Integrity and Regeneration of Mechanotransduction Machinery Regulate Aminoglycoside Entry and Sensory Cell Death

Abstract

Sound perception requires functional hair cell mechanotransduction (MET) machinery, including the MET channels and tip-link proteins. Prior work showed that uptake of ototoxic aminoglycosides (AG) into hair cells requires functional MET channels. In this study, we examined whether tip-link proteins, including Cadherin 23 (Cdh23), regulate AG entry into hair cells. Using time-lapse microscopy on cochlear explants, we found rapid uptake of gentamicin-conjugated Texas Red (GTTR) into hair cells from three-day-old Cdh23+/+ and Cdh23v2J/+ mice, but failed to detect GTTR uptake in Cdh23v2J/v2J hair cells. Pre-treatment of wildtype cochleae with the calcium chelator 1,2-bis(o-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid (BAPTA) to disrupt tip-links also effectively reduced GTTR uptake into hair cells. Both Cdh23v2J/v2J and BAPTA-treated hair cells were protected from degeneration caused by gentamicin. Six hours after BAPTA treatment, GTTR uptake remained reduced in comparison to controls; by 24 hours, drug uptake was comparable between untreated and BAPTA-treated hair cells, which again became susceptible to cell death induced by gentamicin. Together, these results provide genetic and pharmacologic evidence that tip-links are required for AG uptake and toxicity in hair cells. Because tip-links can spontaneously regenerate, their temporary breakage offers a limited time window when hair cells are protected from AG toxicity.

Expression of prestin in OHCs is reduced in Spag6 gene knockout mice

“…For hair cells counting, we used the cell counter tool in Image J software to accumulate the myosinVIIa-positive hair cells within the 400 μm length in the middle turn of the cochlea [17]. As for the quantification of fluorescence intensity of prestin, we also performed as previous study [17].…”

An integrated view of cisplatin-induced nephrotoxicity and ototoxicity

“…Unlike most cells, sensory hair cells have mechano-electrical transduction (MET) channels that are uniquely gated by tip links between individual stereocilia in their apically-located hair bundle (Assad et al, 1991; Hudspeth, 1986; Hudspeth and Corey, 1977; Pickles et al, 1984). Data from electrophysiological and time-lapse imaging studies, as well as drug uptake studies in hair cells with dysfunctional MET channels strongly suggest that the ototoxic aminoglycosides (molecular diameter ~0.81 nm; molecular mass ~445–477 for gentamicin) can permeate through the MET channel pore of at least 1.25 nm (Alharazneh et al, 2011; Farris et al, 2004; Marcotti et al, 2005; Vu et al, 2013). A recent study demonstrated that functional MET channels are required for cytosolic uptake of fluorescently-tagged cisplatin, and for cisplatin-induced hair cell death (Thomas et al, 2013).…”

Identification of ion-channel modulators that protect against aminoglycoside-induced hair cell death

“…Hair cells have a mechanosensory hair bundle located at their apical surface, and there is evidence that the aminoglycosides, known to be permeant blockers of the mechanoelectrical transducer (MET) channels in the hair bundle, enter into hair cells via these large-conductance cation channels (10)(11)(12)(13)(14). Once inside the hair cell, the aminoglycosides cause increases in reactive free radicals and mitochondrial calcium concentration, leading to either apoptosis or necrosis (1,2,15).…”