Prestin as a biochemical marker for early detection of acquired sensorineural hearing loss

Medical Hypotheses xxx (2015) xxx–xxx

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Prestin as a biochemical marker for early detection of acquired sensorineural hearing loss Kourosh Parham Department of Surgery, Division of Otolaryngology-Head and Neck Surgery, UCONN Health, Farmington, CT, United States

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Article history: Received 2 January 2015 Accepted 15 April 2015 Available online xxxx

a b s t r a c t Acquired sensorineural hearing loss and tinnitus can come about through various etiologies such as exposure to excessively loud noise or drugs with ototoxic properties. As such, acquired hearing loss is a common source of morbidity which deleteriously affects the ability to communicate. At present our ability to detect acquired hearing loss and tinnitus at its earliest stages is limited and there are no adjuncts to audiometric evaluation. The earliest cellular targets of noise and ototoxins in the cochlea are the outer hair cells (OHC). I hypothesize that serum assays of OHC specific protein, prestin, will allow detection and quantification of OHC damage before audiometric testing can identify presence of hearing loss. At present, there are no data available to evaluate this hypothesis, but initial evaluation can readily be carried out using existing experimental animal models of ototoxicity and noise-induced hearing loss. Early detection of OHC damage is critical to adoption of measures aimed at ameliorating hearing loss and tinnitus, thus reducing permanent deficits and disability. Ó 2015 Elsevier Ltd. All rights reserved.

Introduction The World Health Organization estimates that 5% of the world’s population – 360 million people – has disabling hearing loss [1]. Almost 50 million Americans have hearing loss, including 1 in 5 teenagers [2]. 60% of veterans returning form Iraq and Afghanistan come home with hearing loss and tinnitus, making hearing complaints, then number 1 war wound [2]. Tinnitus affects 20% of Americans and hearing loss occurs in 90% of these cases [2]. Hearing loss becomes more prevalent with age and those with even mild hearing loss are twice as likely to develop dementia [2]. As a result, hearing loss is a common source of morbidity which deleteriously affects the ability to communicate. At present, we primarily rely on audiometric testing for diagnosis of hearing loss. However, early detection – i.e., before hearing loss becomes measurable by audiometric assessment or disabling – has the potential to prevent progression and substantially reduce a major burden on the society. Hypothesis Biomarkers help increase the accuracy of diagnosis, characterize disease, aid prognosis, predict response to treatment and guide treatment. There are currently over 30 biomarkers in clinical use,

with varying degrees of sensitivity and specificity [3]. For example, there are several serum biomarkers, which are routinely used in clinical oncology, e.g. prostate specific antigen (PSA) for prostate cancer and cancer antigen (CA)-125 for ovarian cancer, and cardiology, e.g., cardiac enzyme creatine kinase (CK) and cardiac proteins, troponins. Lack of serum biomarkers has been an impediment to early diagnosis and management of inner ear disorders (e.g., noise-induced and sudden sensorineural hearing loss, Meniere’s disease, ototoxicity, etc.). We have recently demonstrated that an innerear specific protein may be able to act as a biomarker for vestibular disease [4]. Otolin-1 is a scaffolding protein exclusively expressed in otoconia and cells of the vestibule and the cochlea. We assayed and compared serum samples from control subjects without history of vertigo and subjects with history of benign paroxysmal positional vertigo (BPPV) [4]. We were able to demonstrate that not only otolin-1 was detectable in the serum, but also it was quantifiable with BPPV subjects having significantly higher levels. We now hypothesize that the same approach can be utilized to define biochemical markers for hearing loss. The first requirement for such a biomarker is to identify a protein that is specific enough to be able to hone in on the cochlea, the hearing end organ. Etiologies of hearing loss are diverse and can be broadly classified as congenital or acquired. Acquired hearing loss is far more common, therefore, it is a good starting point for the search for hearing loss biomarkers . It is believed that a variety of different

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Please cite this article in press as: Parham K. Prestin as a biochemical marker for early detection of acquired sensorineural hearing loss. Med Hypotheses (2015), http://dx.doi.org/10.1016/j.mehy.2015.04.015

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ototoxins, as well as, acoustic trauma share common pathways leading to damage and death of cochlear sensory cells through activation of reactive oxygen species [5]. Outer hair cells (OHCs, Fig. 1B–D) are generally more susceptible to damage and are affected gradually from the base of the cochlea to its apex, producing a pattern of hearing loss that begins in high frequencies and progresses to lower frequencies [6]. Inner hair cells (Fig. 1B and C) are the sensory mechanoreceptor responsible for encoding sound wave-induced deflections of the basilar membrane. OHCs, on the other hand, are effector cells that enhance tuning and sensitivity of the cochlea. These functions appear to be directly related to electromotile properties of the OHCs, that is the length of OHCs changes as a function of membrane polarization [7,8]. A protein, named prestin, located in the lateral membrane of the OHCs (Fig. 1D and E) acts as the motor responsible for the voltage-dependent changes [9]. Targeted deletion of prestin results in loss of OHC electromotility in vitro and a 40–60 dB loss of cochlear sensitivity in vivo, without disruption of mechanoelectrical transduction in OHCs [10]. Prestin is believed to be a unique inner ear protein and because of its restricted expression, it has been proposed as a molecular target for delivery of polymerase mediated drug delivery to OHC [11]. An excellent, recent review of the prestin literature is available elsewhere [12]. Since OHCs are early targets for noise-induced damage and ototoxins, it has been proposed that functional measures of OHC function, should be sensitive early indicators of hearing loss [13]. Otoacoustic emissions, discovered in late 1970’s [14], are one such example whose measurement represents a clinical tool which specifically targets OHC function. However, due a number of technical challenges in utilization of otoacoustic emissions, the promise of early detection of hearing loss in the clinical setting has yet to be realized [15]. Based on the above defined roles and characteristics of the OHCs, I propose a novel, adjunctive approach to early detection of hearing loss: serum measurement of inner ear specific protein, prestin, as a biochemical marker. Prestin is an excellent candidate, because of its specific expression in the OHCs [12]. Dead OHCs

from acoustic or ototoxic injury are phagocytosed by supporting cells (Fig. 1C) in the organ of Corti [16]. This process is initiated within minutes of hair cell apoptosis and concurrent with loss of hair cell integrity [17]. With degeneration of OHCs, prestin can be found in phagosomes in the supporting cells [16]. Some of contents of these phagosomes will end up in circulation. Although, there are no reports of prestin in circulation, we have recently demonstrated that another inner ear specific protein, otolin-1, does cross the blood-labyrinthine barrier [4]. Otolin-1 is 70 kDa and prestin is 80 kDa in size. Both are smaller than, for example, methionine (150 kDa) which is a proposed otoprotectant against noise- or toxin-induced hearing loss, believed to act at the level of the OHCs [18]. Therefore, prestin is small enough to cross the blood-labyrinthine barrier and enter circulation. Using sensitive assays, such as enzyme-linked immunosorbent assay (ELISA), even minute quantities of specific proteins can be detected in the serum, as we demonstrated for otolin-1 [4]. There are approximately, 11,000 OHCs in an adult cochlea. Loss of about 10% can produce 2.5–4 dB of decrease in otoacoustic emission amplitudes [19]. Because of extreme sensitivity of the ELISA technique, I suggest that picogram quantities of prestin released into circulation can be detected at a far earlier stage, perhaps from less than 1% OHC loss. To date, there have been no studies of prestin, as a biomarker, specifically. Given the central role of OHCs in sensorineural hearing loss, it is not surprising that the role of prestin in mediating hearing loss has been explored by others. Prestin appears to be involved in mediation of injury in the cochlea in response to loud noise exposure, as genetically induced-prestin dysfunction reduces amount of hearing loss in response to overstimulation [20]. On the other hand, it has also been implicated as a mediator of aminoglycoside-induced apoptosis of OHCs [21]. Furthermore, prestin expression studies suggest dynamic changes in response to agents that influence hearing. Noise-induced hearing loss is associated with elevated prestin gene expression in the remaining OHCs, peaking at the 3rd post-exposure day and return to baseline 4 weeks later [22]. More specifically, there appears to be a base-to-apex gradient in

Fig. 1. (A) Inner ear consisting of cochlea and the semicircular canals. (B) A cross section through the cochlea illustrating the relationship of the organ of Corti to the scala media, tympani and vestibuli. (C) The relationship of the inner and outer hair cells to one another and the supporting cells in the organ of Corti. (D) An outer hair cell with a circle on the lateral membrane where molecular motors (i.e., prestin) are located. (E) 3D structure of prestin (adapted from He et al. [12]).

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prestin mRNA expression [23]. Similarly, after noise-induced hearing loss producing OHC loss in basal cochlea, prestin mRNA expression in residual, apical OHCs increases 7 days to 1 month later [24]. Long-term administration of salicylates increases prestin expression at the mRNA and protein levels, consistent with increase in distortion product otoacoustic emissions [25]. Changes in expression of prestin along the lateral membrane have also been reported in OHCs of the apical turn of the cochlea after radiation exposure, but not in the basal cochlea [26]. Evaluation of the hypothesis I predict a time course of change in serum prestin levels which reflects above dynamic changes after exposure to injurious noise or drugs. Because of inherent homeostatic cellular processes, baseline detection of prestin is expected at low levels. Within hours of hair cell damage from exposure to noise or ototoxins, prestin levels are expected to rise above baseline. About 3–5 days after exposure to the injurious process prestin levels are likely to peak and then decline, however, because of increased expression in the surviving OHCs, levels are expected to remain above baseline for at least 1 month after exposure. A widely accepted guideline for development of biomarkers was introduced by the National Cancer Institute in 2001 [27]. This guideline defines five phases in testing the validity of a cancer biomarker. Although these guidelines were developed for cancer, they are generalizable to other disease states. Phase I, the pre-clinical exploratory stage, leads to identification of potentially useful markers and Phase II, the validation stage, involves studies to determine the capacity of the biomarkers to distinguish between diseased and non-diseased states. Thus Phase II establishes the validity, portability and reproducibility of candidate biomarker and leads to determination of sensitivity and the specificity of the biomarker. Phases III–V involve prospective efforts to identify the potential predictive value to ascertain the disease occurrence, the extent and characteristics of disease detected by the biomarker, determine the false positive rate, and determine the overall impact (i.e., risks and benefits) of controlled screening on the population. Here, by identifying prestin as a candidate biomarker, we have fulfilled Phase I objectives. Phase II can be initiated feasibly by testing above hypotheses in rodent models of ototoxicity (e.g., cisplatin or aminoglycosides) [28] and noise-induced hearing loss [29]. We have initiated these experiments and expect to have early results in the near future. Validation of prestin as a biomarker and reproducibility of this candidate biomarker in different experimental models of hearing loss is expected to lead to clinical trials (Phases III–V). Consequences of the hypothesis Acquired hearing loss and tinnitus are common both in the pediatric and adult populations. For example, platinum-based chemotherapy is an effective treatment option commonly prescribed for a variety of childhood cancers [30]. Early detection of hearing loss in pediatric oncology patients and early intervention are critical to help these patient succeed in achieving developmental milestones [30]. Occupational and recreational noise exposures are the dominant sources of hearing loss and tinnitus in the adult population. For example, military personnel work in high-noise environments, yet we are unable to predict who is susceptible to noise-induced hearing loss and tinnitus [31]. In 2009, US Veterans Affairs’ disability payments for tinnitus and hearing loss exceeded $1.2 billion [31] and is expected to have increased substantially over the past 5 years given that 60% of Iraq and

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Afghanistan veterans suffer from these disabilities. Early identification of individuals at risk will allow intervention before development of disabling hearing loss and tinnitus and substantially reduce morbidity in potential sufferers and financial burden on the society. Conflict of interest statement The author has no financial and/or personal relationships with other people or organizations that could inappropriately influence their work. References [1] Organization WH. Deafness and hearing loss: fact sheet, ; 2014. [accessed December 26, 2014]. [2] Foundation HH. Hearing loss & tinnitus statistics, ; 2014. [3] Yotsukura S, Mamitsuka H. Evaluation of serum-based cancer biomarkers: a brief review from a clinical and computational viewpoint. Crit Rev Oncol/ Hematol 2014. [4] Parham K, Sacks D, Bixby C, Fall P. Inner ear protein as a biomarker in circulation? Otolaryngol Head Neck Surg 2014. [5] Kopke R, Allen KA, Henderson D, Hoffer M, Frenz D, Van de Water T. A radical demise. Toxins and trauma share common pathways in hair cell death. Ann N Y Acad Sci 1999;884:171–91. [6] Schacht J, Talaska AE, Rybak LP. Cisplatin and aminoglycoside antibiotics: hearing loss and its prevention. Anat Rec 2012;295(11):1837–50. [7] Brownell WE, Bader CR, Bertrand D, de Ribaupierre Y. Evoked mechanical responses of isolated cochlear outer hair cells. Science 1985;227(4683):194–6. [8] Zenner HP, Zimmermann U, Schmitt U. Reversible contraction of isolated mammalian cochlear hair cells. Hear Res 1985;18(2):127–33. [9] Zheng J, Shen W, He DZ, Long KB, Madison LD, Dallos P. Prestin is the motor protein of cochlear outer hair cells. Nature 2000;405(6783):149–55. [10] Liberman MC, Gao J, He DZ, Wu X, Jia S, Zuo J. Prestin is required for electromotility of the outer hair cell and for the cochlear amplifier. Nature 2002;419(6904):300–4. [11] Surovtseva EV, Johnston AH, Zhang W, et al. Prestin binding peptides as ligands for targeted polymersome mediated drug delivery to outer hair cells in the inner ear. Int J Pharm 2012;424(1–2):121–7. [12] He DZ, Lovas S, Ai Y, Li Y, Beisel KW. Prestin at year 14: progress and prospect. Hear Res 2014;311:25–35. [13] Harris FP, Probst R. Otoacoustic emissions and audiometric outcomes. In: Robinette M, Glattke T, editors. Otoacoustic emissions: clinical applications. New York: Thieme; 1997. p. 151–79. [14] Kemp DT. Stimulated acoustic emissions from within the human auditory system. J Acoust Soc Am 1978;64(5):1386–91. [15] Dhar S, Hall IJW. Otoacoustic emissions: principles, procedures and protocols. San Diego, CA: Plural Publishing; 2012. [16] Abrashkin KA, Izumikawa M, Miyazawa T, et al. The fate of outer hair cells after acoustic or ototoxic insults. Hear Res 2006;218(1–2):20–9. [17] Bird JE, Daudet N, Warchol ME, Gale JE. Supporting cells eliminate dying sensory hair cells to maintain epithelial integrity in the avian inner ear. J Neurosci 2010;30(37):12545–56. [18] Campbell KC, Meech RP, Klemens JJ, et al. Prevention of noise- and druginduced hearing loss with D-methionine. Hear Res 2007;226(1–2):92–103. [19] Hofstetter P, Ding D, Powers N, Salvi RJ. Quantitative relationship of carboplatin dose to magnitude of inner and outer hair cell loss and the reduction in distortion product otoacoustic emission amplitude in chinchillas. Hear Res 1997;112(1–2):199–215. [20] Cai Q, Wang B, Coling D, et al. Reduction in noise-induced functional loss of the cochleae in mice with pre-existing cochlear dysfunction due to genetic interference of prestin. PLoS ONE 2014;9(12):e113990. [21] Yu L, Jiang XH, Zhou Z, et al. A protective mechanism against antibioticinduced ototoxicity: role of prestin. PLoS ONE 2011;6(2):e17322. [22] Chen GD. Prestin gene expression in the rat cochlea following intense noise exposure. Hear Res 2006;222(1–2):54–61. [23] Mazurek B, Haupt H, Amarjargal N, Yarin YM, Machulik A, Gross J. Upregulation of prestin mRNA expression in the organs of Corti of guinea pigs and rats following unilateral impulse noise exposure. Hear Res 2007;231(1– 2):73–83. [24] Xia A, Song Y, Wang R, et al. Prestin regulation and function in residual outer hair cells after noise-induced hearing loss. PLoS ONE 2013;8(12):e82602. [25] Yu N, Zhu ML, Johnson B, Liu YP, Jones RO, Zhao HB. Prestin up-regulation in chronic salicylate (aspirin) administration: an implication of functional dependence of prestin expression. Cell. Mol. Life Sci.: CMLS 2008;65(15): 2407–18. [26] Yang C, Zhang W, Liu XL, et al. Localization of prestin and expression in the early period after radiation in mice. Eur Arch Otorhinolaryngol 2014;271(12): 3333–40. [27] Pepe MS, Etzioni R, Feng Z, et al. Phases of biomarker development for early detection of cancer. J Natl Cancer Inst 2001;93(14):1054–61.

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Please cite this article in press as: Parham K. Prestin as a biochemical marker for early detection of acquired sensorineural hearing loss. Med Hypotheses (2015), http://dx.doi.org/10.1016/j.mehy.2015.04.015