Genetic Medicine for Hearing Loss: OTOF as Exemplar.

Millions of people worldwide have disabling hearing loss because one of their genes generates an incorrect version of some specific protein the ear requires for hearing. In many of these cases, delivering the correct version of the gene to a specific target cell within the inner ear has the potential to restore cochlear function to enable high-acuity physiologic hearing. Purpose: In this review, we outline our strategy for the development of genetic medicines with the potential to treat hearing loss. We will use the example of otoferlin gene (OTOF)-mediated hearing loss, a sensorineural hearing loss due to autosomal recessive mutations of the OTOF gene.

Divergent Auditory Nerve Encoding Deficits Between Two Common Etiologies of Sensorineural Hearing Loss.

Speech intelligibility can vary dramatically between individuals with similar clinically defined severity of hearing loss based on the audiogram. These perceptual differences, despite equal audiometric-threshold elevation, are often assumed to reflect central-processing variations. Here, we compared peripheral-processing in auditory nerve (AN) fibers of male chinchillas between two prevalent hearing loss etiologies: metabolic hearing loss (MHL) and noise-induced hearing loss (NIHL). MHL results from age-related reduction of the endocochlear potential due to atrophy of the stria vascularis. MHL in the present study was induced using furosemide, which provides a validated model of age-related MHL in young animals by reversibly inhibiting the endocochlear potential. Effects of MHL on peripheral processing were assessed using Wiener-kernel (system identification) analyses of single AN fiber responses to broadband noise, for direct comparison to previously published AN responses from animals with NIHL. Wiener-kernel analyses show that even mild NIHL causes grossly abnormal coding of low-frequency stimulus components. In contrast, for MHL the same abnormal coding was only observed with moderate to severe loss. For equal sensitivity loss, coding impairment was substantially less severe with MHL than with NIHL, probably due to greater preservation of the tip-to-tail ratio of cochlear frequency tuning with MHL compared with NIHL rather than different intrinsic AN properties. Differences in peripheral neural coding between these two pathologies-the more severe of which, NIHL, is preventable-likely contribute to individual speech perception differences. Our results underscore the need to minimize noise overexposure and for strategies to personalize diagnosis and treatment for individuals with sensorineural hearing loss.SIGNIFICANCE STATEMENT Differences in speech perception ability between individuals with similar clinically defined severity of hearing loss are often assumed to reflect central neural-processing differences. Here, we demonstrate for the first time that peripheral neural processing of complex sounds differs dramatically between the two most common etiologies of hearing loss. Greater processing impairment with noise-induced compared with an age-related (metabolic) hearing loss etiology may explain heightened speech perception difficulties in people overexposed to loud environments. These results highlight the need for public policies to prevent noise-induced hearing loss, an entirely avoidable hearing loss etiology, and for personalized strategies to diagnose and treat sensorineural hearing loss.

Loss of LDAH associated with prostate cancer and hearing loss.

Great strides in gene discovery have been made using a multitude of methods to associate phenotypes with genetic variants, but there still remains a substantial gap between observed symptoms and identified genetic defects. Herein, we use the convergence of various genetic and genomic techniques to investigate the underpinnings of a constellation of phenotypes that include prostate cancer (PCa) and sensorineural hearing loss (SNHL) in a human subject. Through interrogation of the subject's de novo, germline, balanced chromosomal translocation, we first identify a correlation between his disorders and a poorly annotated gene known as lipid droplet associated hydrolase (LDAH). Using data repositories of both germline and somatic variants, we identify convergent genomic evidence that substantiates a correlation between loss of LDAH and PCa. This correlation is validated through both in vitro and in vivo models that show loss of LDAH results in increased risk of PCa and, to a lesser extent, SNHL. By leveraging convergent evidence in emerging genomic data, we hypothesize that loss of LDAH is involved in PCa and other phenotypes observed in support of a genotype-phenotype association in an n-of-one human subject.

Loudness Perception of Pure Tones in Parkinson's Disease.

Purpose: Reduced intensity is a hallmark of speech production in Parkinson's disease (PD). Previous work has examined the perception of intensity in PD to explain these speech deficits. This study reports loudness ratings of pure tones by individuals with PD and controls, all with normal thesholds for older adults. Method: Twenty individuals with PD and 23 age- and sex-matched controls rated the loudness of pure tones from 1 (very soft) to 7 (uncomfortably loud). Tones at 500, 750, 1000, 2000, and 4000 Hz were presented from 35 to 80 dB HL (or until a rating of 7 was given). A mixed-model analysis of variance was performed on ratings to assess the effects of group, frequency, sound intensity, and ear. Loudness growth slopes were determined for each participant and analyzed by group. Results: The mean loudness growth slopes of the control and PD groups did not significantly differ. Conclusions: No difference was found in loudness growth slopes in response to externally generated tones in PD. This is in contrast with the findings of previous studies of self-generated speech and externally presented speech. The underlying causes for impaired perception and production of loudness in PD require further investigation.

Global Analysis of Protein Expression of Inner Ear Hair Cells.

The mammalian inner ear (IE) subserves auditory and vestibular sensations via highly specialized cells and proteins. Sensory receptor hair cells (HCs) are necessary for transducing mechanical inputs and stimulating sensory neurons by using a host of known and as yet unknown protein machinery. To understand the protein composition of these unique postmitotic cells, in which irreversible protein degradation or damage can lead to impaired hearing and balance, we analyzed IE samples by tandem mass spectrometry to generate an unbiased, shotgun-proteomics view of protein identities and abundances. By using Pou4f3/eGFP-transgenic mice in which HCs express GFP driven by Pou4f3, we FACS purified a population of HCs to analyze and compare the HC proteome with other IE subproteomes from sensory epithelia and whole IE. We show that the mammalian HC proteome comprises hundreds of uniquely or highly expressed proteins. Our global proteomic analysis of purified HCs extends the existing HC transcriptome, revealing previously undetected gene products and isoform-specific protein expression. Comparison of our proteomic data with mouse and human databases of genetic auditory/vestibular impairments confirms the critical role of the HC proteome for normal IE function, providing a cell-specific pool of candidates for novel, important HC genes. Several proteins identified exclusively in HCs by proteomics and verified by immunohistochemistry map to human genetic deafness loci, potentially representing new deafness genes. SIGNIFICANCE STATEMENT: Hearing and balance rely on specialized sensory hair cells (HCs) in the inner ear (IE) to convey information about sound, acceleration, and orientation to the brain. Genetically and environmentally induced perturbations to HC proteins can result in deafness and severe imbalance. We used transgenic mice with GFP-expressing HCs, coupled with FACS sorting and tandem mass spectrometry, to define the most complete HC and IE proteome to date. We show that hundreds of proteins are uniquely identified or enriched in HCs, extending previous gene expression analyses to reveal novel HC proteins and isoforms. Importantly, deafness-linked proteins were significantly enriched in HCs, suggesting that this in-depth proteomic analysis of IE sensory cells may hold potential for deafness gene discovery.

Translational issues in cochlear synaptopathy.

Understanding the biology of the previously underappreciated sensitivity of cochlear synapses to noise insult, and its clinical consequences, is becoming a mission for a growing number of auditory researchers. In addition, several research groups have become interested in developing therapeutic approaches that can reverse synaptopathy and restore hearing function. One of the major challenges to realizing the potential of synaptopathy rodent models is that current clinical audiometric approaches cannot yet reveal the presence of this subtle cochlear pathology in humans. This has catalyzed efforts, both from basic and clinical perspectives, to investigate novel means for diagnosing synaptopathy and to determine the main functional consequences for auditory perception and hearing abilities. Such means, and a strong concordance between findings in pre-clinical animal models and clinical studies in humans, are important for developing and realizing therapeutics. This paper frames the key outstanding translational questions that need to be addressed to realize this ambitious goal.

Auditory Brainstem Response Latency in Noise as a Marker of Cochlear Synaptopathy.

Evidence from animal and human studies suggests that moderate acoustic exposure, causing only transient threshold elevation, can nonetheless cause "hidden hearing loss" that interferes with coding of suprathreshold sound. Such noise exposure destroys synaptic connections between cochlear hair cells and auditory nerve fibers; however, there is no clinical test of this synaptopathy in humans. In animals, synaptopathy reduces the amplitude of auditory brainstem response (ABR) wave-I. Unfortunately, ABR wave-I is difficult to measure in humans, limiting its clinical use. Here, using analogous measurements in humans and mice, we show that the effect of masking noise on the latency of the more robust ABR wave-V mirrors changes in ABR wave-I amplitude. Furthermore, in our human cohort, the effect of noise on wave-V latency predicts perceptual temporal sensitivity. Our results suggest that measures of the effects of noise on ABR wave-V latency can be used to diagnose cochlear synaptopathy in humans. SIGNIFICANCE STATEMENT: Although there are suspicions that cochlear synaptopathy affects humans with normal hearing thresholds, no one has yet reported a clinical measure that is a reliable marker of such loss. By combining human and animal data, we demonstrate that the latency of auditory brainstem response wave-V in noise reflects auditory nerve loss. This is the first study of human listeners with normal hearing thresholds that links individual differences observed in behavior and auditory brainstem response timing to cochlear synaptopathy. These results can guide development of a clinical test to reveal this previously unknown form of noise-induced hearing loss in humans.

A murine model of neurofibromatosis type 2 that accurately phenocopies human schwannoma formation.

Neurofibromatosis type 2 (NF2) is an autosomal dominant genetic disorder resulting from germline mutations in the NF2 gene. Bilateral vestibular schwannomas, tumors on cranial nerve VIII, are pathognomonic for NF2 disease. Furthermore, schwannomas also commonly develop in other cranial nerves, dorsal root ganglia and peripheral nerves. These tumors are a major cause of morbidity and mortality, and medical therapies to treat them are limited. Animal models that accurately recapitulate the full anatomical spectrum of human NF2-related schwannomas, including the characteristic functional deficits in hearing and balance associated with cranial nerve VIII tumors, would allow systematic evaluation of experimental therapeutics prior to clinical use. Here, we present a genetically engineered NF2 mouse model generated through excision of the Nf2 gene driven by Cre expression under control of a tissue-restricted 3.9kbPeriostin promoter element. By 10 months of age, 100% of Postn-Cre; Nf2(flox/flox) mice develop spinal, peripheral and cranial nerve tumors histologically identical to human schwannomas. In addition, the development of cranial nerve VIII tumors correlates with functional impairments in hearing and balance, as measured by auditory brainstem response and vestibular testing. Overall, the Postn-Cre; Nf2(flox/flox) tumor model provides a novel tool for future mechanistic and therapeutic studies of NF2-associated schwannomas.

Is noise-induced cochlear neuropathy key to the generation of hyperacusis or tinnitus?

Perceptual abnormalities such as hyperacusis and tinnitus often occur after acoustic overexposure. Although such exposure can also result in permanent threshold elevation, some individuals with noise-induced hyperacusis or tinnitus show clinically normal thresholds. Recent work in animals has shown that a "neuropathic" noise exposure can cause immediate, permanent degeneration of the cochlear nerve despite complete threshold recovery and lack of hair cell damage (Kujawa SG, Liberman MC. J Neurosci 29: 14077-14085, 2009; Lin HW, Furman AC, Kujawa SG, Liberman MC. J Assoc Res Otolaryngol 12: 605-616, 2011). Here we ask whether this noise-induced primary neuronal degeneration results in abnormal auditory behavior, based on the acoustic startle response (ASR) and prepulse inhibition (PPI) of startle. Responses were measured in mice exposed either to a "neuropathic" noise or to a lower-intensity, "nonneuropathic" noise and in unexposed control mice. Mice with cochlear neuropathy displayed hyperresponsivity to sound, evidenced by enhanced ASR and PPI, while exposed mice without neuronal loss showed control-like responses. Gap PPI tests, often used to assess tinnitus, revealed limited gap detection deficits in mice with cochlear neuropathy only for certain gap-startle latencies, inconsistent with the presence of tinnitus "filling in the gap." Despite significantly reduced wave 1 of the auditory brainstem response, representing cochlear nerve activity, later peaks were unchanged or enhanced, suggesting compensatory neural hyperactivity in the auditory brainstem. Considering the rapid postexposure onset of both cochlear neuropathy and exaggerated startle-based behavior, the results suggest a role for cochlear primary neuronal degeneration, per se, in the central neural excitability that could underlie the generation of hyperacusis.