A combined genome-wide association and molecular study of age-related hearing loss in H. sapiens

Abstract

Background: Sensorineural hearing loss is one of the most common sensory deficiencies. However, the molecular contribution to age-related hearing loss is not fully elucidated.

Methods: We performed genome-wide association studies (GWAS) for hearing loss-related traits in the UK Biobank (N = 362,396) and selected a high confidence set of ten hearing-associated gene products for staining in human cochlear samples: EYA4, LMX1A, PTK2/FAK, UBE3B, MMP2, SYNJ2, GRM5, TRIOBP, LMO-7, and NOX4.

Results: All proteins were found to be expressed in human cochlear structures. Our findings illustrate cochlear structures that mediate mechano-electric transduction of auditory stimuli, neuronal conductance, and neuronal plasticity to be involved in age-related hearing loss.

Conclusions: Our results suggest common genetic variation to influence structural resilience to damage as well as cochlear recovery after trauma, which protect against accumulated damage to cochlear structures and the development of hearing loss over time.

Keywords: Age-related hearing loss; GWAS; Human gene expression; Structured illumination microscopy.

Fig. 1

Overview of the human cochlea and organ of Corti. Red is used as a contrast to better visualize the inner and outer spiral bundle, tunnel spiral bundle, and basal fibers. The cells of Claudius were not labeled but are located above the cells of Boettcher. Image adapted with permission from Liu et al. [117]

Fig. 2

Expression pattern of LMO7 in the human cochlea. A Super-resolution structured illumination microscopy (SR-SIM) shows LMO7 (green) is expressed in the cuticular plates of the inner and outer hair cells. B Higher magnification of two tangentially cut inner hair cells. Parvalbumin is expressed in the cell bodies and LMO7 in the cuticular plates. C Close-up view of the cuticular plate of an inner hair cell. Actin staining was observed in the stereocilia and cuticular plates. LMO7 is only expressed in the cuticular plates and to a lesser degree in the cytoplasm, particularly near the tight junction between the inner hair cell and phalangeal supporting cell. D Corresponding transmission electron microscopy (TEM) of a human inner hair cell. The stereocilia contain central rootlets that extend into the cuticular plate. A large number of mitochondria face the apical pole and the cuticular plate. The cuticular plate extends laterally against the junctional area, as shown in C. This figure is a modified version of a previously published TEM image [117] and is used here with permission. IHC = inner hair cell, OHCs = outer hair cells, Pv = Parvalbumin, cut. = cuticula, TJ = tight junction, SC = stereocilia, Ph = phalangeal cell, r = stereociliar rootlet

Fig. 3

Expression pattern of TRIOBP in the human cochlea. A SR-SIM image that illustrates TRIOBP expression in the organ of Corti. Expression is present in inner and outer hair cell bodies, cuticula, stereocilia bundles, and nerve fibers. B Confocal microscopy of human spiral ganglion cell bodies expressing TRIOBP. Expression is not seen in the central axons. C Higher magnification shows TRIOBP expression in outer hair cell stereocilia and cuticula. D Corresponding image shows co-expression of actin and TRIOBP in the stereocilia bundle and cuticular plate. IHC = inner hair cell, OHCs = outer hair cells, n = afferent/efferent neurons, TUJ1 = neuron-specific class III beta-tubulin. Nuclei are stained with DAPI (blue)

Fig. 4

Expression pattern of EYA4 in the human cochlea. A EYA4 expression is observed in membranous labyrinth supporting cell nuclei: within the organ of Corti, the inner and outer sulcus, cells of Claudius, and spiral limbus interdental cells. Parvalbumin stains the inner and outer hair cells red. Nuclei were stained with DAPI. B Close-up of the organ of Corti reveals EYA4 expression in the outer hair cells as well as supporting cells: phalangeal cells, pillar cells, Deiters cells, Hensen cells, and Boettcher cells. Cells beneath the basilar membrane lack EYA4 expression. C EYA4 is expressed in the spiral ganglion cells. ICs = inderdental cells, IS = inner sulcus, OC = organ of Corti, Cl = cells of Claudius, IHC = inner hair cell, OHCs = outer hair cells, HCs = Hensen cells, Ph = phalangeal cells

Fig. 5

Expression pattern of SYNJ2 and MMP2 in the human cochlea. A SR-SIM reveals expression of SYNJ2 in the outer spiral bundles, observed here beneath the outer hair cells, which express parvalbumin. B Co-staining of MMP2 and SYNJ2 reveal co-expression within in the outer spiral bundles. C Confocal microscopy of outer and inner hair cells showing lack of SYNJ2 expression. Inner hair cells are only weakly stained for SYNJ2, which is believed to be unspecific. D Spiral ganglion neurons exhibit MMP2 and SYNJ2 expression. E Higher magnification of the area outlined in C. SYNJ2 and MMP2 can be observed to colocalize in ~ 100 nm molecular aggregates. Pv = parvalbumin, OHCs = outer hair cells, IHCs = inner hair cells, OSBs = outer spiral bundles

Fig. 6

Expression pattern of UBE3B in the human cochlea. A UBE3B is expressed together with parvalbumin in human spiral ganglion cell bodies in the basal modiolus. B Same image as A but showing UBE3B staining without the neuronal marker (Pv) and DAPI. Pv = parvalbumin. C Low power image of a radial section of the cochlear duct. Hair cells and neural elements stain positive for parvalbumin. UBE3B was not detected

Fig. 7

Expression pattern of PTK2/FAK in the human cochlea. A FAK expression was observed in both afferent and efferent neurons that innervate the hair cells. Actin was also present in the hair cell stereocilia and cuticular plates. The outer hair cells are highlighted with broken lines. B Low-power confocal image shows FAK staining of basal tunnel fibers and the tunnel spiral bundle. Actin-positive areas of the head and foot regions of the pillar cells are shown. Pillar cells contain invaginated afferent neurons (basal tunnel fibers) that innervate the outer hair cells. The inner hair cell and pillar cells are highlighted with broken lines. C Expression of FAK was present in the cytoplasm of spiral ganglion cells. D–F False color imaging reveals a gradient expression of FAK from the cochlear base to the apex. High expression intensity was observed in the cochlear base (D), which gradually decreases in neurons that supply the second (E) and third cochlear turns (F). OHCs = outer hair cells, T1 = type I spiral ganglion cells, or alternatively, large spiral ganglion cells. DC = Deiters cells, OPC = outer pillar cells, BTF = basal tunnel fibers, TSB = tunnel spiral bundles, HC = Hensen cells, OSBs = outer spiral bundles, IHC = inner hair cell

Fig. 8

Expression pattern of GRM5 in the human spiral ganglion. A GRM5 was observed in spiral ganglion cells. B–D A gradient expression of GRM5 was observed: from high expression in the basal turn (B) of the cochlea, which diminished gradually towards the midturn (C) and cochlear apex (D)

Fig. 9

Expression pattern of LMX1A in the human cochlea. A Expression of LMX1A and actin in the organ of Corti. LMX1A was expressed in both the inner and outer pillar cells, as well as the reticular lamina and Hensen cells. Weak staining is visible in Deiters cells, which suggests the presence of LMX1A also in these cells. Actin expression was observed in the pillar cell feet and heads. Actin was also visible in the stereocilia of the hair cells. B LMX1A was also expressed in the spiral ganglion neurons, with increased expression in the peripheral regions of the cell body cytoplasm. IPC = inner pillar cell, OPC = outer pillar cell, RL = reticular lamina, OHC = outer hair cell, DC = Deiters cell, HC = Hensen cells

Fig. 10

Expression pattern of NOX4 in the human cochlea. A Confocal microscopy reveals strong NOX4 expression in supporting cells of the organ of Corti. Particularly strong expression was observed in Hensen cells, pillar cells, and the cell membrane of the outer hair cells. In contrast, NOX4 expression was negative in the inner hair cell. B NOX4 is expressed in the interdental cells of the spiral limbus, as well as the inner sulcus cells. C Spiral ganglion cells also express NOX4. D Expression of NOX4 in the outer sulcus cells and inferior region of the spiral prominence. E NOX4 expression was also observed in the apical cell membrane of the marginal cells of the stria vascularis. IHC = inner hair cell, OHCs = outer hair cells, HCs = Hensen cells, PiC = pillar cell, IS = inner sulcus, SP = spiral prominence, OS = outer sulcus