Date Published: June 11, 2019
Publisher: Public Library of Science
Author(s): Hsiao-Chun Lin, Yin Ren, Andrew C. Lysaght, Shyan-Yuan Kao, Konstantina M. Stankovic, Kourosh Parham.
The vast majority of hearing loss, the most common sensory impairment, and vertigo, which commonly causes falls, both reflect underlying dysfunction of inner ear cells. Perilymph sampling can thus provide molecular cues to hearing and balance disorders. While such “liquid biopsy” of the inner ear is not yet in routine clinical practice, previous studies have uncovered alterations in perilymph in patients with certain types of hearing loss. However, the proteome of perilymph from patients with intact hearing has been unknown. Furthermore, no complete characterization of perilymph from patients with vestibular dysfunction has been reported. Here, using liquid-chromatography with tandem mass spectrometry, we analyzed samples of normal perilymph collected from three patients with skull base meningiomas and intact hearing. We identified 228 proteins that were common across the samples, establishing a greatly expanded proteome of the previously inferred normal human perilymph. Further comparison to perilymph obtained from three patients with vestibular dysfunction with drop attacks due to Meniere’s disease showed 38 proteins with significantly differential abundance. The abundance of four protein candidates with previously unknown roles in inner ear biology was validated in murine cochleae by immunohistochemistry and in situ hybridization: AACT, HGFAC, EFEMP1, and TGFBI. Together, these results motivate future work in characterizing the normal human perilymph and identifying biomarkers of inner ear disease.
Hearing loss is the most common sensory impairment in humans and it currently disables 466 million people across the globe; this number is expected to rise to 900 million by 2050 . Nearly two-thirds of the population aged over 70 in the United states is affected by disabling hearing loss . A vast majority of this burden is due to sensorineural hearing loss (SNHL), which originates from defects in the cochlea, the spiraling organ of the inner ear (Fig 1). In addition, a third of the general population in the U.S. report vestibular symptoms such as vertigo, a persistent spinning sensation [3,4]. The majority of vertigo originates from the balance organs within the inner ear (Fig 1). When both the hearing and balance parts of the inner ear are affected, this can lead to audiovestibular pathologies such as in Meniere’s disease (MD), which is characterized by fluctuating hearing loss, vertigo, tinnitus and aural fullness. When vertiginous attacks become incapacitating and hearing loss turns profound, surgical removal of the inner ear’s vestibular organs (via labyrinthectomy) provides an effective treatment for vertigo when conservative medical therapy fails. In addition, labyrinthectomy also provides a rare opportunity to access the inner ear tissue in living humans.
Amongst the existing diagnostic armamentarium for patients with hearing and vestibular disorders, there are few tests that shed light on the underlying molecular pathophysiology. The perilymph fluid represents a promising resource for biomarker discovery and ultimately, the development of new diagnostic and prognostic tools, largely because it is much more highly enriched for molecules secreted by inner ear cells than plasma or CSF. Nevertheless, a systematic comparative analysis between the perilymph proteome in patients with loss of audiovestibular function and those with normal hearing have not been performed. This is largely due to the rare ability to instrument the inner ear with normal hearing, and partly due to inherent technical difficulties in extracting sufficient samples during surgical procedures, as well as limitations in post-hoc analysis techniques to quantitatively assess the composition of perilymph.
In this report, we have assembled the first proteome of normal human perilymph, collected from people with intact hearing, and performed comparative proteomic analysis of perilymph from patients undergoing labyrinthectomies for disabling vestibular dysfunction due to MD with drop attacks. Using tandem LC-MS/MS, we have significantly expanded the landscape of the known perilymph proteome to over 200 proteins, more than doubling candidate proteins found in previous efforts. We further validated 4 novel protein candidates that had not been previously described in the adult inner ear. We analyzed and defined their precise spatial distribution in the adult murine cochlea by immunohistochemistry (AACT) or fluorescent in-situ hybridization (Efemp1, Hgfac, Tgfbi). Findings from this work will motivate the future development of diagnostic and prognostic assays for hearing loss and vestibular dysfunction based on proteomic markers from inner ear fluids.