala vestibuli

Outer ear Ossicle

Scala media Tectorial membrane

Tympanic membrane

Stria vascularis Outer hair cell

Cochlea

Inner hair cell

Cochlear implants Electrical medical devices that are implanted into the cochlea as a substitute for the function of damaged hair cells.

Organ of Corti

Nerve fibre

c

Scala tympani

Basilar membrane

d

The end-organ in the inner ear that functions in sound perception.

Outer hair cells (OHCs). Sensory cells for sound perception that are required to amplify input sound signals for their subsequent detection and processing by inner hair cells.

Inner hair cells Sensory cells that transmit sound information via neurons to the nervous system.

Tectorial membrane An acellular membrane of the inner ear that covers the mechanically sensitive hair bundles of hair cells and is required for the transmission of mechanical signals that are evoked by sound stimuli.

Basilar membrane A stiff acellular structure of the inner ear that underlies the sensory epithelium and resonates in response to sound-induced mechanical signals.

Tympanic membrane A membrane that separates the external from the middle ear, also referred to as the eardrum.

Ossicles The three bones of the middle ear that transmit sound information from the external ear to the cochlea in the inner ear.

Synapses Structural specializations that allow signal transmission between neurons or from sensory cells, such as hair cells, to neurons.

5 µM

5 µM

Figure 1 | The auditory sensory organ. a | A diagram of the auditory sensory organ in humans. The snail-shaped cochlea, ossicles and nerve fibres are highlighted. b | A cross section of the mammalian cochlea. c | A scanning electron micrograph Nature Reviews | Drug Discovery of the hair bundles of mouse outer and inner hair cells after removal of the tectorial membrane. The image was false-coloured to highlight the hair cells’ mechanosensitive hair bundles, which are clusters of stereocilia (orange-red; stereocilia of outer hair cells but not of inner hair cells form a V-shape on top of each hair cell). d | Magnification of a mouse outer hair cell to reveal the hair bundle structure. Parts a and b are modified from: ©Schwander, M. et al., 2010. Originally published in J. Cell Biol. 190: 9–20. Part c: this cover image was published in Trends Cell Biol., 11, Mechanisms that regulate mechanosensory hair cell differentiation, Copyright Elsevier (2001).

covers the apical surface of the organ of Corti and is attached to the hair bundles of OHCs. The cell bodies of hair cells form specialized adhesive contacts with supporting cells that, in turn, adhere at their basolateral surfaces to the basilar membrane, an extracellular matrix assembly with a different molecular composition from the tectorial membrane9 (FIG. 1b). Hearing is initiated when sound waves that reach the outer ear travel through the ear canal to the tympanic membrane10. Here, the sound energy is transferred, via the bony ossicles of the middle ear, to the oval window at the base of the fluid-filled cochlea. The motions of the oval window are converted into fluid pressure waves, which travel down the cochlear duct and induce vibrations in the basilar membrane. From here, the vibrations are transferred onto the hair cells, leading to the

deflection of their hair bundles, which contain mechanically gated ion channels10. The opening of these transduction channels leads to hair cell depolarization and to the release of neurotransmitters onto afferent neurons, which form synapses with IHCs. The electrical signals are propagated through the nervous system and processed in the brainstem, auditory cortex and higher brain areas. Hair cells along the length of the cochlear duct are tuned to specific frequencies, these are organized so that hair cells at the base of the cochlea respond to the highest frequencies and those at the apex respond to the lowest frequencies9. This tonotopic organization is achieved in part as a consequence of gradual changes in the features of the organ of Corti, such as the width and thickness of the basilar membrane and stereocilia height 9.

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REVIEWS Remarkably, the inner ear amplifies its inputs; this is necessary as sound energy that produces basilar membrane motion would otherwise quickly dissipate owing to viscous damping. The underlying active feedback mechanism is called the cochlear amplifier and depends on OHCs11. When a sound of a specific frequency induces passive basilar membrane motion at a specific position along the cochlear duct, OHCs are activated locally and increase the vibration of the basilar membrane; these vibrations are detected by IHCs that then activate sensory afferent neurons. The cochlear amplifier has a compressive linearity, which results in a much stronger amplification of soft sounds than loud sounds11; the amplification mechanism also dramatically sharpens the frequency selectivity of the cochlea11.

Sensorineural hearing loss The most common form of hearing impairment is sensorineural hearing loss, which is caused by a range of genetic and environmental factors that damage the inner ear or auditory nerve. Forms of hearing impairment that are not discussed in this Review include conductive hearing loss, which occurs when transmission of sound energy from the external world to the inner ear is impaired, as well as Ménière disease and otitis media (BOX 1).

Hypomorphic alleles Alleles of genes that reduce the activity of the gene or of its gene product.

Reactive oxygen species (ROS). Chemically reactive molecules that contain oxygen and have important functions for cell signalling, but in excessive amounts can cause tissue damage.

Genetic forms. Hearing loss that is inherited in the absence of other phenotypic manifestations is referred to as nonsyndromic. In congenital forms of the disease, approximately three-quarters of the cases are inherited in an autosomal recessive manner 4. In most instances, the onset of hearing loss is prelingual, severe and affects all frequencies, but progressive forms have also been observed. By contrast, dominant forms of hearing loss typically have a later onset and tend to be progressive4. Nonsyndromic and syndromic forms of deafness are highly heterogeneous genetically and may arise from mutations in as many as 500 genes12. To date, mutations in 55 recessive, 30 dominant and 3 X‑linked genes have been associated with nonsyndromic deafness, and mutations in nearly 50 genes have been linked to syndromic forms (see the Hereditary Hearing Loss website). In many instances, distinct mutations in a single gene can lead to different disease outcomes. For example, mutations in the gene encoding the molecular motor myosin VIIa (MYO7A) cause nonsyndromic and syndromic forms of the disease and are inherited either dominantly or recessively 5,8,13. Genes that are linked to hearing loss encode proteins with a wide range of molecular functions, including transcription factors, transporters, cytoskeleton components, molecular motors and ion channels (TABLE 1), which suggests that a large number of molecular pathways can be considered as therapeutic targets. Although most of the genes are known to be affected in fairly few individuals, there are notable exceptions; mutations in gap junction B2 (GJB2; also known as connexin 26) cause half of all cases of genetic deafness14. Mouse models. There is a striking conservation between the genetic pathways that regulate auditory perception in mice and humans4–6,8,12, and mutations in more than

50 genes have been linked to hearing loss in both mice and humans. For example, functional null alleles of the Usher syndrome 1C (USH1C), MYO7A and CDH23 genes cause congenital deafness and progressive vision loss (that is, Usher syndrome type I) in humans; in contrast, predicted hypomorphic alleles spare the visual system from damage but cause auditory impairment, which is frequently progressive. In most cases, mice do not exhibit phenocopies of the visual defect, but they are an excellent model for auditory impairment that is caused by mutations in human genes. Indeed, functional null alleles of the mouse Ush1c, Myo7a and Cdh23 genes cause congenital deafness, whereas hypomorphic mutations cause hearing loss that is frequently progressive5,8. In the mouse, disease mechanisms can be explored by analysing the underlying molecular, cellular and physiological changes (FIGS 2,3). Hearing function can be evaluated with hearing tests that are quantitative and non-invasive (FIG. 2), and the effects of therapeutic interventions on these parameters can be evaluated with precision. Histological examination enables the identification of cellular defects, for example, defects in the morphology of hair bundles (FIG. 3). Targets of genetic damage. Most genes that have been linked to genetic forms of hearing loss are expressed in hair cells (TABLE 1), which indicates that this cell type is particularly sensitive to genetic lesions. Not all of the cellular functions of hair cells are equally affected, and the hair bundle seems to be a particularly vulnerable structure. Over 20 genes have been linked to defects in hair bundle formation, maintenance and transduction5,6. Unsurprisingly, many of the genes that are essential for hair bundle function are actin-associated genes; such as MYO6, MYO7A, MYO15A, espin (ESPN) and TRIOBP (TABLE 1). Other affected genes encode scaffolding proteins, which dictate protein-complex formation, including USH1C and USH1G; others encode components of the mechanotransduction machinery in bundles, such as CDH23, protocadherin-related 15 (PCDH15), transmembrane channel-line 1 (TMC1) and lipoma HMGIC fusion partner-like 5 (LHFPL5) (TABLE 1). Thus, the hair bundle provides an attractive target for therapeutic intervention. Mutations have also been identified in genes of the mitochondrial genome and in nuclear genes that regulate mitochondrial function (TABLE 1). Hair cells have a high demand for energy; they are rich in mitochondria and depend on their efficient energy production15. However, mitochondria are the source of reactive oxygen species (ROS), which can cause damage to cells16. Thus, perturbations in mitochondrial function might impair the energy balance in hair cells and affect the integrity of cellular structures. Other elements that are perturbed in genetic deafness include adhesion complexes between hair cells and supporting cells, ion channels, solute transporters, transcription factors and components of the specialized ribbon synapses of hair cells (TABLE 1). Other inner ear cell types and structures are also affected by mutations. Components of the acellular basilar and tectorial membranes, including collagens and tectorins, are frequently affected by gene mutations



REVIEWS Box 1 | Other major inner-ear diseases Simple hearing loss, which is primarily due to the loss of hair cells, is the main complaint of patients with inner-ear disorders, but other inner-ear disorders exist. Two syndromes that are key targets for pharmacological intervention are subjective tinnitus203,204, which is characterized by a persistent ringing in the ears, and Ménière disease205, which has a complex range of symptoms that include vertigo and hearing loss. Otitis media, which is characterized by inflammation in the middle ear, is treated with both drugs and medical interventions.

Tinnitus Tinnitus can be objective or subjective. In objective tinnitus, an actual tone can be detected from a patient’s ear, which is thought to arise from the oscillatory activity of hair cells, owing to an inadequate control of cochlear amplification. The oscillation of hair cells leads to mechanical forces being directed outwards along the mechanical chain of the ear, which leads to eardrum vibration and sound production. Usually these tones are fixed in amplitude and frequency and cannot be heard by the patient. However, it is likely that the transient tinnitus that is heard by individuals with normal hearing, for example, tinnitus lasting 5–10 seconds and then fading away, is true objective tinnitus that is controlled by feedback within the ear. Subjective tinnitus is very common, varies tremendously in severity and is often associated with hearing loss, although it is also a common side effect of medications (for example, aspirin). Although tones or high-frequency white noise are most commonly reported, tinnitus can present as whining, buzzing, clicking, roaring or in many other forms. Although it is most often just an annoyance, tinnitus can in some cases be extremely psychologically disturbing. There is no cure. Tinnitus is thought to arise from the over-activity of auditory brain regions that functioned to process the sound frequencies that are then lost during hearing loss. For example, high-frequency hearing loss is common after the age of 40– 50 years, and tinnitus often presents itself as sound at the frequencies that are no longer detected by the patient. The limbic system seems to have some control over how notable tinnitus is, with high emotions increasing the severity of symptoms for some patients. Ménière disease Ménière disease (also called endolymphatic hydrops) affects the inner ear and can lead to hearing loss, tinnitus and balance impairment with variable severity. Initially, patients experience fairly short periods of vertigo, tinnitus and hearing loss. The onset of vertigo, and the accompanying hearing loss, is unpredictable and can last from minutes to hours, although prolonged attacks that last for days or weeks may occur. The hearing loss may improve after an episode, but it frequently becomes progressively worse206. Ménière disease is thought to be associated with an excess of fluid in the inner ear. The inner ear contains several fluid-filled compartments. Endolymph is secreted from the stria vascularis into the scala media and bathes the stereocilia of hair cells. Endolymph contains a high concentration of K+, the major ion carrier for the mechanotransduction channel of hair cells. Two other fluid-filled compartments of the inner ear, the scala vestibuli and scala tympani, are separated from the scala media and contain perilymph, which has a low K+ concentration. When the volume of endolymph is too high (for example, owing to the drainage problems that are associated with obstruction of the endolymphatic duct or to excessive fluid secretion from the stria vascularis), scala media membranes become dilated and damaged, and endolymph leaks into other compartments of the inner ear206. Other factors, including ischaemia and even autoimmune injuries, may also underlie Ménière disease205.

Endolymph The fluid within the membranous labyrinth of the inner ear that bathes the stereocilia of hair cells.

Gap junctions Specialized intracellular junctions that connect the cytoplasm of two cells and enable the passage of molecules and ions between them.

Fibrocytes Mesenchymal cells that are distributed throughout the inner ear and are thought to be involved in K+ recycling.

Stria vascularis A secretory epithelium of the inner ear that produces and secretes endolymph and also sets the endocochlear potential.

Otitis media Otitis media is particularly common in childhood. Acute otitis media is usually associated with ear pain and perforation of the eardrum can also occur. The disease is most commonly triggered by upper respiratory tract infection and is caused by abnormal function of the Eustachian tube, owing to inflammation. The resulting negative pressure in the middle ear cavity can trigger liquid accumulation, which can become infected by bacteria and sometimes drain outside the ear207. In otitis media with effusion, which often follows an episode of acute otitis media, fluid accumulates in the middle ear; although it may not cause substantial pain, it can thicken over time, leading to conductive hearing loss, tinnitus and vertigo. Other forms of the disease include chronic suppurative otitis media, which is a persistent infection of the ear that is caused by perforation or tearing of the eardrum, and adhesive otitis media, which is associated with penetration of the ear drum into the middle ear space where it adheres to other structures, such as the ossicles. Bilateral or severe unilateral otitis media is treated with antibiotics; tympanostomy tubes (also known as vents, grommets, ear tubes or pressure equalization tubes) are used to treat recurrent otitis media207.

that cause hearing impairment (TABLE 1). K+ ion recycling, which is crucial for hearing function10, is also affected by genetic mutations. Following deflection of the hair bundle by sound-induced mechanical force, K+ in the endolymph enters hair cells, exits passively through basolateral K+ channels and is then transported, via gap junctions, to supporting cells and fibrocytes. From there, K+ moves to the stria vascularis, which then secretes K+ back into the endolymph17. Mutations in the genes that encode two K+ channel subunits, KCNE1 and KCNQ4,

affect K+ recycling and cause hearing loss. Similarly, mutations in the gap junction genes GJB2, GJB3 and GJB6 cause deafness (TABLE 1). Noise-induced hearing loss. Occupational noise exposure is responsible for approximately 10% of hearing loss in adults18, and hearing loss and tinnitus are also the most common service-related disabilities for military veterans19. Short impulses of high intensity noise (beyond 130 dB), such as a gunshot or explosion, can trigger



REVIEWS Table 1 | Genetic forms of hearing loss Molecular function

Gene

Disease

Refs

MYO3A

DFNB30

211

MYO6

DFNA22

212

DFNB37

213

USH1B

214

DFNB2

215,216

Stereocilia Molecular motor

MYO7A

Cytoskeletal

Adaptor

DFNA11

215

MYO14*

DFNA4

217

MYO15

DFNB3

ACTG1

DFNA20/26

RDX

DFNB24

221

TRIOBP

DFNB28

222,223

TPRN

DFNB79

224,225

ESPN

DFNB36

226

USH1C

USH1C

227,228

DFNB18

229,230

DFNB31

231

WHRN

Membrane

Mechanotransduction

218 219,220

USH2D

232

USH1G (also known as SANS)

USH1G

233,234

PTPRQ

DFNB84A

235

GPR98 (also known as VLGR1)

USH2C

236

STRC

DFNB16

237

ILDR1

DFNB42

238

LOXHD1

DFNB77

208

CDH23

DFNB12

239

USH1D

239,240

PCDH15

USH1F

241,242

TMC1

DFNB7/11 DFNA36

243 243

LHFPL5

DFNB66/67

244,245

GJB2

DFNB1A

246

DFNA3A

246

DFNB91

247

DFNA2B

248

DFNB1B

249

DFNA3B

250

CLDN14

DFNB29

251

MARVELD2

DFNB49

252

TJP2

DFNA51

253

KCNQ1*

JLNS1

254

KCNQ4

DFNA2A

255

KCNE1*

JLNS2

256,257

KCNJ10*

EASTS

258

Cell body Gap junction

GJB3 GJB6 Adherens or tight junctions

Ion channels

sudden hearing loss, which is generally irreversible and associated with structural damage to the auditory system20. Unfortunately, because of the widespread damage it causes, impulse-noise damage is difficult to treat. A more promising target population for therapeutic intervention is patients who are exposed to continuous moderate-to-loud noise that does not cause immediate severe structural damage; such damage induces a temporary threshold shift (TTS) or permanent threshold shift (PTS). Recovery from TTS can occur within 24–48 hours21, but PTS is irreversible. Although it is currently unclear whether the mechanisms that lead to TTS and PTS are similar, both forms of hearing loss can be recapitulated in animal models22. However, threshold shift is not always predictive of notable damage. Liberman and colleagues have shown that even if threshold shifts in response to noise fully recover, they are still associated with substantial permanent damage to auditory synapses23–25. Although it is difficult to detect using clinical measures in humans, this cochlear neuropathy may underlie the difficulties in speech discrimination and temporal processing that occur in some individuals26. Susceptibility to damaging effects of noise differs remarkably among individuals, which indicates that genetic factors might be important in disease aetiology. Gene association studies using candidate-gene approaches have focused mostly on genes that are linked to oxidative stress, K+ recycling and the heat shock response, as well as genes that are linked to inherited hearing loss27. The strongest associations have been obtained for KCNE1 and KCNQ4 (genes involved in K+ recycling), the gene encoding catalase (an enzyme involved in oxidative stress), the genes encoding HSP70 (a protein involved in the heat shock response), PCDH15 (a gene involved in hair cell function) and MYH14 (a gene that encodes an actin-dependent myosin motor)27 (TABLE 2). Intriguingly, HSP70 has been implicated in protecting mice from antibiotic-induced damage28, which raises the potential of this stress pathway for therapeutic intervention. Chemically-induced hearing loss. Various pharmacological agents are ototoxic, that is, they can lead to reversible or irreversible loss of auditory (and vestibular) sensitivity. Such agents include aminoglycoside antibiotics, platinum-containing chemotherapy agents, loop diuretics (such as furosemide) and nonsteroidal anti-inflammatory drugs (such as aspirin), as well as quinine and heavy metals. Aminoglycosides have a broad-spectrum of antibacterial activity, which makes them particularly useful in the treatment of various serious anaerobic Gram-negative bacterial infections. Unfortunately, they can cause substantial hearing loss, with estimates of a 20–50% chance of incidence when treating acute infections29–31. Hair cells are readily damaged by aminoglycosides. Evidence suggests that aminoglycosides are preferentially transported into the inner ear extracellular fluid32 and then enter hair cells through their mechanotransduction channels33. Although the intracellular site of action is not



REVIEWS Table 1 (cont.) | Genetic forms of hearing loss Molecular function

Gene

Disease

Refs

ESRRB

DFNB35

259

EYA4

DFNA10

260

POU3F4

DFNA15

261

miRNA

MIR96

DFNA50

262

Mitochondria

PNPT1

DFNB70

263

MT‑TL1

MELAS, MIDD

264,265

MT-TK

MERFF, MIDD

266,267,268

MT‑TS1

PMEAH

Nucleus Transcription

Nonsyndromic

Synapse

Extracellular matrix

269 187,270

MT-TE

MIDD

271

MT‑RNR1

Nonsyndromic

272

MSRB3

DFNB74

273

OTOF

DFNB9

274

COMT2 (also known as LRTOMT)

DFNB63

TECTA*

DFNA8/12

277

OTOA*

DFNB22

278

COCH*

DFNA9

279

OTOGL*

DFNB84B

280

OTOG*

DFNB18B

281

COL4A3, COL4A4 and COL4A5*

Alport syndrome

COL11A2*

DFNB13

275,276

282,283 284

ACTG1, actin G1; CLDN14, claudin 14; COCH, cochlin; COL, collagen; DFNA, autosomal dominant hearing loss; DFNB, autosomal recessive hearing loss; EASTS, epilepsy, ataxia, sensorineural deafness and tubulopathy syndrome; ESPN, espin; ESRRB, oestrogen-related receptor B; JLNS, Jervell and Lange-Nielsen syndrome; KCN, K+ channel; MAH, progressive myoclonic epilepsy, ataxia, hearing impairment; MELAS, mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes; MERRF, myoclonic epilepsy and ragged red fibres syndrome; MIDD, maternally inherited diabetes and deafness; MSRB3, methionine sulfoxide reductase B3; MYO, myosin; OTOA, otoanchorin; OTOF, otoferlin; OTOG, otogelin; OTOGL, otogelin-like; PMEAH, progressive myoclonic epilepsy, ataxia and hearing impairment; PTPRQ, protein tyrosine phosphatase receptor Q; RDX, radixin; STRC, stereocilin; TECTA, tectorin A; TJP2, tight junction protein 2; USH, Usher syndrome; WHRN, whirlin. *These genes are not necessarily expressed in hair cells but affect hair cell function.

Temporary threshold shift A temporary, recoverable loss in sensitivity to sound.

Permanent threshold shift Permanent loss in the sensitivity to sound.

Aminoglycoside antibiotics Antibacterial drugs directed against Gram-negative bacteria that inhibit protein synthesis.

Apoptosis Programmed cell death that relies on the activation of a cascade of events, which are genetically encoded.

firmly established, the similarity of mitochondrial ribosomes to their bacterial counterparts34 and the maternal inheritance of susceptibility to aminoglycosides35 both point strongly to the involvement of mitochondria36. Reinforcing this association, mutations in the mitochondrial genome have been linked to susceptibility to aminoglycoside-induced hearing loss37. Platinum compounds, such as cisplatin and carboplatin, are highly effective against a wide range of malignancies but these drugs are ototoxic, particularly in children38. Although the mechanism of action of platinum drugs in causing cell death — formation of cross-linked adducts with genomic DNA, which leads to apoptosis — is assumed to be similar in both hair cells and cancer cells, hair cells seem to be unusually sensitive owing to their selective transport of the drugs38. Platinum compounds are transported across the blood–labyrinth

barrier into the extracellular fluids of the ear, and then into hair cells via apical trafficking pathways38. Several transport proteins, including low-density lipoprotein receptor-related protein 2 (LRP2), solute carrier family 22 member 2 (SLC22A2) and SLC31A1, have been proposed to mediate the selective uptake of certain molecules into hair cells39–41, but conclusive evidence of their roles in platinum-drug transport is lacking. Various candidate genes have been investigated that might either prevent or contribute to platinum-mediated ototoxicity, including glutathione S-transferase M3 (GSTM3), glutathione S-transferase P1 (GSTP1), SLC31A1, xerderma pigmentosum, complementation group C (XPC) and LRP2 (TABLE 3), but studies have not in general revealed an association38. A wider screen investigating 2,000 single nucleotide polymorphisms (SNPs) in more than 200 genes encoding molecules that are involved in drug metabolism or transport revealed two candidate genes, thipurine S-methyltransferase (TPMT) and catechol-O-methyltransferase (COMT), as possible determinants of platinum-mediated ototoxity 42 (TABLE 3); a later study confirmed the association of TMPT and COMT, and provided evidence for the association of ATP-binding cassette, sub-family C (CFTR/MRP), member 3 (ABCC3) (REF. 43). However, confounding factors make these linkages tenuous38. Age-related hearing loss. Age-related hearing loss (ARHL) is characterized by symmetric sensorineural hearing loss that starts primarily with high frequencies. It is the most common form of sensory impairment in older people and can have detrimental effects on quality of life44,45. The age of onset, progression and severity show substantial variation. Approximately 35% of individuals over 65 years of age are affected, and 50% of all octogenarians suffer from ARHL46,47. The three main types of ARHL have been correlated with specific locations of pathological changes in the ear: sensory, which exhibits hearing loss of high-frequency sound, hair cell loss and subsequent neuronal degeneration; neural, which exhibits loss of word discrimination and primary neuronal degeneration; and metabolic, or striatal, which exhibits a flat pure-tone audiogram and atrophy of the stria vascularis48–50. Additional types include mechanical (changes in the basilar membrane), mixed and undetermined ARHL50,51. Although ARHL is difficult to subdivide by histopathologic criteria52, this classification scheme nevertheless highlights its complexity, as it can arise as a result of damage to different parts of the ear. Although hearing loss has been considered to be part of a natural ageing process, not all humans suffer from ARHL; heritability studies suggest that the source of variability is both genetic and environmental 53. Studies with the Framingham cohort demonstrated that heritability in metabolic ARHL is 53% between sisters and 35% between mothers and sisters54. In a Swedish study of twins, the heritability was 47%55, and a Danish study reported heritability of 40% in twins 75 years of age and older56. Genome-wide association studies (GWAS) for ARHL have been carried out in



REVIEWS a Auditory brain stem response 120

I

II

III

C57BL/6J Sba/Sba

100

ABR threshold (dB)

8 mV IV

80

dB

ABR (mV)

90

70

0

2

4

6

8

80 60 40 20 0

10

C57BL/6J

Time (ms)

+/Sba Sba/Sba Mouse line

b Distortion product otoacoustic emission 70

f1

f2

80 dB DPOAE threshold (dB)

DPOAE level (SPL dB)

2f1–f2

L1 = L2 = 70 dB

L1 = L2 = 45 dB

0

10

20

30

40

50

60 50 40 30 20 10 0

Frequency (kHz)

(SNPs). Variation in DNA sequences in which a single nucleotide differs in the genome between members of a species.

Audiogram A graph that represents the response of the auditory system to a range of standardized frequencies and intensities, which is used to determine the sensitivity of the auditory system to sound.

Genome-wide association studies (GWAS). A methodology that scans markers across the entire genome to identify genetic variations that are associated with genetic traits, including susceptibility to hearing loss.

8

10

12

14

16

20

28

Frequency (kHz) +/Sba

Single nucleotide polymorphisms

6

+/Sba

Sba/Sba

Sba/Sba

Figure 2 | Evaluation of hearing function in mice. a | A measurement of the auditory brain stem response (ABR) is Nature Reviews |intensity Drug Discovery shown. Mice are anaesthetized and a loudspeaker is inserted into the ear canal. Sounds of decreasing are applied. Electrodes are attached externally behind the ear to measure neuronal activity. The ABR assay establishes auditory thresholds, which are the lowest sound pressure levels (in decibels, dB) that evoke a neuronal signal. Left panel, representative ABR recordings show that in homozygous samba (Sba/Sba) mice lines, a model for the disease autosomal recessive hearing loss 77 (DFNB77), no ABR can be evoked. Peak I represents auditory nerve activity and peaks II–IV represent nerve activity in the brain along the auditory pathway. Right panel, quantifications for control C57BL/6J mice, +/Sba mice and Sba/Sba mutants; auditory thresholds are elevated in Sba/Sba mice. b | Compromised outer hair cell (OHC) function leads to defects in the amplification of acoustic signals, which can be determined by measurements of the distortion product otoacoustic emission (DPOAE). Loudspeakers are inserted into the ear canal and two sound signals of different frequencies (f1 and f2) are applied. OHCs detect and amplify these signals, thereby generating a signal at the cubic distortion frequency (2f1–f2) that is emitted from the ear and can be recorded. Left panel, DPOAE response spectra from 3‑week-old wild-type and Sba/Sba mice at a single stimulus condition (median primary frequency = 16 kHz). The cubic distortion product (2f1–f2), which is present in the wild-type mice (red arrows), is absent in the mutants (grey arrows). Right panel, quantified DPOAE thresholds in 3‑week-old mice reveal elevated thresholds in Sba/Sba mice at all analysed frequencies. Note that the y-axis ends at 70dB. DPOAE thresholds that are beyond 70dB are cut off at 70dB and represent a profound lack of response to all tested sound intensities. SPL, sound pressure level. Error bars are mean ± standard deviation. Parts a and b: adapted with permission from REF. 208, Elsevier.

European populations57–59. Highly associated SNPs in the gene encoding metabotropic glutamate receptor type 7 (GRM7) have been identified60 (TABLE 4); a population follow‑up study in the United States confirmed this association61. In a Finnish population, a highly associated SNP was identified in the gene encoding the RAS GTPase-activating-like protein (IQGAP2)58 (TABLE 4), but the association has yet to be confirmed in other populations. Other candidate-based genetic studies have established associations between ARHL and

various genes, including those that are linked to oxidative stress, such as arylamine N‑acetyltransferase 2 (NAT2)62–64 and GST genes63; transcription factors, such as grainyhead-like protein 2 homologue (GRHL2)59; potassium homeostasis molecules, such as KCNQ465; the vasoactive peptide endothelin (EDN1)66; mitochondrial uncoupling protein 2 (UCP2)67; and mitochondrial DNA mutations68–70 (TABLE 4). Further studies in different populations will be important in defining the significance of these genetic variations.



REVIEWS Control

Myo7a mutant

F-actin

8 µm

SEM

5 µm

Figure 3 | Analysis of hair bundle morphology in mouse models. After the inner ear is dissected and the sensory epithelium is exposed, hair cell stereocilia can be visualized by Nature Reviews | Drug Discovery staining with fluorescence-labelled phalloidin (green, upper panels) or by scanning electron microscopy (SEM) (lower panels). Note the disorganization of the stereociliary bundles in a mouse with a mutation in myosin VIIa (Myo7a), a model for Usher syndrome 1C (USH1C) and autosomal recessive hearing loss 2 (DFNB2) diseases in humans. Figure adapted with permission from REF. 209, Society for Neuroscience.

Studies in mice support the notion of genetic predispositions to ARHL. Among 80 tested inbred mouse strains, 19 strains showed early-onset hearing loss, whereas 16 strains exhibited hearing loss at an older age71,72. These studies were subsequently extended73,74 and the results were meta-analysed75. In total, 18 ARHL loci (Ahl) have been mapped (see the Jackson Laboratory Hereditary Hearing Impairment in Mice website), and 6 of the affected genes have been identified. These encode the tip-link component CDH23 (encoded by Ahl1), ATP-citrate synthase (encoded by Ahl4), the scaffolding protein GIPC3 (encoded by Ahl5), the actin-bundling protein fascin 2 (FSCN2; encoded by Ahl8), the G‑protein coupled receptor 98 (GPR98; also known as VLGR1) and the protein encoded by the mitochondrial Table 2 | Genetic susceptibility and noise-induced hearing loss Gene

Function

Population

KCNQ4

K+ ion recycling

Swedish and Polish

285,286

KCNE1

K+ ion recycling

Swedish and Polish

285,286

CAT

Oxidative stress

Swedish and Polish

287

PCDH15

Hair cell development and mechanotransduction

Swedish and Polish

288

MYH14

Myosin motor

Swedish and Polish

HSP70

Stress response

Swedish, Polish and Chinese

CAT, catalase; HSP70, heat-shock protein 70 family; MYH14, myosin heavy chain 14.

Refs

288 289,290

tRNA-Arg gene. Although there was no overlap with genes that have been identified in humans, studies in both mice and humans have probably only scratched the surface of identifying the genes associated with ARHL; it seems likely that hundreds of genes could be involved. Regardless, the evaluation of human-population polymorphisms in the genes that have been associated with ARHL in mice may be informative. These studies demonstrate the great variability in the genetic landscape of ARHL and indicate that functional defects in multiple molecular and cellular processes can contribute to the disease. The mouse is a useful model for the disease, and different inbred mouse strains have been used to model various forms of ARHL. For example, C57BL/6J mice exhibit neural ARHL that is accompanied by broad degenerative changes in the cochlea and auditory nerve76. By contrast, CBA/CaJ inbred mice retain hair cells and neurons but show a substantial decline in endocochlear potential, thus making this strain a model for striatal ARHL76. Other inbred strains show distinct pathological changes in the organ of Corti, spiral ganglion neurons or the lateral wall77. Diagnosing hearing loss. In humans, the diagnosis of hearing loss is still fairly imprecise. The two most common tests are conventional audiometry, which tests thresholds in the 250–8,000 Hz range, and distortion product otoacoustic emissions (DPOAE) testing, which examines the function of OHCs. Although it is much less commonly used, extended high frequency audiometry, which tests the range from 8,000–16,000 Hz, can be useful in detecting early signs of hearing loss78. With the advent of next-generation sequencing and the identification of many mutations that cause hearing loss, genetic screening for common mutations is now feasible and carried out in some clinical environments79,80. As sequencing costs continue to drop, and the expertise for sequence analysis spreads, genetic testing is likely to play an increasing part in the diagnosis of hearing loss. Eventually, knowledge of the common genetic variants that predispose individuals to hearing loss will allow susceptible individuals to protect their hearing.

Regenerative therapy: hair cell regeneration Most hearing loss results from irreversible damage to hair cells. Unfortunately, because cochlear hair cells do not regenerate in mammals, hearing loss that results from the death of hair cells is permanent81. However, regeneration and transplantation strategies are being pursued for hair cell replacement. The regeneration strategy derives from the observation that the inability to replace hair cells after noise or ototoxin damage is not universal; birds, fish and amphibians all readily regenerate their hair cells, and even the adult mammalian vestibular system shows limited hair cell regeneration81. The right cocktail of reagents might plausibly trigger the regeneration of mammalian cochlear hair cells from precursor cells that are already present in the organ of Corti. By contrast, the transplantation strategy uses either inner-ear stem cells or pluripotent stem cells that are coaxed into a hair cell lineage, derived either from embryonic stem cells or induced



REVIEWS Table 3 | Genetic susceptibility and platinum-induced ototoxicity Gene

Function

Population

Refs

GSTM3

Detoxification and protection from ROS

German

291

GSTP1

Detoxification and protection from ROS

Norwegian

292

SLC31A1

Copper transport

Chinese

293

XPC

DNA repair

LRP2

Endocytosis

German

TPMT

Thiopurine metabolism

Canadian

42,43

COMT

Neurotransmitter metabolism and hormone metabolism

Canadian

42,43

ABCC3

ABC transporter

Canadian

42,43

294 40

GST, glutathione S-transferase; LRP2, Low-density lipoprotein-related receptor 2; ROS, reactive oxygen species; SLC31A1, solute carrier 31A1; TPMT, thiopurine S-methyltransferase; XPC, xeroderma pigmentosum complementation 3.

pluripotent stem cells; in either case, cells would be transplanted into the inner ear to replace lost hair cells82. Some progress has been achieved with both strategies, although neither is close to becoming a therapeutic reality 83.

Serotypes Groups of viruses or microorganisms that can be distinguished by shared specific antigens, which are determined by serological tests.

Pillar cells Supporting cells within the sensory epithelium of the inner ear that form the walls of a fluid-filled tunnel between the inner and outer hair cells.

Deiters’ cells Supporting cells within the sensory epithelium of the inner ear that sit on the basilar membrane and hold the base of outer hair cells. They also form apical processes that extend next to the apical surfaces of outer hair cells.

ATOH1‑dependent hair cell production. Evidence suggests that hair cell differentiation is controlled by a single master switch, the basic-loop-helix transcription factor atonal 1 (ATOH1; see FIG. 4). ATOH1 is located downstream of the genes encoding sex determining region Y-box 2 (SOX2)84, EYA1 and SIX1 (REF. 85), and its expression is necessary for the differentiation of hair cells during development 86. Moreover, the transcription factors POU4F3 and GFI1, which are also necessary for differentiation and are needed to maintain the hair cell phenotype87–89, require ATOH1 for their expression90–92. The expression of ATOH1 in some cell types of the inner ear is sufficient to induce the formation of hair cells93–95. Moreover, the conversion of inner ear cells to hair cells by ATOH1 can, in some cases, lead to functional recovery in both auditory 96,97 and vestibular 98,99 systems. Together, these data suggest that delivering ATOH1 to cells of the damaged inner ear might drive those cells sufficiently towards a fully fledged hair cell fate that could restore hearing and balance. Unfortunately, despite seeming to be a promising approach, there is relatively little evidence that ATOH1 transfection can produce substantial numbers of hair cells in adult animals; moreover, the continued expression of ATOH1 in hair cells leads to notable cell death100,101. Because ATOH1 may mediate only one of several parallel pathways that are used for hair cell differentiation85, targeting other molecules, as well as confining ATOH1 activation to a narrow time window, is likely to be necessary in order to stimulate hair cell formation. Although efforts are underway to modulate ATOH1 pharmacologically, the activation of ATOH1 function could have unintended consequences. Given that ATOH1 has other functions within the body, systemically applied drugs might have substantial side effects. Instead, local applications of reagents to the inner ear, for example using viruses, may be a safer approach. In

animal models, adenovirus vectors have been used for ATOH1 delivery 96,102. Viral transfection using adenoassociated virus (AAV) is also promising; in animal models, gene delivery by AAV has been very successful in the retina103 and in the inner ear 104. Multiple AAV serotypes that target different spectra of inner-ear cell types are available and potentially enable cell-selective ATOH1 expression105. ATOH-intersecting pathways. ATOH1 activation leads to the expression of Notch-family ligands of the Delta family in hair cells, which in turn activates Notch signalling and repression of the hair cell-differentiation programme in supporting cells106. Targeting the Notch pathway could therefore be useful to augment ATOH1 function. Indeed, in early postnatal mice, the inhibition of Notch signalling permitted hair cell regeneration following ototoxic damage107. WNT signalling is similarly intertwined with ATOH1 action. ATOH1 must be activated by the canonical WNT effector β-catenin at the appropriate time for hair cell differentiation to proceed108–110. WNT ligands are thought to be secreted from cells that are outside the sensory epithelium111, and their production may cease in the adult ear. Alternatively, structural changes that occur in the cochlea during early postnatal development could present a physical barrier to WNT ligand diffusion. Thus, exogenous activation of canonical WNT signalling may be another necessary step in the triggering of hair cell regeneration. In addition, control of the cell cycle is essential for hair cell differentiation. Although supporting cells can transdifferentiate into hair cells in chicks112 and mice107, the supporting cells are depleted in the process. The triggering of supporting-cell division may, therefore, be necessary to maintain the population of supporting cells in the cochlea and to maximize the number of hair cells that are produced. Multiple cell-cycle inhibitors that are expressed as progenitors differentiate into hair cells, and gene knockout of some of these inhibitors increases the proliferation and population of hair cells113–115. These observations suggest that strategies to overcome the blocks to cell division may enhance ATOH1‑dependent reprogramming. Targets for cellular conversion. A key question is whether the differentiated supporting cells in the cochlea (for example, pillar cells, Deiters’ cells and phalangeal cells) are viable targets for conversion to hair cells — directly or through de‑differentiation — or whether other fairly de‑differentiated cells that can be directly targeted are present in the cochlea. In other organs, the WNT pathway component, LGR5, is a marker of stem cells116. In the neonatal mouse cochlea, LGR5‑positive inner pillar cells and Deiters’ cells from the third row are proposed to be the sources of most of the new hair cells that grow following ototoxic damage117. Nevertheless, although LGR5‑positive Deiters’ cells exist into adulthood118, the refractoriness of adult cochleas to hair cell regeneration shows that LGR5 expression alone is insufficient to drive the process of differentiation.



REVIEWS Table 4 | Genetic susceptibility and age-related hearing loss Gene

Function

Population

Refs

GRM7

Neuronal excitability and synaptic transmission

European and American

60,61

IQGAP2

GTPase activating-like protein

Finnish and Sami

NAT2

Detoxification and ROS

Turkish

GSTM1 GSTT1

Detoxification and ROS

American

63

GRHL2

Transcription factor

European

59

KCNQ4

K+ ion recycling

American

65

EDN1

Vasoactive peptide

Japanese

66

UCP2

Mitochondrial uncoupling protein

Japanese

Cdh23

Tip link component

Mice

295

Cs (also known as Ahl4)

Citrate synthase

Mice

296

Gipc3

Scaffolding protein

Mice

297

Fscn2

Actin bundling protein

Mice

298

Gpr98 (also known as Vlgr1)

Ankle link component

Mice

299

tRNA-Arg (also known as mt-Tr)

Arginine tRNA

Mice

300

58 62,64

67

EDN1, endothelin 1; Gipc3, glypican 3; GRM7, glutamate receptor; GST, glutathione S-transferase; KCN, K+ channel; NAT2, N-acetyltransferase 2; ROS, reactive oxygen species; UCP2, mitochondrial uncoupling protein 2.

The block to regeneration. Because pathways that promote hair cell regeneration may be suppressed in mammals, targeting of the suppressive regulatory mechanisms could overcome the block to regeneration119. The comprehensive molecular profiling of responses to hair cell damage in regenerating and non-regenerating animals may highlight these pathways. Although multiple factors could together be responsible for poor hair cell regeneration in mammals, including Notch inhibition, absent WNT signalling and active cell-cycle inhibitors, the mechanisms blocking regeneration in adult mammals might not be molecular 119. Interesting suggestions include the barrier to diffusion that is presented by the basilar and tectorial membranes120, as well as a mechanical block to regeneration that is mediated by the greatly expanded apical actin network of supporting cells119.

Regenerative therapy: transplanted stem cells Another major strategy for the restoration of hearing loss that is due to the loss of hair cells would be to introduce stem cells into the damaged ear, with the aim that they differentiate into bona fide hair cells once they are in the right environment. Stem cells could conceivably be derived from pluripotent stem cells, such as embryonic stem cells121 or induced pluripotent stem cells122; alternatively, stem cells could be isolated from the inner ear. Although recent reports have suggested that hair celllike cells can be made in vitro123,124, for therapy it is most likely that stem cells would be guided into progenitors in vitro. Once introduced into the ear, the progenitor cells may then differentiate into hair cells. Introduction of the appropriate environmental cues might also be necessary. Inner-ear-resident stem cells. Multipotent stem cells that can differentiate into hair cell-like cells apparently do exist in the adult inner ear. Initial reports indicated

that cells exhibiting stem cell-like properties could be isolated from the mammalian inner ear and that, when transferred to cultures or transplanted into the ear, they developed hair cell characteristics125,126. Indeed in humans, stem cell-like cells have been isolated from the inner ears of fetuses and adults127,128. However, routinely isolating human inner-ear stem cells is difficult, and also raises ethical issues, which makes this an approach that is unlikely to be clinically useful. Induction from pluripotent stem cells. Embryonic or induced pluripotent stem cells have been induced to develop into cells that have many crucial characteristics of hair cells, including mechanotransduction and basolateral currents123,124,129 (FIG. 4). The key to successful shepherding to the hair cell-like state was the stepwise delivery of signals that mimic those that are present in normal hair cell development, although the total yield of hair cells was low. The use of a three-dimensional culture matrix has led to a remarkably robust generation of hair cell-like cells124 (FIG. 4). Although the hair cell-like cells took on characteristics of developing hair cells, with most resembling vestibular hair cells, these are highly promising results. Promising approaches and pitfalls. Even if an appropriate stem-cell population that could produce hair cells could be isolated or generated, the delivery of cells into the inner ear represents an enormous challenge. Most of the cells that are transplanted into the inner ear are not successfully integrated into the organ of Corti and many die130. Regardless, the successful formation of hair celllike cells from stem cells is a promising development for drug screening. With sufficient scaling, cell-based assays could be developed to test the potency of drugs to stimulate or block hair cell development and regeneration.



REVIEWS a

b

↓ ATOH1 ↑ Notch

SOX2

Hair cell or supporting cell decision

SOX2 EYA1–SIX1 Progenitor cell

Supporting cell

↑ ATOH1 ↓ Notch

0 ESC or iPS cell

POU4F3 GFI1

5

Days in vitro 8

Floating

Adherent

DKK1 SIS3 IGF1

bFGF

20 Adherent

Cell state

No growth factors Growth or (or stromal cell differentiation conditioning) factor

Hair cell WNT

c

d

e

Days in vitro 0

ESCs

0 1 DE

NE

BMP

g FGF

Non-sensory

Cranial placodes

Sensory

ME

3

4

NNE

PPE

f

TGFβ

BMPi and FGF

EPI

5

8

OEPD Endogenous WNT

Otic vesicles

8–14

Hair cells, support cells and neurons

14–20

5 µm

5 µm MYO7A

SOX2

MYO7A

F-actin

Inner ear organoid

18–30

Figure 4 | Production of hair cells. a | ATOH1 is the master switch for hair cell development. Progenitor cells in the inner ear express sex determining region Y-box 2 (SOX2) and EYA1–SIX1, which can together trigger the expression of Natureand Reviews | Druglevels Discovery ATOH1. ATOH1 and Notch signal reciprocally in adjacent cells, with cells having high ATOH1 low Notch becoming hair cells and cells that have low ATOH1 and high Notch levels becoming supporting cells. SOX2 continues to be expressed in supporting cells, but the presence of ATOH1 in hair cells leads to expression of the transcription factors POU4F3 and GFI1, which control differentiation of the hair cell. Ectopic expression of ATOH1 is sufficient to drive production of new hair cells; the inhibition of Notch facilitates formation of hair cells. WNT (and other signalling molecules) control developmental progression towards hair cells in several ways, including the direct regulation of ATOH1 expression through the control of β‑catenin. b | The procedure used by Oshima, Heller and colleagues to make hair cell-like cells123. c, d | Micrographs of hair cells created with the Oshima protocol. e | Protocol used by Koehler and Hashino124,210 to make inner ear organoids containing many hair cell-like cells. Dashed arrows indicate alternative differentiation pathways. f | Example of an inner ear organoid showing domains of non-sensory and sensory cells. SOX2 marks supporting cells and MYO7A marks hair cells. g | Intermediate-magnification view of the sensory region of an organoid; staining with phalloidin (green) to reveal actin in stereocilia and with MYO7A‑specific antibodies (red) to mark hair cell-like cells. Hair bundles are very prominent. bFGF, basic fibroblast growth factor; BMP, bone morphogenetic protein; DE, definitive ectoderm; DKK1, Dickkopf-related protein 1; EPI, epidermis; ESC, embryonic stem cell; FGF, fibroblast growth factor; IGF1, insulin-like growth factor I; iPS cell, induced pluripotent stem cell; ME, mesendoderm; NE, neural ectoderm; NNE, non-neural ectoderm; OEPD, otic-epibranchial placode domain; PPE, preplacodal ectoderm; SIS3, selective inhibitor of SMAD3; TGFβ, transforming growth factor-β. Parts a, c and d: Reprinted from Cell, 141, Oshima, k., Shin, K., Diensthuber, M., Peng, A. W., Ricci, A. J. & Heller, S., Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells, 704–716, Copyright (2010), with permission from Elsevier. Part e is modified from, and parts f and g are reproduced from, REF. 210, Nature Publishing Group.



REVIEWS Although hair cells are an important target, progress towards therapy is more promising for the restoration of auditory neurons. Human embryonic stem cells could be induced to develop into otic neural progenitors or into otic epithelial progenitors — from which a small subpopulation of hair cell-like cells and neurons could be generated131. Importantly, when transplanted into animals with spiral ganglion neuron damage, the cells not only incorporated into the ganglion but also generated substantial functional recovery 131. Although it is a promising approach, regenerative therapy must be considered in the context of different forms of hearing loss. Although in many forms of hearing loss hair cells are affected, other structures, such as the auditory nerve or the stria vascularis, can also be involved23,132. Clearly, one treatment may not fit all. At present, cochlear implants work well for congenital prelingual hearing loss, but regenerative therapy might eventually lead to a greater restoration of hearing acuity. Progressive, age-related and noise-induced hearing loss (NIHL) each pose specific challenges; patients either show widespread damage (that is, trauma) or come to the clinic only when hearing loss is substantial and associated with secondary degenerative changes. Indeed, it will be challenging to restore function in a cochlea that has sustained substantial degenerative changes.

Gene therapy Hearing loss arising from inherited mutations in key genes might be alleviated by gene therapy, as indicated by mouse studies discussed below. Successes in gene therapy for disease in other organs, such as the retina133, have suggested plausible therapeutic strategies that can be applied to the inner ear. Gene therapy is unlikely to be as widely applicable to hearing loss as a viable hair cell-regeneration strategy would be, but nonetheless, it may be very effective in some cases134,135. Functional rescue by restoring genes. In most examples of recessive inherited deafness, the restoration of hearing will require delivery of the missing gene or gene product. In a recent model, a null allele of solute carrier family 27 (Slc17a8; also known as Vglut3) was rescued using wild-type Slc17a8 that was delivered selectively to inner hair cells by AAV1 (REF. 104). Although the viruses used for rescue were delivered when the mice were less than 2 weeks of age, functional rescue nevertheless lasted for more than a year in some cases. The extension of these studies to humans will not be simple, but it is certainly plausible. Functional rescue by inhibition or editing. As many forms of deafness are dominantly inherited, a potential therapeutic strategy is to selectively inhibit the dominant gene product to enable the remaining wildtype copy to restore function. In a proof‑of‑principle experiment, a dominant mutant form of the gap junction gene Gjb2 was expressed in mouse cochlea, thus elevating hearing thresholds; however, if an siRNA specifically targeting the dominant negative construct was co‑transfected, hearing thresholds were restored136.

This experiment indicates that knockdown of dominant gene products might provide a potential therapeutic route for some forms of hearing loss. In a more clinically relevant test, antisense oligonucleotides were used to correct splicing defects in a mouse model for the 216Ala mutation in the human USH1C gene137. This mutation is produced when a cryptic 5′ splicing site is generated that is favoured over the wildtype 5′ splice site at the end of exon 3, which leads to a frameshift and a truncation of the protein product of USH1C (that is, harmonin). The systemic delivery of an antisense oligonucleotide that corrects splicing defects led to partial structural and functional recovery in the cochlea, which lasted for at least several months137. Although hereditary deafness that arises from correctable splicing errors is uncommon, this study represents a notable success. In utero gene therapy. Although still a distant goal for human gene therapy, animal models for in utero gene therapy are being developed. Initial experiments demonstrated that when plasmids were delivered to the otocyst of an embryonic day 11 (E11) mouse they directed protein expression in a substantial fraction of hair cells, even as late as 30 days after delivery 95. In a proof‑of‑principle experiment, in utero electroporation was used both to knockdown Gjb6 with short hairpin RNAs and to rescue the auditory deficit by reintroducing Gjb6 (REF. 138). Correcting genetic defects in human embryos using similar in utero techniques may someday prove viable135, although technical and ethical issues are considerable. Challenges. Although mouse models are valuable for testing possible therapeutic strategies, the delayed onset of functional maturation in mice (hearing develops during the second postnatal week) and humans (hearing develops during the third trimester of pregnancy) makes it difficult to directly translate the proof‑of‑principle studies from mice to humans. In addition, for mutations that cause profound deafness, gene delivery strategies may never be successful, as the loss of critical hair cell genes often leads to a complete loss of hair cells and all morphological specializations in the ear. Delivery of the wild-type gene must occur in the context of a normal hair cell or progenitor, which limits the time window when gene therapy is effective. Progressive hearing loss, which can take decades to develop, would be a better target for this approach, although recessive progressive hearing loss is rare compared with dominant progressive hearing loss4.

Pharmacological approaches Hearing loss would be ideally treated with orally delivered pharmacological compounds. Although the blood– labyrinth barrier (located within the inner ear) could limit the access of some drugs, direct drug delivery to the inner ear, either by the non-invasive intratympanic or the invasive intracochlear route, is also a feasible alternative option139. Patients who are the most likely to respond well to pharmacological treatment of hearing



REVIEWS • GFHL • NIHL • DIHL • ARHL

• Structural/ functional damage • ROS • Apoptosis

cancer research, strategies have been developed to interfere with the binding of transcription factors to DNA; conversely, transcription factors with desired DNA binding specificities have been engineered143. Without a doubt, repair of damaged structures in the hair cell must rely on the activation of endogenous programmes of cellular homeostasis.

• Regenerative therapy • Gene therapy • Antioxidants • Anti-apoptotics Tectorial membrane Outer hair cell

Inner hair cell Synaptic defects Nerve fibre

K+

Basilar membrane • GFHL • NIHL • ARHL

• Neurotrophin • Neurotransmitter receptor modulators

K+

• GFHL • NIHL • ARHL • Gap junctional modulators • Potassium channel modulator

Figure 5 | Common defects in hearing loss and potential therapeutic approaches. The organ of Corti is prominently affected in various forms of hearing loss. Hair cells are Nature Reviews | Drug Discovery frequently affected in genetic forms of hearing loss (GFHL), noise-induced hearing loss (NIHL), drug-induced hearing loss (DIHL) and age-related hearing loss (ARHL). Genetic defects or environmental insults may affect hair cell structural integrity and/or lead to the production of reactive oxygen species (ROS), which ultimately cause cell death by apoptosis. Ribbon synapses between inner hair cells and afferent neurons, as well as K+ recycling, are thought to be frequently affected in GFHL, NIHL and ARHL. Potential therapeutic approaches are shown.

loss are those with limited pathological changes or slow progressive hearing loss that is accompanied by the gradual decline of cellular structure and function, such as patients with ARHL. Provided that the damage is limited, environmentally induced hearing loss might also be a good target for pharmacological intervention. Some forms of noise exposure lead to an impairment of hearing function that is only transient (that is, a TTS occurs), which indicates that there are intrinsic protective and regenerative processes which can restore auditory function following injury 20,22. Although no Food and Drug Administration (FDA)-approved drug has yet reached the market to treat hearing loss, several potential target areas for drug discovery are discussed below (FIG. 5). Small molecule and regenerative approaches. Hair cell damage following decades of exposure to chronic occupational noise is prevalent in humans; damage to OHCs is greatest, whereas loss of IHCs is more limited140. In one study, degenerative changes in the auditory nerve corresponded in severity with loss of OHCs in humans141; a similar pattern of degenerative changes has been observed in rodents142. It is difficult to envision a therapeutic approach that relies on small molecules to repair hair cells or their intricately structured hair bundle. Perhaps regenerative approaches can be enhanced with pharmacological approaches; for example, small molecules could be developed to regulate the expression of genes that are important for hair cell development and function, such as the transcription factors ATOH1 and POU4F3. In

Potassium recycling and homeostasis. Disruption of the mechanisms that mediate recycling of K+ ions in the cochlea leads to hearing loss. Mutations and polymorphisms in gap-junction proteins and K+ channels have been implicated in congenital hearing loss, NIHL and ARHL (TABLES 1–4). Targeting of ion channels or gapjunctional pathways that are involved in K+ transport may, therefore, be useful strategies for pharmacological intervention. To modulate their activity, voltage-gated potassium channels have been targeted with venom peptides, antibodies or small molecules144. For example, ezogabine enhances KCNQ ion channel activity and is FDA-approved as an antiepileptic drug 145. Similarly, modulators of gap junctions have been described. Rotigaptide is a small peptide modulator of gap junction function in cardiac muscle146 and has been clinically tested in cardiac arrhythmias. Tonabersat also targets gap junctions and has been evaluated for migraine treatment 147. Other modulators of gap junction function have been tested for applications in other areas, such as as inflammation148, but there are no published reports describing their use for treating hearing loss. Mitochondrial function and reactive oxygen species. The overproduction of ROS may be involved in the pathogenesis of sensorineural hearing loss. Mitochondria play a pivotal role in ROS generation, and mutations in genes that affect mitochondrial function have been linked to genetic forms of hearing loss (TABLE 1). ROS that are generated in mitochondria are hypothesized to damage key cellular components, including nuclear and mitochondrial DNA, as well as proteins and membranes149. In animal models, noise exposure induces ROS generation in the inner ear, which persists at high levels for 7–10 days20, as well as hair cell loss. As the intensity of noise and the length of exposure increase, damage becomes more extensive and irreversible. Damage includes impaired blood flow to the cochlea, fused hair cell stereocilia and degeneration of supporting structures and nerve fibres; degenerative changes are also observed in the stria vascularis20. Many of these consequences are thought to arise from ROS. Therapy for ROS-mediated NIHL is most likely to be successful during the early stages of disease progression, when structural damage and hair cell loss is limited. A few clinical and military trials have been carried out for TTS, in which administration of nutritional supplements, such as Mg 2+ or vitamin B12, before moderate noise exposure showed some beneficial effects150. It is more desirable and practical, however, to develop a therapy to treat patients following noise exposure. Animal models have been useful for assessing whether counteracting the effects of ROS might be of therapeutic



REVIEWS value. Antioxidants, such as glutathione, d‑methionine, ebselen, resveratrol and ascorbic acid, all attenuated NIHL in animal models when given before noise exposure20. In some studies, compounds such as A1 adenosine receptor agonists, ferulic acid, d‑methionine and N‑acetylcysteine attenuated NIHL when given up to 3 days after noise exposure151–154. Interestingly, polymorphisms in the gene encoding catalase have been linked to an increased susceptibility to NIHL in humans27, and mice that are heterozygous for a mutation in Sod1 show an increased vulnerability to NIHL155. These genetic findings provide additional evidence that antioxidant enzymes might be crucial for maintaining normal hearing under loud noise conditions. Similar to their importance in NIHL, ROS have been proposed to play a major part in ARHL. Studies in animal models support a role for ROS in ARHL. For example, age-related loss of hair cells was accelerated in Sod1 mutant mice155–157, and mice with defects in vitamin C synthesis showed increased hearing thresholds and loss of spiral ganglion neurons158. Conversely, C57BL/6 mice overexpressing catalase in their mitochondria had decreased oxidative DNA damage and slowed progression of ARHL159. Finally, a diet containing specific antioxidants slowed down ARHL in rats160–163. Thus, targeting members of antioxidant pathways, including the enzymes that are involved in glutathione metabolism and in the breakdown of superoxide anions and hydrogen peroxidase, could be feasible options for the treatment of NIHL and ARHL. Interestingly, analysis of human archival temporal bone samples revealed that a 4,977-bp deletion, called the ‘common deletion’, was markedly more frequent in the cochlea of patients with ARHL compared with unaffected individuals164. This further suggests that mitochondria, which are important in regulating ROS levels, have an important role in ARHL.

Auditory brain stem response Electrical potentials that are measured with electrodes placed on the scalp while the ear is stimulated with sound of defined intensity and frequency using a loudspeaker.

Cell death. A hallmark of both NIHL and ARHL is the death of hair cells. Several animal studies show protection against, or enhanced recovery from, NIHL when apoptotic cascades are blocked, for example, by the inhibition of mitogen-activated protein kinase (MAPK) or Jun N-terminal kinase (JNK)165–168. ARHL was slowed in mice lacking BAK, a mitochondrial protein that is important for apoptosis regulation162. In a clinical study, thymidylate kinase (TMK; also known as AM111), a membrane-permeable, anti-apoptotic JNK ligand, seemed to prevent some hearing loss that occurred post trauma169, which reinforces the therapeutic value of interfering with apoptosis. Indeed, drugs that target apoptotic pathways have been used in some preclinical and clinical studies for other diseases, including cancer 170,171. Unfortunately, many of these drugs actually enhance apoptosis and thus would not be useful for the treatment of hearing loss. Chemically induced hair cell damage. Efforts aimed at preventing aminoglycoside ototoxicity have focused on intercepting or preventing the damaging effects of ROS. In animal models, protection against aminoglycoside ototoxicity has been demonstrated by iron chelators, as

well as by a wide array of antioxidants (such as lipoic acid, α‑tocopherol, ebselen, d‑methionine and salicylates)172. Few of these compounds have been tested in humans, although favourable results were seen in a randomized double-blind trial with salicylate173. In two small studies, N‑acetylcysteine174 and vitamin E175 were shown to be effective and ineffective at preventing hear loss, respectively. However, it is a concern that protection against ototoxicity may reduce the clinical effectiveness of aminoglycosides. A more effective strategy may be to chemically dissociate the ototoxic effects from the therapeutic effects by changing the aminoglycoside structure. Aminoglycosides are thought to be ototoxic owing to their entry into hair cells via transduction channels, so modification of the structure to prevent channel permeation is an appropriate strategy. Several promising drugs have been developed36,176–178 and, although none have made it to the clinic so far, this remains an area of active investigation. To prevent platinum-induced otoxicity, several drugs have been proposed as possible protective agents, including antioxidants, ROS scavengers and anti-inflammatory drugs (as described for the aminoglycosides). However, some of these may interfere with the beneficial effects of platinum drugs179–183. Moreover, none of these reagents has been tested in humans. However, sodium thiosulphate, which is already FDA-approved for cyanide poisoning, has been shown to be effective in preventing ototoxicity in both children and adults, without reducing cancer chemotherapeutic effects184–186. Alternative platinum-based drugs with reduced ototoxicity have also been developed, although their effectiveness against tumours may be decreased correspondingly 38. As with aminoglycosides, the development of chemical modification strategies that lead to reduced uptake into hair cells without impacting therapeutic effectiveness is a highly anticipated approach. Maintaining synapses. As noted above, noise damage that leads to the complete recovery of auditory thresholds (that is, true TTS) can produce degenerative changes in the cochlear nerve and accelerate the onset of ARHL152,187. The observation that even TTS can lead to structural changes at the synapse provides an interesting pathway for intervention. IHCs form ribbon synapses onto afferent neurons, and up to 30% of these synapses are lost following noise damage; nerve fibres with low and medium spontaneous firing rates are predominantly affected25. The selective loss of these fibres, which typically have high auditory thresholds, might explain why standard tests (such as measurement of the auditory brain stem response that emphasize sensitivity, show apparent full recovery of hearing function. High-threshold fibres are particularly important for speech perception and their loss compromises hearing in a noisy environment 25,188. Synaptic loss is not necessarily accompanied by the immediate death of neurons, so delivery of molecules that promote the sprouting of the terminals of nerve fibres and increase synaptic innervation might be of clinical value. Neurotrophins, such as BDNF and NT3,



REVIEWS regulate these processes in the auditory system and stimulate the growth and sprouting of auditory nerve fibres in vivo189. Targeting neurotrophins, other growth factors or their receptors might be viable treatment options for some forms of NIHL. OHCs form synapses with efferent neurons. Efferent neurons provide sound-evoked feedback to reduce cochlear amplification190. Afferent neuron synaptic loss that is caused by continuous moderate-level noise is increased in animals whose efferent neurons have been surgically lesioned24. One major component of the efferent nervous system is cholinergic and depends on the α‑9 nicotinic acetylcholine receptor (CHRNA9) and CHRNA10. Overexpression of CHRNA9 in OHCs, which receive efferent innervation, markedly reduces acoustic injury from noise that normally causes either temporary or permanent damage191. Expression of a mutant CHRNA9 that increases the magnitude and duration of efferent cholinergic effects is also protective192. Surgical de‑efferentation also accelerated the age-dependent amplitude reduction in cochlear neural responses and increased the loss of synapses between hair cells and cochlear nerve fibres193. Because efferent feedback has such an important role in protecting the auditory system, pharmacological activation of efferent neurons might, therefore, prevent both NIHL and ARHL. Stria vascularis. An age-dependent decline in the function of the stria vascularis is one mechanism that may be specific to ARHL. The stria vascularis is important for K+ recycling and for establishing the endocochlear potential17, which is essential for high-sensitivity hearing; some forms of ARHL show degenerative changes in the stria vascularis47,77. Studies in animal models show that a decline in the stria vascularis that leads to changes in the endocochlear potential contributes substantially to some forms of ARHL47,77. Reduced vascularization of the stria vascularis that correlates with ageing has been described47,77, which suggests that interventions that maintain the vasculature might be of clinical significance.

Future approaches to drug discovery Ultimately, it will be desirable to set up high-throughput screening efforts for drug discovery, using complex chemical libraries to identify compounds that can protect against hearing lost. Larval zebrafish have already proven successful for large-scale screening of pharmacological agents. Although these fish lack an organ of Corti, they do have a sophisticated inner ear and, even more importantly, surface-exposed hair cells in their lateral line system. Larvae that are aliquoted into wells of microtitre plates can be incubated with water-soluble compounds; screening can be carried out by optical methods, such as by the assessment of dye entry into hair cells. For example, by examining 10,600 compounds with such an assay, several benzothiophene carboxamides were identified that prevented aminoglycoside-induced hair cell death194. A subsequent screen of 1,040 FDA-approved compounds yielded 7 additional protective compounds, one of which (9‑amino‑1,2,3,4‑tetrahydroacridine; tacrine) also protected against aminoglycoside toxicity

in mice utricles195. Otoprotective compounds are not the only drugs for which zebrafish screens are well suited. Several screens for drugs that kill hair cells identified compounds that could be ototoxic in humans196–198. Hair cell regeneration could also be aided by pharmacological agents; a zebrafish screen of 1,680 compounds identified 2 enhancers and 6 inhibitors of regeneration199. Many compounds identified in zebrafish screens will eventually prove unsuitable in the mammalian inner ear, however, as access to hair cells in the inner ear is far more difficult in mammals. Nevertheless, throughput with this model system is fairly high and thus it is a powerful first step in identifying modulators of inner ear function. Mice provide valuable preclinical models and also enable the screening of small molecules in a targeted manner, especially because quantitative assays for hearing function are available (FIG. 2). For example, an organotypic culture system that permits the analysis of hair cell development and function with concomitant cell transfection, including that of genetically encoded Ca2+ indicators, opens the door for screening efforts200. Although the throughput of mouse inner-ear culture systems will necessarily be kept low by the difficulty of dissection, such systems will always be valuable for translating the compounds that have been identified in zebrafish or similar organisms to the mammal199. A particularly promising route is to base screening efforts on hair cells that have been generated in vitro from embryonic and induced pluripotent stem cells123,124,129, which would enable the generation of hair cells from patient-derived cells, or the use of clustered regularly interspaced short palindromic repeat (CRISPR) technology to create disease models201. An initial limitation is that in vitro hair cell generation has only been achieved robustly in rodents. First attempts with human cells have shown potential202, however, and there is every reason to assume that this goal can be achieved in the very near future.

Conclusions Despite the enormous financial and personal toll that hearing loss takes on people and society, and despite the wide possible market for therapeutics that could prevent or ameliorate the disease, investment in research and drug discovery for hearing loss has greatly lagged that of other disease areas. Modern technological revolutions in sequencing, bioinformatics, imaging and gene modification have enabled previously unthinkable experimental approaches to become routine, which has enabled remarkable scientific and therapeutic opportunities. We need to encourage the development of comprehensive programmes that aim to discover drugs that protect against hearing loss or stimulate regeneration after damage; such programmes would not only have a wide-ranging positive impact on health but would also offer opportunities for substantial financial returns. It is remarkable that so few pharmaceutical or biotechnology companies have invested in this area. Even more remarkable is the scientific potential — we are only now beginning to unlock the mechanisms that allow the ear to perform so admirably.



REVIEWS 1. Thompson, D. C. et al. Universal newborn hearing screening: summary of evidence. JAMA 286, 2000–2010 (2001). 2. Lin, F. R., Thorpe, R., Gordon-Salant, S. & Ferrucci, L. Hearing loss prevalence and risk factors among older adults in the United States. J. Gerontol. A Biol. Sci. Med. Sci. 66, 582–590 (2011). 3. Hong, O., Kerr, M. J., Poling, G. L. & Dhar, S. Understanding and preventing noise-induced hearing loss. Dis. Mon. 59, 110–118 (2013). 4. Angeli, S., Lin, X. & Liu, X. Z. Genetics of hearing and deafness. Anat. Rec. (Hoboken) 295, 1812–1829 (2012). 5. Richardson, G. P., de Monvel, J. B. & Petit, C. How the genetics of deafness illuminates auditory physiology. Annu. Rev. Physiol. 73, 311–334 (2011). 6. Dror, A. A. & Avraham, K. B. Hearing impairment: a panoply of genes and functions. Neuron 68, 293–308 (2010). 7. Raviv, D., Dror, A. A. & Avraham, K. B. Hearing loss: a common disorder caused by many rare alleles. Ann. NY Acad. Sci. 1214, 168–179 (2010). 8. Schwander, M., Kachar, B. & Müller, U. The cell biology of hearing. J. Cell Biol. 190, 9–20 (2010). 9. Mann, Z. F. & Kelley, M. W. Development of tonotopy in the auditory periphery. Hear. Res. 276, 2–15 (2011). 10. Hudspeth, A. J. How hearing happens. Neuron 19, 947–950 (1997). 11. Hudspeth, A. J. Making an effort to listen: mechanical amplification in the ear. Neuron 59, 530–545 (2008). 12. Steel, K. P. in Genes and Common Diseases: Genetics in Modern Medicine (eds Wright, A. F. & Hastie, N. D.) 505–515 (Cambridge Univ. Press, 2007). 13. Friedman, T. B., Schultz, J. M., Ahmed, Z. M., Tsilou, E. T. & Brewer, C. C. Usher syndrome: hearing loss with vision loss. Adv. Otorhinolaryngol 70, 56–65 (2011). 14. Kemperman, M. H., Hoefsloot, L. H. & Cremers, C. W. Hearing loss and connexin 26. J. R. Soc. Med. 95, 171–177 (2002). 15. Shin, J. B. et al. Hair bundles are specialized for ATP delivery via creatine kinase. Neuron 53, 371–386 (2007). 16. Barnham, K. J., Masters, C. L. & Bush, A. I. Neurodegenerative diseases and oxidative stress. Nature Rev. Drug Discov. 3, 205–214 (2004). 17. Kikuchi, T., Adams, J. C., Miyabe, Y., So, E. & Kobayashi, T. Potassium ion recycling pathway via gap junction systems in the mammalian cochlea and its interruption in hereditary nonsyndromic deafness. Med. Electron. Microsc. 33, 51–56 (2000). 18. Dobie, R. A. The burdens of age-related and occupational noise-induced hearing loss in the United States. Ear Hear. 29, 565–577 (2008). 19. CDC. Severe hearing impairment among military veterans — United States, 2010. MMWR Morb. Mortal. Wkly Rep. 60, 955–958 (2011). 20. Oishi, N. & Schacht, J. Emerging treatments for noiseinduced hearing loss. Expert Opin. Emerg. Drugs 16, 235–245 (2011). 21. Humes, L. E. & Joellenbeck, L. Noise and Military Service: Implications for Hearing Loss and Tinnitus. (National Academies Press, 2005). 22. Clark, W. W. Recent studies of temporary threshold shift (TTS) and permanent threshold shift (PTS) in animals. J. Acoust. Soc. Am. 90, 155–163 (1991). 23. Kujawa, S. G. & Liberman, M. C. Adding insult to injury: cochlear nerve degeneration after ‘temporary’ noise-induced hearing loss. J. Neurosci. 29, 14077–14085 (2009). 24. Maison, S. F., Usubuchi, H. & Liberman, M. C. Efferent feedback minimizes cochlear neuropathy from moderate noise exposure. J. Neurosci. 33, 5542–5552 (2013). 25. Furman, A. C., Kujawa, S. G. & Liberman, M. C. Noise-induced cochlear neuropathy is selective for fibers with low spontaneous rates. J. Neurophysiol. 110, 577–586 (2013). 26. Plack, C. J., Barker, D. & Prendergast, G. Perceptual consequences of ‘hidden’ hearing loss. Trends Hear. 18, 2331216514550621 (2014). 27. Sliwinska-Kowalska, M. & Pawelczyk, M. Contribution of genetic factors to noise-induced hearing loss: a human studies review. Mutat. Res. 752, 61–65 (2013). 28. Taleb, M. et al. Hsp70 inhibits aminoglycoside-induced hearing loss and cochlear hair cell death. Cell Stress Chaperones 14, 427–437 (2009).

29. Moore, R. D., Smith, C. R. & Lietman, P. S. Risk factors for the development of auditory toxicity in patients receiving aminoglycosides. J. Infect. Dis. 149, 23–30 (1984). 30. Lerner, S. A., Schmitt, B. A., Seligsohn, R. & Matz, G. J. Comparative study of ototoxicity and nephrotoxicity in patients randomly assigned to treatment with amikacin or gentamicin. Am. J. Med. 80, 98–104 (1986). 31. Fausti, S. A., Frey, R. H., Henry, J. A., Olson, D. J. & Schaffer, H. I. Early detection of ototoxicity using highfrequency, tone-burst-evoked auditory brainstem responses. J. Am. Acad. Audiol. 3, 397–404 (1992). 32. Li, H. & Steyger, P. S. Systemic aminoglycosides are trafficked via endolymph into cochlear hair cells. Sci. Rep. 1, 159 (2011). 33. Marcotti, W., van Netten, S. M. & Kros, C. J. The aminoglycoside antibiotic dihydrostreptomycin rapidly enters mouse outer hair cells through the mechano-electrical transducer channels. J. Physiol. 567, 505–521 (2005). This paper describes the mechanisms by which dihydrostreptomycin enters hair cells to cause hair cell death. 34. Agrawal, R. K. & Sharma, M. R. Structural aspects of mitochondrial translational apparatus. Curr. Opin. Struct. Biol. 22, 797–803 (2012). 35. Hu, D. N. et al. Genetic aspects of antibiotic induced deafness: mitochondrial inheritance. J. Med. Genet. 28, 79–83 (1991). 36. Matt, T. et al. Dissociation of antibacterial activity and aminoglycoside ototoxicity in the 4‑monosubstituted 2‑deoxystreptamine apramycin. Proc. Natl Acad. Sci. USA 109, 10984–10989 (2012). 37. Ding, Y., Leng, J., Fan, F., Xia, B. & Xu, P. The role of mitochondrial DNA mutations in hearing loss. Biochem. Genet. 51, 588–602 (2013). 38. Langer, T., am Zehnhoff-Dinnesen, A., Radtke, S., Meitert, J. & Zolk, O. Understanding platinum-induced ototoxicity. Trends Pharmacol. Sci. 34, 458–469 (2013). 39. Ciarimboli, G. et al. Organic cation transporter 2 mediates cisplatin-induced oto- and nephrotoxicity and is a target for protective interventions. Am. J. Pathol. 176, 1169–1180 (2010). 40. Riedemann, L. et al. Megalin genetic polymorphisms and individual sensitivity to the ototoxic effect of cisplatin. Pharmacogenom. J. 8, 23–28 (2008). 41. Xu, X. et al. Prediction of copper transport protein 1 (CTR1) genotype on severe cisplatin induced toxicity in non-small cell lung cancer (NSCLC) patients. Lung Cancer 77, 438–442 (2012). 42. Ross, C. J. et al. Genetic variants in TPMT and COMT are associated with hearing loss in children receiving cisplatin chemotherapy. Nature Genet. 41, 1345–1349 (2009). 43. Pussegoda, K. et al. Replication of TPMT and ABCC3 genetic variants highly associated with cisplatininduced hearing loss in children. Clin. Pharmacol. Ther. 94, 243–251 (2013). 44. Dalton, D. S. et al. The impact of hearing loss on quality of life in older adults. Gerontologist 43, 661–668 (2003). 45. Heine, C. & Browning, C. J. Communication and psychosocial consequences of sensory loss in older adults: overview and rehabilitation directions. Disabil Rehabil. 24, 763–773 (2002). 46. Gates, G. A. & Mills, J. H. Presbycusis. Lancet 366, 1111–1120 (2005). 47. Yamasoba, T. et al. Current concepts in age-related hearing loss: epidemiology and mechanistic pathways. Hear. Res. 303, 30–38 (2013). 48. Schuknecht, H. F. Presbycusis. Trans. Am. Laryngol. Rhinol. Otol. Soc. 401–418; discussion 419–420 (1955). 49. Schuknecht, H. F. Further observations on the pathology of presbycusis. Arch. Otolaryngol. 80, 369–382 (1964). 50. Schuknecht, H. F. & Gacek, M. R. Cochlear pathology in presbycusis. Ann. Otol. Rhinol. Laryngol. 102, 1–16 (1993). 51. Nelson, E. G. & Hinojosa, R. Age-related histopathologic changes in the human cochlea. Otolaryngol. Head Neck Surg. 133, 817 (2005). 52. Ohlemiller, K. K. Age-related hearing loss: the status of Schuknecht’s typology. Otolaryngol. Head Neck Surg. 12, 439–443 (2004). This paper provides a critical review that highlights the uses and limitations of the definition of the pathology of ARHL using Schuknecht’s typology.


53. Huang, Q. & Tang, J. Age-related hearing loss or presbycusis. Eur. Arch. Otorhinolaryngol 267, 1179–1191 (2010). 54. Gates, G. A., Couropmitree, N. N. & Myers, R. H. Genetic associations in age-related hearing thresholds. Arch. Otolaryngol. Head Neck Surg. 125, 654–659 (1999). 55. Karlsson, K. K., Harris, J. R. & Svartengren, M. Description and primary results from an audiometric study of male twins. Ear Hear. 18, 114–120 (1997). 56. Christensen, K., Frederiksen, H. & Hoffman, H. J. Genetic and environmental influences on self-reported reduced hearing in the old and oldest old. J. Am. Geriatr. Soc. 49, 1512–1517 (2001). 57. Huyghe, J. R. et al. Genome-wide SNP-based linkage scan identifies a locus on 8q24 for an age-related hearing impairment trait. Am. J. Hum. Genet. 83, 401–407 (2008). 58. Van Laer, L. et al. A genome-wide association study for age-related hearing impairment in the Saami. Eur. J. Hum. Genet. 18, 685–693 (2010). 59. Van Laer, L. et al. The grainyhead like 2 gene (GRHL2), alias TFCP2L3, is associated with age-related hearing impairment. Hum. Mol. Genet. 17, 159–169 (2008). 60. Friedman, R. A. et al. GRM7 variants confer susceptibility to age-related hearing impairment. Hum. Mol. Genet. 18, 785–796 (2009). This paper describes a gene association study that links polymorphisms in GRM7 to ARHL. GRM7 is, so far, the only gene that seems to achieve genome-wide significance in gene association studies for ARHL. 61. Newman, D. L. et al. GRM7 variants associated with age-related hearing loss based on auditory perception. Hear. Res. 294, 125–132 (2012). This study reinforces the link between polymorphisms in GRM7 and ARHL. 62. Van Eyken, E. et al. Contribution of the N‑acetyltransferase 2 polymorphism NAT2*6A to age-related hearing impairment. J. Med. Genet. 44, 570–578 (2007). 63. Bared, A. et al. Antioxidant enzymes, presbycusis, and ethnic variability. Otolaryngol. Head Neck Surg. 143, 263–268 (2010). 64. Unal, M. et al. N‑acetyltransferase 2 gene polymorphism and presbycusis. Laryngoscope 115, 2238–2241 (2005). 65. Arnett, J. et al. Autosomal dominant progressive sensorineural hearing loss due to a novel mutation in the KCNQ4 gene. Arch. Otolaryngol. Head Neck Surg. 137, 54–59 (2011). 66. Uchida, Y., Sugiura, S., Nakashima, T., Ando, F. & Shimokata, H. Endothelin‑1 gene polymorphism and hearing impairment in elderly Japanese. Laryngoscope 119, 938–943 (2009). 67. Sugiura, S., Uchida, Y., Nakashima, T., Ando, F. & Shimokata, H. The association between gene polymorphisms in uncoupling proteins and hearing impairment in Japanese elderly. Acta Otolaryngol. 130, 487–492 (2010). 68. Seidman, M. D. et al. Association of mitochondrial DNA deletions and cochlear pathology: a molecular biologic tool. Laryngoscope 106, 777–783 (1996). 69. Bai, U., Seidman, M. D., Hinojosa, R. & Quirk, W. S. Mitochondrial DNA deletions associated with aging and possibly presbycusis: a human archival temporal bone study. Am. J. Otol. 18, 449–453 (1997). 70. Markaryan, A., Nelson, E. G. & Hinojosa, R. Quantification of the mitochondrial DNA common deletion in presbycusis. Laryngoscope 119, 1184–1189 (2009). 71. Zheng, Q. Y., Johnson, K. R. & Erway, L. C. Assessment of hearing in 80 inbred strains of mice by ABR threshold analyses. Hear. Res. 130, 94–107 (1999). In this study, the comprehensive testing of auditory function in inbred mouse strains reveals genetic background-dependent differences in auditory sensitivity and in susceptibility to ARHL. 72. Johnson, K. R., Zheng, Q. Y. & Noben-Trauth, K. Strain background effects and genetic modifiers of hearing in mice. Brain Res. 1091, 79–88 (2006). 73. Johnson, K. R., Longo-Guess, C., Gagnon, L. H., Yu, H. & Zheng, Q. Y. A locus on distal chromosome 11 (ahl8) and its interaction with Cdh23ahl underlie the early onset, age-related hearing loss of DBA/2J mice. Genomics 92, 219–225 (2008). 74. Zheng, Q. Y., Ding, D., Yu, H., Salvi, R. J. & Johnson, K. R. A locus on distal chromosome 10 (ahl4) affecting age-related hearing loss in A/J mice. Neurobiol. Aging 30, 1693–1705 (2009).


REVIEWS 75. Ohmen, J. et al. Genome-wide association study for age-related hearing loss (AHL) in the mouse: a metaanalysis. J. Assoc. Res. Otolaryngol. 15, 335–352 (2014). 76. Ohlemiller, K. K. Mechanisms and genes in human strial presbycusis from animal models. Brain Res. 1277, 70–83 (2009). 77. Fetoni, A. R., Picciotti, P. M., Paludetti, G. & Troiani, D. Pathogenesis of presbycusis in animal models: a review. Exp. Gerontol. 46, 413–425 (2011). 78. Mehrparvar, A. H. et al. Conventional audiometry, extended high-frequency audiometry, and DPOAE for early diagnosis of NIHL. Iran. Red Crescent Med. J. 16, e9628 (2014). 79. Shearer, A. E. & Smith, R. J. Genetics: advances in genetic testing for deafness. Curr. Opin. Pediatr. 24, 679–686 (2012). 80. Lin, X. et al. Applications of targeted gene capture and next-generation sequencing technologies in studies of human deafness and other genetic disabilities. Hear. Res. 288, 67–76 (2012). 81. Rubel, E. W., Furrer, S. A. & Stone, J. S. A brief history of hair cell regeneration research and speculations on the future. Hear. Res. 297, 42–51 (2013). 82. Hu, Z. & Ulfendahl, M. The potential of stem cells for the restoration of auditory function in humans. Regen. Med. 8, 309–318 (2013). 83. Geleoc, G. S. & Holt, J. R. Sound strategies for hearing restoration. Science 344, 1241062 (2014). 84. Kiernan, A. E. et al. Sox2 is required for sensory organ development in the mammalian inner ear. Nature 434, 1031–1035 (2005). This study identifies Sox2 as an upstream regulator for the expression of Atoh1, which is essential for hair cell development. This paper thus provides the first insights into the transcriptional programme that is necessary to specify differentiation of pluripotent cells into hair cells in vitro. 85. Ahmed, M. et al. Eya1–Six1 interaction is sufficient to induce hair cell fate in the cochlea by activating Atoh1 expression in cooperation with Sox2. Dev. Cell 22, 377–390 (2012). This paper identifies EYA1–SIX1 as an additional component of the transcriptional network that also contains Atoh1 and Sox2 and regulates the generation of hair cells. 86. Bermingham, N. A. et al. Math1: an essential gene for the generation of inner ear hair cells. Science 284, 1837–1141 (1999). This paper demonstrates that Atoh1 (also known as Math1) is essential for the development of hair cells, which is a crucial finding for work focusing on generating hair cells in vitro. 87. Erkman, L. et al. Role of transcription factors Brn‑3.1 and Brn‑3.2 in auditory and visual system development. Nature 381, 603–606 (1996). References 87 and 88 both demonstrate that POU4F3 (also known as BRN3.1) is essential for the terminal differentiation of hair cells, an important finding for generating hair cells in vitro. 88. Xiang, M. et al. Essential role of POU-domain factor Brn‑3c in auditory and vestibular hair cell development. Proc. Natl Acad. Sci. USA 94, 9445–9450 (1997). 89. Wallis, D. et al. The zinc finger transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear hair cell differentiation and survival. Development 130, 221–232 (2003). 90. Hu, X. et al. Sonic hedgehog (SHH) promotes the differentiation of mouse cochlear neural progenitors via the Math1–Brn3.1 signaling pathway in vitro. J. Neurosci. Res. 88, 927–935 (2010). 91. Masuda, M. et al. Regulation of POU4F3 gene expression in hair cells by 5ʹ DNA in mice. Neuroscience 197, 48–64 (2011). 92. Chonko, K. T. et al. Atoh1 directs hair cell differentiation and survival in the late embryonic mouse inner ear. Dev. Biol. 381, 401–410 (2013). 93. Zheng, J. L. & Gao, W. Q. Overexpression of Math1 induces robust production of extra hair cells in postnatal rat inner ears. Nature Neurosci. 3, 580–586 (2000). This paper is the first to demonstrate that overexpression of Atoh1 can induce the production of extra hair cells in the inner ear. 94. Kawamoto, K., Ishimoto, S., Minoda, R., Brough, D. E. & Raphael, Y. Math1 gene transfer generates new cochlear hair cells in mature guinea pigs in vivo. J. Neurosci. 23, 4395–4400 (2003).

95. Gubbels, S. P., Woessner, D. W., Mitchell, J. C., Ricci, A. J. & Brigande, J. V. Functional auditory hair cells produced in the mammalian cochlea by in utero gene transfer. Nature 455, 537–541 (2008). 96. Izumikawa, M. et al. Auditory hair cell replacement and hearing improvement by Atoh1 gene therapy in deaf mammals. Nature Med. 11, 271–276 (2005). This study shows that the overexpression of Atoh1 in the cochlea of deaf guinea pigs leads to the generation of hair cells and to improved hearing function. 97. Yang, S. M. et al. Regeneration of stereocilia of hair cells by forced Atoh1 expression in the adult mammalian cochlea. PLoS ONE 7, e46355 (2012). 98. Baker, K., Brough, D. E. & Staecker, H. Repair of the vestibular system via adenovector delivery of Atoh1: a potential treatment for balance disorders. Adv. Otorhinolaryngol 66, 52–63 (2009). 99. Schlecker, C. et al. Selective atonal gene delivery improves balance function in a mouse model of vestibular disease. Gene Ther. 18, 884–890 (2011). 100. Liu, Z. et al. Age-dependent in vivo conversion of mouse cochlear pillar and Deiters’ cells to immature hair cells by Atoh1 ectopic expression. J. Neurosci. 32, 6600–6610 (2012). 101. Cai, T., Seymour, M. L., Zhang, H., Pereira, F. A. & Groves, A. K. Conditional deletion of Atoh1 reveals distinct critical periods for survival and function of hair cells in the organ of Corti. J. Neurosci. 33, 10110–10122 (2013). 102. Izumikawa, M., Batts, S. A., Miyazawa, T., Swiderski, D. L. & Raphael, Y. Response of the flat cochlear epithelium to forced expression of Atoh1. Hear. Res. 240, 52–56 (2008). 103. Vandenberghe, L. H. & Auricchio, A. Novel adenoassociated viral vectors for retinal gene therapy. Gene Ther. 19, 162–168 (2012). 104. Akil, O. et al. Restoration of hearing in the VGLUT3 knockout mouse using virally mediated gene therapy. Neuron 75, 283–293 (2012). In this study, it is demonstrated that hearing function can be restored in VGLUT3 knockout mice by reintroducing VGLUT3 cDNA into inner hair cells using AAV viral vectors. 105. Luebke, A. E., Rova, C., Von Doersten, P. G. & Poulsen, D. J. Adenoviral and AAV-mediated gene transfer to the inner ear: role of serotype, promoter, and viral load on in vivo and in vitro infection efficiencies. Adv. Otorhinolaryngol. 66, 87–98 (2009). 106. Driver, E. C., Sillers, L., Coate, T. M., Rose, M. F. & Kelley, M. W. The Atoh1‑lineage gives rise to hair cells and supporting cells within the mammalian cochlea. Dev. Biol. 376, 86–98 (2013). 107. Mizutari, K. et al. Notch inhibition induces cochlear hair cell regeneration and recovery of hearing after acoustic trauma. Neuron 77, 58–69 (2013). 108. Shi, F., Cheng, Y. F., Wang, X. L. & Edge, A. S. β-catenin up‑regulates Atoh1 expression in neural progenitor cells by interaction with an Atoh1 3ʹ enhancer. J. Biol. Chem. 285, 392–400 (2010). 109. Jacques, B. E. et al. A dual function for canonical Wnt/β-catenin signaling in the developing mammalian cochlea. Development 139, 4395–4404 (2012). 110. Shi, F. et al. β-catenin is required for hair-cell differentiation in the cochlea. J. Neurosci. 34, 6470–6479 (2014). 111. Munnamalai, V. & Fekete, D. M. Wnt signaling during cochlear development. Semin. Cell Dev. Biol. 24, 480–489 (2013). 112. Roberson, D. W., Alosi, J. A. & Cotanche, D. A. Direct transdifferentiation gives rise to the earliest new hair cells in regenerating avian auditory epithelium. J. Neurosci. Res. 78, 461–471 (2004). 113. Lowenheim, H. et al. Gene disruption of p27(Kip1) allows cell proliferation in the postnatal and adult organ of corti. Proc. Natl Acad. Sci. USA 96, 4084–4088 (1999). 114. Chen, P. & Segil, N. p27(Kip1) links cell proliferation to morphogenesis in the developing organ of Corti. Development 126, 1581–1590 (1999). 115. Sage, C. et al. Proliferation of functional hair cells in vivo in the absence of the retinoblastoma protein. Science 307, 1114–1118 (2005). 116. Barker, N. et al. Identification of stem cells in small intestine and colon by marker gene Lgr5. Nature 449, 1003–1007 (2007).


117. Bramhall, N. F., Shi, F., Arnold, K., Hochedliner, K. & Edge, A. S. B. Lgr5‑positive supporting cells generate new hair cells in the postnatal cochlea. Stem Cell Reports 2, 311–322 (2014). This study demonstrates that LGR5‑positive supporting cells in the neonatal cochlea can differentiate into hair cells following ototoxic damage. 118. Chai, R. et al. Dynamic expression of Lgr5, a Wnt target gene, in the developing and mature mouse cochlea. J. Assoc. Res. Otolaryngol. 12, 455–469 (2011). 119. Burns, J. C. & Corwin, J. T. A historical to present-day account of efforts to answer the question: ‘what puts the brakes on mammalian hair cell regeneration?’. Hear. Res. 297, 52–67 (2013). 120. Walters, B. J. & Zuo, J. Postnatal development, maturation and aging in the mouse cochlea and their effects on hair cell regeneration. Hear. Res. 297, 68–83 (2013). 121. Thomson, J. A. et al. Embryonic stem cell lines derived from human blastocysts. Science 282, 1145–1147 (1998). 122. Okita, K., Ichisaka, T. & Yamanaka, S. Generation of germline-competent induced pluripotent stem cells. Nature 448, 313–317 (2007). 123. Oshima, K. et al. Mechanosensitive hair cell-like cells from embryonic and induced pluripotent stem cells. Cell 141, 704–716 (2010). This paper reports that hair cell-like cells can be generated in vitro by the transdifferentiation of embryonic stem cell and induced pluripotent stem cells. 124. Koehler, K. R., Mikosz, A. M., Molosh, A. I., Patel, D. & Hashino, E. Generation of inner ear sensory epithelia from pluripotent stem cells in 3D culture. Nature 500, 217–221 (2013). This manuscript describes a new method for the generation of inner ear sensory epithelia containing hair cells in vitro using threedimensional cultures. The yield of hair cells seems much larger than in previous attempts to generate hair cells. 125. Malgrange, B. et al. Proliferative generation of mammalian auditory hair cells in culture. Mech. Dev. 112, 79–88 (2002). 126. Li, H., Liu, H. & Heller, S. Pluripotent stem cells from the adult mouse inner ear. Nature Med. 9, 1293–1299 (2003). 127. Chen, W. et al. Human fetal auditory stem cells can be expanded in vitro and differentiate into functional auditory neurons and hair cell-like cells. Stem Cells 27, 1196–1204 (2009). 128. Hu, Z. et al. Generation of human inner ear prosensory-like cells via epithelial-to‑mesenchymal transition. Regen Med. 7, 663–673 (2012). 129. Li, H., Roblin, G., Liu, H. & Heller, S. Generation of hair cells by stepwise differentiation of embryonic stem cells. Proc. Natl Acad. Sci. USA 100, 13495–13500 (2003). 130. Okano, T. & Kelley, M. W. Stem cell therapy for the inner ear: recent advances and future directions. Trends Amplif. 16, 4–18 (2012). 131. Chen, W. et al. Restoration of auditory evoked responses by human ES‑cell-derived otic progenitors. Nature 490, 278–282 (2012). This study describes the in vitro generation of otic neuroprogenitors that can differentiate into hair cells and sensory neurons. Upon implantation of the progenitors in a model of auditory neuropathy, auditory function was substantially improved. 132. Hirose, K. & Liberman, M. C. Lateral wall histopathology and endocochlear potential in the noise-damaged mouse cochlea. J. Assoc. Res. Otolaryngol. 4, 339–352 (2003). 133. Petrs-Silva, H. & Linden, R. Advances in gene therapy technologies to treat retinitis pigmentosa. Clin. Ophthalmol. 8, 127–136 (2014). 134. Sacheli, R., Delacroix, L., Vandenackerveken, P., Nguyen, L. & Malgrange, B. Gene transfer in inner ear cells: a challenging race. Gene Ther. 20, 237–247 (2013). 135. Sun, H., Huang, A. & Cao, S. Current status and prospects of gene therapy for the inner ear. Hum. Gene Ther. 22, 1311–1322 (2011). 136. Maeda, Y., Fukushima, K., Nishizaki, K. & Smith, R. J. In vitro and in vivo suppression of GJB2 expression by RNA interference. Hum. Mol. Genet. 14, 1641–1650 (2005).


REVIEWS 137. Lentz, J. J. et al. Rescue of hearing and vestibular function by antisense oligonucleotides in a mouse model of human deafness. Nature Med. 19, 345–350 (2013). This study describes how antisense technology can be used to suppress a splice-site mutation to recover auditory function in an animal model for human deafness. 138. Miwa, T., Minoda, R., Ise, M., Yamada, T. & Yumoto, E. Mouse otocyst transuterine gene transfer restores hearing in mice with connexin 30 deletion-associated hearing loss. Mol. Ther. 21, 1142–1150 (2013). 139. Rivera, T., Sanz, L., Camarero, G. & Varela-Nieto, I. Drug delivery to the inner ear: strategies and their therapeutic implications for sensorineural hearing loss. Curr. Drug Deliv. 9, 231–242 (2012). 140. Schuknecht, H. F. Pathology of the Ear. (Harvard Univ. Press, 1974). 141. Nakamoto, Y., Iino, Y. & Kodera, K. [Temporal bone histopathology of noise-induced hearing loss]. Nihon Jibiinkoka Gakkai Kaiho 108, 172–181 (2005). 142. Wang, Y., Hirose, K. & Liberman, M. C. Dynamics of noise-induced cellular injury and repair in the mouse cochlea. J. Assoc. Res. Otolaryngol. 3, 248–268 (2002). 143. Yan, C. & Higgins, P. J. Drugging the undruggable: transcription therapy for cancer. Biochim. Biophys. Acta 1835, 76–85 (2013). 144. Wulff, H., Castle, N. A. & Pardo, L. A. Voltage-gated potassium channels as therapeutic targets. Nature Rev. Drug Discov. 8, 982–1001 (2009). 145. Amabile, C. M. & Vasudevan, A. Ezogabine: a novel antiepileptic for adjunctive treatment of partial-onset seizures. Pharmacotherapy 33, 187–194 (2013). 146. Shiroshita-Takeshita, A., Sakabe, M., Haugan, K., Hennan, J. K. & Nattel, S. Model-dependent effects of the gap junction conduction-enhancing antiarrhythmic peptide rotigaptide (ZP123) on experimental atrial fibrillation in dogs. Circulation 115, 310–318 (2007). 147. Cao, Y. & Zheng, O. J. Tonabersat for migraine prophylaxis: a systematic review. Pain Physician 17, 1–8 (2014). 148. O’Carroll, S. J. et al. The use of connexin-based therapeutic approaches to target inflammatory diseases. Methods Mol. Biol. 1037, 519–546 (2013). 149. Waris, G. & Ahsan, H. Reactive oxygen species: role in the development of cancer and various chronic conditions. J. Carcinog. 5, 14 (2006). 150. Daniel, E. Noise and hearing loss: a review. J. Sch. Health 77, 225–231 (2007). 151. Wong, A. C. et al. Post exposure administration of A(1) adenosine receptor agonists attenuates noiseinduced hearing loss. Hear. Res. 260, 81–88 (2010). 152. Fetoni, A. R. et al. In vivo protective effect of ferulic acid against noise-induced hearing loss in the guineapig. Neuroscience 169, 1575–1588 (2010). 153. Kopke, R. D. et al. NAC for noise: from the bench top to the clinic. Hear. Res. 226, 114–125 (2007). 154. Bielefeld, E. C. et al. Noise protection with N‑acetyll‑cysteine (NAC) using a variety of noise exposures, NAC doses, and routes of administration. Acta Otolaryngol. 127, 914–919 (2007). 155. Ohlemiller, K. K. et al. Targeted deletion of the cytosolic Cu/Zn‑superoxide dismutase gene (Sod1) increases susceptibility to noise-induced hearing loss. Audiol Neurootol 4, 237–246 (1999). 156. McFadden, S. L., Ding, D., Reaume, A. G., Flood, D. G. & Salvi, R. J. Age-related cochlear hair cell loss is enhanced in mice lacking copper/zinc superoxide dismutase. Neurobiol. Aging 20, 1–8 (1999). 157. Keithley, E. M. et al. Cu/Zn superoxide dismutase and age-related hearing loss. Hear. Res. 209, 76–85 (2005). 158. Kashio, A. et al. Effect of vitamin C depletion on agerelated hearing loss in SMP30/GNL knockout mice. Biochem. Biophys. Res. Commun. 390, 394–398 (2009). 159. Schriner, S. E. et al. Extension of murine life span by overexpression of catalase targeted to mitochondria. Science 308, 1909–1911 (2005). 160. Seidman, M. D. Effects of dietary restriction and antioxidants on presbyacusis. Laryngoscope 110, 727–738 (2000). 161. Seidman, M. D., Khan, M. J., Tang, W. X. & Quirk, W. S. Influence of lecithin on mitochondrial DNA and age-related hearing loss. Otolaryngol. Head Neck Surg. 127, 138–144 (2002). 162. Someya, S. et al. Age-related hearing loss in C57BL/6J mice is mediated by Bak-dependent mitochondrial apoptosis. Proc. Natl Acad. Sci. USA 106, 19432–19437 (2009).

163. Heman-Ackah, S. E., Juhn, S. K., Huang, T. C. & Wiedmann, T. S. A combination antioxidant therapy prevents age-related hearing loss in C57BL/6 mice. Otolaryngol. Head Neck Surg. 143, 429–434 (2010). 164. Markaryan, A., Nelson, E. G. & Hinojosa, R. Detection of mitochondrial DNA deletions in the cochlea and its structural elements from archival human temporal bone tissue. Mutat. Res. 640, 38–45 (2008). 165. Meltser, I., Tahera, Y. & Canlon, B. Differential activation of mitogen-activated protein kinases and brain-derived neurotrophic factor after temporary or permanent damage to a sensory system. Neuroscience 165, 1439–1446 (2010). 166. Wang, J. et al. A peptide inhibitor of c‑Jun N‑terminal kinase protects against both aminoglycoside and acoustic trauma-induced auditory hair cell death and hearing loss. J. Neurosci. 23, 8596–8607 (2003). 167. Shim, H. J., Kang, H. H., Ahn, J. H. & Chung, J. W. Retinoic acid applied after noise exposure can recover the noise-induced hearing loss in mice. Acta Otolaryngol. 129, 233–238 (2009). 168. Ahn, J. H., Kang, H. H., Kim, Y. J. & Chung, J. W. Antiapoptotic role of retinoic acid in the inner ear of noiseexposed mice. Biochem. Biophys. Res. Commun. 335, 485–490 (2005). 169. Suckfuell, M., Canis, M., Strieth, S., Scherer, H. & Haisch, A. Intratympanic treatment of acute acoustic trauma with a cell-permeable JNK ligand: a prospective randomized phase I/II study. Acta Otolaryngol. 127, 938–942 (2007). 170. Wang, K. Molecular mechanisms of hepatic apoptosis. Cell Death Dis. 5, e996 (2014). 171. Fischer, U. & Schulze-Osthoff, K. New approaches and therapeutics targeting apoptosis in disease. Pharmacol. Rev. 57, 187–215 (2005). 172. Rybak, L. P. & Whitworth, C. A. Ototoxicity: therapeutic opportunities. Drug Discov. Today 10, 1313–1321 (2005). 173. Sha, S. H., Qiu, J. H. & Schacht, J. Aspirin to prevent gentamicin-induced hearing loss. N. Engl. J. Med. 354, 1856–1857 (2006). 174. Feldman, L. et al. Gentamicin-induced ototoxicity in hemodialysis patients is ameliorated by N‑acetylcysteine. Kidney Int. 72, 359–363 (2007). 175. Kharkheli, E., Kevanishvili, Z., Maglakelidze, T., Davitashvili, O. & Schacht, J. Does vitamin E prevent gentamicin-induced ototoxicity? Georgian Med. News 146, 14–17 (2007). 176. Hainrichson, M. et al. Branched aminoglycosides: biochemical studies and antibacterial activity of neomycin B derivatives. Bioorg. Med. Chem. 13, 5797–5807 (2005). 177. Armstrong, E. S. & Miller, G. H. Combating evolution with intelligent design: the neoglycoside ACHN‑490. Curr. Opin. Microbiol. 13, 565–573 (2010). 178. Pokrovskaya, V., Nudelman, I., Kandasamy, J. & Baasov, T. Aminoglycosides redesign strategies for improved antibiotics and compounds for treatment of human genetic diseases. Methods Enzymol. 478, 437–462 (2010). 179. Shin, Y. S. et al. A novel synthetic compound, 3‑amino‑3-(4‑fluoro-phenyl)-1H‑quinoline‑2,4‑dione, inhibits cisplatin-induced hearing loss by the suppression of reactive oxygen species: in vitro and in vivo study. Neuroscience 232, 1–12 (2012). 180. Ozkiris, M., Kapusuz, Z., Karacavus, S. & Saydam, L. The effects of lycopene on cisplatin-induced ototoxicity. Eur. Arch. Otorhinolaryngol 270, 3027–3033 (2013). 181. Sagit, M., Korkmaz, F., Akcadag, A. & Somdas, M. A. Protective effect of thymoquinone against cisplatininduced ototoxicity. Eur. Arch. Otorhinolaryngol. 270, 2231–2237 (2013). 182. Simsek, G. et al. Protective effects of resveratrol on cisplatin-dependent inner-ear damage in rats. Eur. Arch. Otorhinolaryngol. 270, 1789–1793 (2013). 183. Shin, Y. S. et al. Novel synthetic protective compound, KR‑22335, against cisplatin-induced auditory cell death. J. Appl. Toxicol. 34, 191–204 (2014). 184. Brock, P. R. et al. Platinum-induced ototoxicity in children: a consensus review on mechanisms, predisposition, and protection, including a new international society of pediatric oncology Boston ototoxicity scale. J. Clin. Oncol. 30, 2408–2417 (2012). 185. Doolittle, N. D. et al. Delayed sodium thiosulfate as an otoprotectant against carboplatin-induced hearing loss in patients with malignant brain tumors. Clin. Cancer Res. 7, 493–500 (2001).


186. Neuwelt, E. A. et al. Toxicity profile of delayed high dose sodium thiosulfate in children treated with carboplatin in conjunction with blood–brain-barrier disruption. Pediatr. Blood Cancer 47, 174–182 (2006). 187. Reid, F. M., Vernham, G. A. & Jacobs, H. T. A novel mitochondrial point mutation in a maternal pedigree with sensorineural deafness. Hum. Mutat. 3, 243–247 (1994). 188. Bharadwaj, H. M., Verhulst, S., Shaheen, L., Liberman, M. C. & Shinn-Cunningham, B. G. Cochlear neuropathy and the coding of supra-threshold sound. Front. Syst. Neurosci. 8, 26 (2014). 189. Fritzsch, B., Pirvola, U. & Ylikoski, J. Making and breaking the innervation of the ear: neurotrophic support during ear development and its clinical implications. Cell Tissue Res. 295, 369–382 (1999). 190. Rabbitt, R. D. & Brownell, W. E. Efferent modulation of hair cell function. Otolaryngol. Head Neck Surg. 19, 376–381 (2011). 191. Maison, S. F., Luebke, A. E., Liberman, M. C. & Zuo, J. Efferent protection from acoustic injury is mediated via α9 nicotinic acetylcholine receptors on outer hair cells. J. Neurosci. 22, 10838–10846 (2002). This study demonstrates the importance of efferent neurons, which connect to OHCs, for the prevention of NIHL. 192. Taranda, J. et al. A point mutation in the hair cell nicotinic cholinergic receptor prolongs cochlear inhibition and enhances noise protection. PLoS Biol. 7, e18 (2009). 193. Liberman, M. C., Liberman, L. D. & Maison, S. F. Efferent feedback slows cochlear aging. J. Neurosci. 34, 4599–4607 (2014). 194. Owens, K. N. et al. Identification of genetic and chemical modulators of zebrafish mechanosensory hair cell death. PLoS Genet. 4, e1000020 (2008). This study describes the usefulness of the lateral line organ of zebrafish, which contains hair cells that sense fluid motion, for identifying genetic mutations and pharmacological compounds that prevent hair cell death induced by ototoxic agents. 195. Ou, H. C. et al. Identification of FDA-approved drugs and bioactives that protect hair cells in the zebrafish (Danio rerio) lateral line and mouse (Mus musculus) utricle. J. Assoc. Res. Otolaryngol. 10, 191–203 (2009). 196. Ton, C. & Parng, C. The use of zebrafish for assessing ototoxic and otoprotective agents. Hear. Res. 208, 79–88 (2005). 197. Chiu, L. L., Cunningham, L. L., Raible, D. W., Rubel, E. W. & Ou, H. C. Using the zebrafish lateral line to screen for ototoxicity. J. Assoc. Res. Otolaryngol. 9, 178–190 (2008). 198. Hirose, Y., Simon, J. A. & Ou, H. C. Hair cell toxicity in anti-cancer drugs: evaluating an anti-cancer drug library for independent and synergistic toxic effects on hair cells using the zebrafish lateral line. J. Assoc. Res. Otolaryngol. 12, 719–728 (2011). 199. Namdaran, P., Reinhart, K. E., Owens, K. N., Raible, D. W. & Rubel, E. W. Identification of modulators of hair cell regeneration in the zebrafish lateral line. J. Neurosci. 32, 3516–3528 (2012). 200. Xiong, W., Wagner, T., Yan, L., Grillet, N. & Müller, U. Using injectoporation to deliver genes to mechano sensory hair cells. Nature Protoc. 9, 2438–2449 (2014). This study describes a new method that enables the culturing of explants of the inner-ear sensory epithelia that are suitable for gene transfer into hair cells and for analysing the functional properties of hair cells by electrophysiology. 201. Hsu, P. D., Lander, E. S. & Zhang, F. Development and applications of CRISPR‑Cas9 for genome engineering. Cell 157, 1262–1278 (2014). 202. Ronaghi, M. et al. Inner ear hair cell-like cells from human embryonic stem cells. Stem Cells Dev. 23, 1275–1284 (2014). 203. Baguley, D., McFerran, D. & Hall, D. Tinnitus. Lancet 382, 1600–1607 (2013). 204. Langguth, B., Kreuzer, P. M., Kleinjung, T. & De Ridder, D. Tinnitus: causes and clinical management. Lancet Neurol. 12, 920–930 (2013). 205. Berlinger, N. T. Meniere’s disease: new concepts, new treatments. Minn. Med. 94, 33–36 (2011). 206. Semaan, M. T., Alagramam, K. N. & Megerian, C. A. The basic science of Meniere’s disease and endolymphatic hydrops. Otolaryngol. Head Neck Surg. 13, 301–307 (2005).


REVIEWS 207. Li, J. D. et al. Panel 4: recent advances in otitis media in molecular biology, biochemistry, genetics, and animal models. Otolaryngol. Head Neck Surg. 148, E52–E63 (2013). 208. Grillet, N. et al. Mutations in LOXHD1, an evolutionarily conserved stereociliary protein, disrupt hair cell function in mice and cause progressive hearing loss in humans. Am. J. Hum. Genet. 85, 328– 337 (2009). 209. Schwander, M. et al. A novel allele of myosin VIIa reveals a critical function for the C‑terminal FERM domain for melanosome transport in retinal pigment epithelial cells. J. Neurosci. 29, 15810–15818 (2009). 210. Koehler, K. R. & Hashino, E. 3D mouse embryonic stem cell culture for generating inner ear organoids. Nature Protoc. 9, 1229–1244 (2014). 211. Walsh, T. et al. From flies’ eyes to our ears: mutations in a human class III myosin cause progressive nonsyndromic hearing loss DFNB30. Proc. Natl Acad. Sci. USA 99, 7518–7523 (2002). 212. Melchionda, S. et al. MYO6, the human homologue of the gene responsible for deafness in Snell’s waltzer mice, is mutated in autosomal dominant nonsyndromic hearing loss. Am. J. Hum. Genet. 69, 635–640 (2001). 213. Ahmed, Z. M. et al. Mutations of MYO6 are associated with recessive deafness, DFNB37. Am. J. Hum. Genet. 72, 1315–1322 (2003). 214. Weil, D. et al. Defective myosin VIIA gene responsible for Usher syndrome type 1B. Nature 374, 60–61 (1995). 215. Liu, X. Z. et al. Mutations in the myosin VIIA gene cause non-syndromic recessive deafness. Nature Genet. 16, 188–190 (1997). 216. Weil, D. et al. The autosomal recessive isolated deafness, DFNB2, and the Usher 1B syndrome are allelic defects of the myosin-VIIA gene. Nature Genet. 16, 191–193 (1997). 217. Donaudy, F. et al. Multiple mutations of MYO1A, a cochlear-expressed gene, in sensorineural hearing loss. Am. J. Hum. Genet. 72, 1571–1577 (2003). 218. Wang, A. et al. Association of unconventional myosin MYO15 mutations with human nonsyndromic deafness DFNB3. Science 280, 1447–1451 (1998). 219. Zhu, M. et al. Mutations in the γ-actin gene (ACTG1) are associated with dominant progressive deafness (DFNA20/26). Am. J. Hum. Genet. 73, 1082–1091 (2003). 220. van Wijk, E. et al. A mutation in the gamma actin 1 (ACTG1) gene causes autosomal dominant hearing loss (DFNA20/26). J. Med. Genet. 40, 879–884 (2003). 221. Khan, S. Y. et al. Mutations of the RDX gene cause nonsyndromic hearing loss at the DFNB24 locus. Hum. Mutat. 28, 417–423 (2007). 222. Shahin, H. et al. Mutations in a novel isoform of TRIOBP that encodes a filamentous-actin binding protein are responsible for DFNB28 recessive nonsyndromic hearing loss. Am. J. Hum. Genet. 78, 144–152 (2006). 223. Riazuddin, S. et al. Mutations in TRIOBP, which encodes a putative cytoskeletal-organizing protein, are associated with nonsyndromic recessive deafness. Am. J. Hum. Genet. 78, 137–143 (2006). 224. Li, Y. et al. Mutations in TPRN cause a progressive form of autosomal-recessive nonsyndromic hearing loss. Am. J. Hum. Genet. 86, 479–484 (2010). 225. Rehman, A. U. et al. Targeted capture and nextgeneration sequencing identifies C9orf75, encoding taperin, as the mutated gene in nonsyndromic deafness DFNB79. Am. J. Hum. Genet. 86, 378–388 (2010). 226. Naz, S. et al. Mutations of ESPN cause autosomal recessive deafness and vestibular dysfunction. J. Med. Genet. 41, 591–595 (2004). 227. Verpy, E. et al. A defect in harmonin, a PDZ domaincontaining protein expressed in the inner ear sensory hair cells, underlies Usher syndrome type 1C. Nature Genet. 26, 51–55 (2000). 228. Bitner-Glindzicz, M. et al. A recessive contiguous gene deletion causing infantile hyperinsulinism, enteropathy and deafness identifies the Usher type 1C gene. Nature Genet. 26, 56–60 (2000). 229. Ouyang, X. M. et al. Mutations in the alternatively spliced exons of USH1C cause non-syndromic recessive deafness. Hum. Genet. 111, 26–30 (2002). 230. Ahmed, Z. M. et al. Nonsyndromic recessive deafness DFNB18 and Usher syndrome type IC are allelic mutations of USHIC. Hum. Genet. 110, 527–531 (2002).

231. Mburu, P. et al. Defects in whirlin, a PDZ domain molecule involved in stereocilia elongation, cause deafness in the whirler mouse and families with DFNB31. Nature Genet. 34, 421–428 (2003). 232. Ebermann, I. et al. A novel gene for Usher syndrome type 2: mutations in the long isoform of whirlin are associated with retinitis pigmentosa and sensorineural hearing loss. Hum. Genet. 121, 203–211 (2007). 233. Mustapha, M. et al. A novel locus for Usher syndrome type I, USH1G, maps to chromosome 17q24‑25. Hum. Genet. 110, 348–350 (2002). 234. Weil, D. et al. Usher syndrome type I G (USH1G) is caused by mutations in the gene encoding SANS, a protein that associates with the USH1C protein, harmonin. Hum. Mol. Genet. 12, 463–471 (2003). 235. Schraders, M. et al. Mutations in PTPRQ are a cause of autosomal-recessive nonsyndromic hearing impairment DFNB84 and associated with vestibular dysfunction. Am. J. Hum. Genet. 86, 604–610 (2010). 236. Weston, M. D., Luijendijk, M. W., Humphrey, K. D., Moller, C. & Kimberling, W. J. Mutations in the VLGR1 gene implicate G‑protein signaling in the pathogenesis of Usher syndrome type II. Am. J. Hum. Genet. 74, 357–366 (2004). 237. Verpy, E. et al. Mutations in a new gene encoding a protein of the hair bundle cause non-syndromic deafness at the DFNB16 locus. Nature Genet. (2001). 238. Borck, G. et al. Loss‑of‑function mutations of ILDR1 cause autosomal-recessive hearing impairment DFNB42. Am. J. Hum. Genet. 88, 127–137 (2011). 239. Bork, J. M. et al. Usher syndrome 1D and nonsyndromic autosomal recessive deafness DFNB12 are caused by allelic mutations of the novel cadherinlike gene CDH23. Am. J. Hum. Genet. 68, 26–37 (2001). 240. Bolz, H. et al. Mutation of CDH23, encoding a new member of the cadherin gene family, causes Usher syndrome type 1D. Nature Genet. 27, 108–112 (2001). 241. Ahmed, Z. M. et al. Mutations of the protocadherin gene PCDH15 cause Usher syndrome type 1F. Am. J. Hum. Genet. 69, 25–34 (2001). 242. Alagramam, K. N. et al. Mutations in the novel protocadherin PCDH15 cause Usher syndrome type 1F. Hum. Mol. Genet. 10, 1709–1718 (2001). 243. Kurima, K. et al. Dominant and recessive deafness caused by mutations of a novel gene, TMC1, required for cochlear hair-cell function. Nature Genet. 30, 277–284 (2002). 244. Kalay, E. et al. Mutations in the lipoma HMGIC fusion partner-like 5 (LHFPL5) gene cause autosomal recessive nonsyndromic hearing loss. Hum. Mutat. 27, 633–639 (2006). 245. Shabbir, M. I. et al. Mutations of human TMHS cause recessively inherited non-syndromic hearing loss. J. Med. Genet. 43, 634–640 (2006). 246. Kelsell, D. P. et al. Connexin 26 mutations in hereditary non-syndromic sensorineural deafness. Nature 387, 80–83 (1997). 247. Liu, X. Z. et al. Mutations in connexin31 underlie recessive as well as dominant non-syndromic hearing loss. Hum. Mol. Genet. 9, 63–67 (2000). 248. Xia, J. H. et al. Mutations in the gene encoding gap junction protein β‑3 associated with autosomal dominant hearing impairment. Nature Genet. 20, 370–373 (1998). 249. del Castillo, I. et al. A deletion involving the connexin 30 gene in nonsyndromic hearing impairment. N. Engl. J. Med. 346, 243–249 (2002). 250. Grifa, A. et al. Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus. Nature Genet. 23, 16–18 (1999). 251. Wilcox, E. R. et al. Mutations in the gene encoding tight junction claudin‑14 cause autosomal recessive deafness DFNB29. Cell 104, 165–172 (2001). 252. Riazuddin, S. et al. Tricellulin is a tight-junction protein necessary for hearing. Am. J. Hum. Genet. 79, 1040– 1051 (2006). 253. Walsh, T. et al. Genomic duplication and overexpression of TJP2/ZO‑2 leads to altered expression of apoptosis genes in progressive nonsyndromic hearing loss DFNA51. Am. J. Hum. Genet. 87, 101–109 (2010). 254. Neyroud, N. et al. A novel mutation in the potassium channel gene KVLQT1 causes the Jervell and LangeNielsen cardioauditory syndrome. Nature Genet. 15, 186–189 (1997). 255. Kubisch, C. et al. KCNQ4, a novel potassium channel expressed in sensory outer hair cells, is mutated in dominant deafness. Cell 96, 437–446 (1999).


256. Tyson, J. et al. IsK and KvLQT1: mutation in either of the two subunits of the slow component of the delayed rectifier potassium channel can cause Jervell and Lange-Nielsen syndrome. Hum. Mol. Genet. 6, 2179–2185 (1997). 257. Schulze-Bahr, E. et al. KCNE1 mutations cause jervell and Lange-Nielsen syndrome. Nature Genet. 17, 267–268 (1997). 258. Reichold, M. et al. KCNJ10 gene mutations causing EAST syndrome (epilepsy, ataxia, sensorineural deafness, and tubulopathy) disrupt channel function. Proc. Natl Acad. Sci. USA 107, 14490–14495 (2010). 259. Collin, R. W. et al. Mutations of ESRRB encoding estrogen-related receptor beta cause autosomalrecessive nonsyndromic hearing impairment DFNB35. Am. J. Hum. Genet. 82, 125–138 (2008). 260. Wayne, S. et al. Mutations in the transcriptional activator EYA4 cause late-onset deafness at the DFNA10 locus. Hum. Mol. Genet. 10, 195–200 (2001). 261. Vahava, O. et al. Mutation in transcription factor POU4F3 associated with inherited progressive hearing loss in humans. Science 279, 1950–1954 (1998). 262. Mencia, A. et al. Mutations in the seed region of human miR‑96 are responsible for nonsyndromic progressive hearing loss. Nature Genet. 41, 609–613 (2009). 263. von Ameln, S. et al. A mutation in PNPT1, encoding mitochondrial-RNA-import protein PNPase, causes hereditary hearing loss. Am. J. Hum. Genet. 91, 919–927 (2012). 264. Goto, Y., Nonaka, I. & Horai, S. A mutation in the tRNA(Leu)(UUR) gene associated with the MELAS subgroup of mitochondrial encephalomyopathies. Nature 348, 651–653 (1990). 265. van den Ouweland, J. M. et al. Mutation in mitochondrial tRNA(Leu)(UUR) gene in a large pedigree with maternally transmitted type II diabetes mellitus and deafness. Nature Genet. 1, 368–371 (1992). 266. Shoffner, J. M. et al. Myoclonic epilepsy and raggedred fiber disease (MERRF) is associated with a mitochondrial DNA tRNA(Lys) mutation. Cell 61, 931–937 (1990). 267. Zeviani, M. et al. A MERRF/MELAS overlap syndrome associated with a new point mutation in the mitochondrial DNA tRNA(Lys) gene. Eur. J. Hum. Genet. 1, 80–87 (1993). 268. Kameoka, K. et al. Novel mitochondrial DNA mutation in tRNA(Lys) (8296A—>G) associated with diabetes. Biochem. Biophys. Res. Commun. 245, 523–527 (1998). 269. Jaksch, M. et al. Progressive myoclonus epilepsy and mitochondrial myopathy associated with mutations in the tRNA(Ser(UCN)) gene. Ann. Neurol. 44, 635–640 (1998). 270. Tiranti, V. et al. Maternally inherited hearing loss, ataxia and myoclonus associated with a novel point mutation in mitochondrial tRNASer(UCN) gene. Hum. Mol. Genet. 4, 1421–1427 (1995). 271. Hao, H., Bonilla, E., Manfredi, G., DiMauro, S. & Moraes, C. T. Segregation patterns of a novel mutation in the mitochondrial tRNA glutamic acid gene associated with myopathy and diabetes mellitus. Am. J. Hum. Genet. 56, 1017–1025 (1995). 272. Prezant, T. R. et al. Mitochondrial ribosomal RNA mutation associated with both antibiotic-induced and non-syndromic deafness. Nature Genet. 4, 289–294 (1993). 273. Ahmed, Z. M. et al. Functional null mutations of MSRB3 encoding methionine sulfoxide reductase are associated with human deafness DFNB74. Am. J. Hum. Genet. 88, 19–29 (2011). 274. Yasunaga, S. et al. A mutation in OTOF, encoding otoferlin, a FER‑1‑like protein, causes DFNB9, a nonsyndromic form of deafness. Nature Genet. 21, 363–369 (1999). 275. Du, X. et al. A catechol-O‑methyltransferase that is essential for auditory function in mice and humans. Proc. Natl Acad. Sci. USA 105, 14609–14614 (2008). 276. Ahmed, Z. M. et al. Mutations of LRTOMT, a fusion gene with alternative reading frames, cause nonsyndromic deafness in humans. Nature Genet. 40, 1335–1340 (2008). 277. Verhoeven, K. et al. Mutations in the human α-tectorin gene cause autosomal dominant non-syndromic hearing impairment. Nature Genet. 19, 60–62 (1998).


REVIEWS 278. Zwaenepoel, I. et al. Otoancorin, an inner ear protein restricted to the interface between the apical surface of sensory epithelia and their overlying acellular gels, is defective in autosomal recessive deafness DFNB22. Proc. Natl Acad. Sci. USA 99, 6240–6245 (2002). 279. Robertson, N. G. et al. Mutations in a novel cochlear gene cause DFNA9, a human nonsyndromic deafness with vestibular dysfunction. Nature Genet. 20, 299–303 (1998). 280. Yariz, K. O. et al. Mutations in OTOGL, encoding the inner ear protein otogelin-like, cause moderate sensorineural hearing loss. Am. J. Hum. Genet. 91, 872–882 (2012). 281. Schraders, M. et al. Mutations of the gene encoding otogelin are a cause of autosomal-recessive nonsyndromic moderate hearing impairment. Am. J. Hum. Genet. 91, 883–889 (2012). 282. Barker, D. F. et al. Identification of mutations in the COL4A5 collagen gene in Alport syndrome. Science 248, 1224–1227 (1990). 283. Mochizuki, T. et al. Identification of mutations in the α3(IV) and α4(IV) collagen genes in autosomal recessive Alport syndrome. Nature Genet. 8, 77–81 (1994). 284. McGuirt, W. T. et al. Mutations in COL11A2 cause non-syndromic hearing loss (DFNA13). Nature Genet. 23, 413–419 (1999). 285. Van Laer, L. et al. The contribution of genes involved in potassium-recycling in the inner ear to noise-induced hearing loss. Hum. Mutat. 27, 786–795 (2006). 286. Pawelczyk, M. et al. Analysis of gene polymorphisms associated with K+ ion circulation in the inner ear of patients susceptible and resistant to noise-induced hearing loss. Ann. Hum. Genet. 73, 411–421 (2009). 287. Konings, A. et al. Association between variations in CAT and noise-induced hearing loss in two independent noise-exposed populations. Hum. Mol. Genet. 16, 1872–1883 (2007).

288. Konings, A. et al. Candidate gene association study for noise-induced hearing loss in two independent noiseexposed populations. Ann. Hum. Genet. 73, 215–224 (2009). 289. Konings, A. et al. Variations in HSP70 genes associated with noise-induced hearing loss in two independent populations. Eur. J. Hum. Genet. 17, 329–335 (2009). 290. Yang, M. et al. Association of hsp70 polymorphisms with risk of noise-induced hearing loss in Chinese automobile workers. Cell Stress Chaperones 11, 233–239 (2006). 291. Peters, U. et al. Glutathione S‑transferase genetic polymorphisms and individual sensitivity to the ototoxic effect of cisplatin. Anticancer Drugs 11, 639–643 (2000). 292. Oldenburg, J., Kraggerud, S. M., Cvancarova, M., Lothe, R. A. & Fossa, S. D. Cisplatin-induced long-term hearing impairment is associated with specific glutathione S‑transferase genotypes in testicular cancer survivors. J. Clin. Oncol. 25, 708–714 (2007). 293. Xu, X. et al. Genetic polymorphism of copper transporter protein 1 is related to platinum resistance in Chinese non-small cell lung carcinoma patients. Clin. Exp. Pharmacol. Physiol. 39, 786–792 (2012). 294. Caronia, D. et al. Common variations in ERCC2 are associated with response to cisplatin chemotherapy and clinical outcome in osteosarcoma patients. Pharmacogenom. J. 9, 347–353 (2009). 295. Noben-Trauth, K., Zheng, Q. Y. & Johnson, K. R. Association of cadherin 23 with polygenic inheritance and genetic modification of sensorineural hearing loss. Nature Genet. 35, 21–23 (2003). 296. Johnson, K. R., Gagnon, L. H., Longo-Guess, C. & Kane, K. L. Association of a citrate synthase missense mutation with age-related hearing loss in A/J mice. Neurobiol. Aging 33, 1720–1729 (2012).


297. Charizopoulou, N. et al. Gipc3 mutations associated with audiogenic seizures and sensorineural hearing loss in mouse and human. Nature Commun. 2, 201 (2011). 298. Shin, J. B. et al. The R109H variant of fascin‑2, a developmentally regulated actin crosslinker in hair-cell stereocilia, underlies early-onset hearing loss of DBA/2J mice. J. Neurosci. 30, 9683–9694 (2010). 299. Skradski, S. L. et al. A novel gene causing a mendelian audiogenic mouse epilepsy. Neuron 31, 537–544 (2001). 300. Johnson, K. R., Zheng, Q. Y., Bykhovskaya, Y., Spirina, O. & Fischel-Ghodsian, N. A nuclearmitochondrial DNA interaction affecting hearing impairment in mice. Nature Genet. 27, 191–194 (2001).

Acknowledgements

This work was supported by NIH grants R01 DC002368 (P.B-G.), R01 DC011034 (P.B-G.), R01 DC005965 (U.M.) and RO1 DC007704 (U.M.); fellowship support from the Hearing Health Foundation (P.B-G); the Dorris Neuroscience Center (U.M.), the Skaggs Institute for Chemical Biololgy (U.M.) and the California Institute of Regenerative Medicine (U.M.).

Competing interests statement

The authors declare no competing interests.

FURTHER INFORMATION Hereditary Hearing Impairment in Mice: http://hearingimpairment.jax.org Hereditary Hearing Loss: http://hereditaryhearingloss.org OMIM: http://omim.org ALL LINKS ARE ACTIVE IN THE ONLINE PDF


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