But no matter the mechanism, ototoxic drugs have to enter the hair cell to induce cytotoxicity. Traditionally, this was thought to occur via endocytosis. However, Marcotti and colleagues demonstrated that the ototoxic aminoglycosides could enter hair cells via the mechanoelectrical transduction (MET) channel (J Physiol 2005;567[Pt 2]:505-521).
The gating of this MET channel's depolarizing current is regulated by tip links between adjacent stereocilia and is essential for auditory perception. Numerous investigators have defined the characteristics of the MET channel and associated proteins, as well as the transduction current, which can be disrupted genetically or pharmacologically to block MET channel function.
Integrity and Regeneration of Mechanotransduction Machinery Regulate Aminoglycoside Entry and Sensory Cell Death
Vu AA, Nadaraja GS, et al PLOS ONE 2013;8(1):e54794
These manipulations of the MET complex are utilized by Vu et al to demonstrate that aminoglycoside entry into hair cells via MET channels is the predominant mechanism leading to acute hair cell cytotoxicity in vitro.
The authors first demonstrate that fluorescently tagged gentamicin is rapidly taken up by hair cells from wild-type 3-day-old mice but not by hair cells from Cdh23 v2J mice, which express dysfunctional cadherin 23 that abolishes tip-link gating of the MET channel. The reduced uptake of aminoglycosides by Cdh23 v2J/v2J hair cells was sufficient to protect them from a one-hour incubation with gentamicin, which typically kills wild-type hair cells with functional tip links in vitro.
Stereociliary tip-link integrity and function can also be pharmacologically disrupted by calcium chelation. Upon restoration of calcium, hair cells can regenerate tip links and regain MET channel activity within 24 hours. The authors exploited this knowledge to demonstrate that pharmacological disruption of tip links reduced hair cell uptake of fluorescently tagged gentamicin and protected hair cells against acute gentamicin-induced cell death.
If cochlear explants were allowed to recover for 24 hours after calcium chelation, hair cell uptake of fluorescently tagged gentamicin was restored, corresponding to the time course of tip-link regeneration. In addition, 24 hours after cessation of calcium chelation, treated hair cells were equally sensitive to acute gentamicin treatment as untreated hair cells were.
The authors’ primary interpretation of these data is that tip-link gating of the aminoglycoside-permeant MET channel is crucial for gentamicin entry into hair cells and subsequent hair cell degeneration. They hypothesize that, since hair cells can regenerate tip links, transient pharmacological ablation of tip links may represent a therapeutic target to prevent permanent aminoglycoside-induced ototoxicity clinically.
This elegant paper also raises new questions about the nature of aminoglycoside-induced ototoxicity. Clearly, aminoglycoside entry into hair cells through MET channels is required for acute hair cell cytotoxicity, while entry by endocytosis or permeation via other aminoglycoside-permeant channels expressed by hair cells (e.g., TRPA1 and TRPV4) may occur in chronic exposure in vivo.
This is an important distinction that will enable new research to partially differentiate between various intracellular mechanisms of aminoglycoside-induced hair cell death, such as cytoplasmic generation of reactive oxygen species and lysis of mitochondria and other organelles, as well as to investigate whether endocytotic uptake of aminoglycosides can lead to cytotoxicity. Slower mechanisms of aminoglycoside-induced hair cell cytotoxicity have been described (Hear Res 2009;253[1-2]:32-41) and may contribute to mechanisms of ototoxic synergy or to continued progression of hearing loss after cessation of drug treatment.
Presumably, Cdh23 v2J/v2J hair cells had intact endocytotic mechanisms. If so, the acute gentamicin incubation period was not sufficient to induce hair cell degeneration over the subsequent two days in vitro.
Could a longer incubation time induce hair cell cytotoxicity via endocytotic mechanisms and endoplasmic reticular stress in cochlear explants lacking functional tip links? Alternatively, could activation of other aminoglycoside-permeant cation channels expressed by hair cells also trigger gentamicin entry and hair cell degeneration, particularly across the basolateral membrane of hair cells? These questions are natural extensions of the pioneering work accomplished by Vu and colleagues in the quest to understand the complex mechanisms of ototoxicity and how to prevent its lifelong consequences of hearing loss, deafness, and vestibular deficits.
Read past Journal Club columns in a special collection on this HJ website: http://bit.ly/HJJournalClub.© 2013 Lippincott Williams & Wilkins, Inc.