Mechanisms of aminoglycoside ototoxicity and drug-damage repair in sensory hair cells: towards the design of otoprotective strategies

Our sense of hearing depends on the correct functioning of hair cells, the cells in the inner ear that convert motion into electrical signals that the brain interprets as sound. The aminoglycoside antibiotics, of which gentamicin is the most widely used, are administered to seriously ill patients in hospital that are at risk from life-threatening infections, in order to try to eradicate the bacteria causing those infections. These antibiotics have the unfortunate side effect of causing hearing loss in an estimated 20-50% of patients. Because of their high efficacy compared to other antibiotics and broad anti-bacterial spectrum the use of these antibiotics is however on the increase and likely to continue. It would therefore be very beneficial to try and develop strategies for preventing the hearing loss caused by these drugs. In our recent research we have identified how aminoglycoside antibiotics enter hair cells in the inner ear, eventually killing them and thus leading to deafness. First, they enter the hair cells via their mechano-electrical transducer channels - these are situated on top of the bundles of 'hairs' or stereocilia on the hair cells, and these channels open and close as sound waves move the stereocilia. Once inside the hair cells, they remain trapped and cannot get out. Then they enter the energy producing machinery in the cell, the mitochondria, by a currently unknown route. We will investigate whether entry is via ion channels in the inner membrane of the mitochondria. After the aminoglycosides have entered them, these mitochondria release proteins (pro-apoptotic factors) that ultimately kill the cells. Before the hair cells are killed though, we found that they form bleb-like membrane protrusions on their surface, on and around the hair bundles. We will investigate whether this is a direct effect of the aminoglycosides or whether this depends on the aminoglycosides first entering the mitochondria. We also found that the hair cells can recover from these membrane 'blebs', provided the exposure to the aminoglycosides is sufficiently short. This indicates the presence of a damage-repair process in the hair cells, which we wish to investigate with a view to making it more effective. One of the main aims of our research is to develop compounds that can compete with the aminoglycoside antibiotics for entry into the hair cells, and that may therefore prevent hair-cell damage and death. If any of these compounds are found to be clinically safe, we hope this may eventually lead to the development of drugs that, when co-administered with aminoglycoside antibiotics, prevent deafness in patients.

Aminoglycoside antibiotics are increasingly used in the clinic to combat serious infections but can kill sensory hair cells in the inner ear causing deafness in a significant number of patients. As permeant blockers of the hair cell's mechanoelectrical transduction (MET) channels the aminoglycosides enter and accumulate in hair cells via these channels disrupting apical plasma membrane homeostasis and mitochondrial function. These immediate effects lead to hair-cell death, but can be reversed by a previously-unrecognised damage-repair pathway if drug exposure times are restricted in duration. The aims are to develop strategies for preventing, or reversing, the early effects of aminoglycosides on hair cells. We will (i) fully define the pore properties of the hair cell's MET channel, developing novel, high-affinity blockers of this channel that may prevent aminoglycoside entry into hair cells, (ii) elucidate how aminoglycoside enter mitochondria and disrupt function and (iii) fully characterise the damage-repair process. An interdisclipinary approach combining patch-clamp recording of MET channel activity in mouse cochlear hair cells, chemical biology and drug design, and high-throughput screening of chemical libraries in zebrafish larvae will be used to find novel, potentially oto-protective, MET channel blockers. Characterisation of ion channels present in the mitochondrial inner membrane together with measurements of mitochondrial potential will reveal how aminoglycosides target the mitochondrial matrix and disrupt mitochondrial function. Confocal immunofluorescence and serial section electron microscopy, combined with functional analysis in mouse cochlear cultures and zebrafish lateral line organs using viral constructs, morpholinos and transgenes, will be used to characterise the molecular and cellular basis of the damage-repair mechanism. These studies will provide strategies for preventing the unwanted side effects of a clinically useful class of antibiotic.

Potential beneficiaries of this research:
Academia: International research groups involved in hearing research will benefit from the increased insight into hair cell function that our programme will provide, by being able to build upon our findings.
Medicine: Clinicians (e.g. those specializing in internal medicine, paediatrics, acute care, surgery) will benefit from our research into otoprotective compounds initially by being made more aware of the risks for hearing loss in administering aminoglycoside antibiotics. If our approach proves successful they will be able eventually to administer these antibiotics more safely.
Industry: We will consider developing a partnership within the pharmaceutical industry towards realizing the commercial application of any promising otoprotective compounds we may develop.
General public: We will engage with the general public by raising awareness of the risks of exposure to aminoglycoside antibiotics. If we succeed in creating clinically safe and applicable otoprotective compounds we will contribute to health and well-being by preventing deafness in infants, children and adults exposed to aminoglycoside antibiotics.