Live imaging the phagocytic activity of inner ear supporting cells in response to hair cell death

Abstract

Hearing loss and balance disorders affect millions of people worldwide. Sensory transduction in the inner ear requires both mechanosensory hair cells (HCs) and surrounding glia-like supporting cells (SCs). HCs are susceptible to death from aging, noise overexposure, and treatment with therapeutic drugs that have ototoxic side effects; these ototoxic drugs include the aminoglycoside antibiotics and the antineoplastic drug cisplatin. Although both classes of drugs are known to kill HCs, their effects on SCs are less well understood. Recent data indicate that SCs sense and respond to HC stress, and that their responses can influence HC death, survival, and phagocytosis. These responses to HC stress and death are critical to the health of the inner ear. Here we have used live confocal imaging of the adult mouse utricle, to examine the SC responses to HC death caused by aminoglycosides or cisplatin. Our data indicate that when HCs are killed by aminoglycosides, SCs efficiently remove HC corpses from the sensory epithelium in a process that includes constricting the apical portion of the HC after loss of membrane integrity. SCs then form a phagosome, which can completely engulf the remaining HC body, a phenomenon not previously reported in mammals. In contrast, cisplatin treatment results in accumulation of dead HCs in the sensory epithelium, accompanied by an increase in SC death. The surviving SCs constrict fewer HCs and display impaired phagocytosis. These data are supported by in vivo experiments, in which cochlear SCs show reduced capacity for scar formation in cisplatin-treated mice compared with those treated with aminoglycosides. Together, these data point to a broader defect in the ability of the cisplatin-treated SCs, to preserve tissue health in the mature mammalian inner ear. . 4 SCs perform many functions, including providing critical trophic factors, preventing excitotoxicity, and mediating regeneration in those systems (non-mammalian vertebrates) capable of replacing lost HCs. 5-11 When HCs die, SCs also preserve the integrity and function of the remaining tissue by forming scars and clearing dead HCs. 2,12-17 Maintaining a fluid barrier at the surface of the sensory epithelium after damage is necessary to preserve the electro-chemical gradient that drives HC depolarization and therefore sensory transduction after the onset of hearing (reviewed in Wangemann). 18 Several major stressors cause HC death, 19-22 including aging, noise trauma, and exposure to therapeutic drugs with ototoxic side effects. When a HC is killed by noise or aminoglycoside antibiotics, surrounding SCs form a filamentous actin (F-actin) cable that constricts the HC at its apex. 2,[12][13][14][15][16][17] This process separates the apical portion of the cell, including the stereocilia bundle, from the HC body and preserves a sealed reticular lamina. 23 In the chick utricle, following the apical constriction of dead HCs, the SCs engulf and phagocytose the remaining HC corpse. 15 Additional data from the chick indicate that the ototoxi...

Supporting cells remove and replace sensory receptor hair cells in a balance organ of adult mice

“…However, it is also possible that the actin spike does not play a role in HC clearance. Because we did not detect stereocilia in translocated HCs, we presume that the apical portion of each HC is cleaved by SCs and ejected apically before translocation, similar to what occurs after aminoglycoside toxicity (Meiteles and Raphael, 1994; Li et al, 1995; Bird et al, 2010; Monzack et al, 2015). However, it is also possible that stereocilia proteins are degraded in HCs prior to, or during, translocation.

10.7554/eLife.18128.025Figure 10.Model of HC turnover in adult mouse utricles under normal conditions.( A ) Model of HC clearance.

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Two cell populations participate in clearance of damaged hair cells from the sensory epithelia of the inner ear

“…Rather than being extruded intact from the sensory epithelium, dying hair cells are eliminated via a two-step mechanism that involves the extrusion of the apical portion of the hair cell, followed by the phagocytosis of the remaining basal portion of the hair cell (Bird et al, 2010). In essence, this process is similar to that which occurs in the vestibular organs of mammals (Monzack et al, 2015). The supporting cells that surround a dying hair cell first enlarge their apical surfaces, causing the upper portion of the hair cell, including the stereocilia bundle and cuticular plate to be pinched between adjacent supporting cells and then ejected from the epithelium.…”

“…However, in addition to repairing the epithelial barrier, inner ear supporting cells can also serve as ‘amateur’ phagocytes, and often play a critical role in the removal of cellular debris. Time-lapse imaging studies of the injured mouse utricle demonstrate that hair cell death leads to the formation of a phagosome by surrounding supporting cells (Monzack et al, 2015). Supporting cells that surround a dying hair cell quickly assemble a cup-shaped, actin structure that pushes the apical portion of the hair cell towards the luminal surface of the epithelium, and then encloses the remaining basal portion and pushes it toward the basement membrane.…”

Intercellular Ca2+ signalling in the adult mouse cochlea

“…Any such deterioration was identified by visible changes in cell morphology and loss of cytoplasmic fluorescence (Monzack et al . ). All imaging experiments were conducted at room temperature (21–23°C).…”

“…The tissue could be imaged by confocal microscopy for up to 6 h with no apparent deterioration of the supporting cells. Any such deterioration was identified by visible changes in cell morphology and loss of cytoplasmic fluorescence (Monzack et al 2015). All imaging experiments were conducted at room temperature (21-23°C).…”