Blockade of c-Jun N-terminal kinase pathway attenuates gentamicin-induced cochlear and vestibular hair cell death

Abstract

The ototoxic action of aminoglycoside antibiotics leading to the loss of hair cells of the inner ear is well documented. However, the molecular mechanisms are poorly defined. We have previously shown that in neomycin-exposed organotypic cultures of the cochlea, the c-Jun N-terminal kinase (JNK) pathway – associated with stress, injury and apoptosis – is activated in hair cells and leads to their death. We have also shown that hair cell death can be attenuated by CEP-1347, an inhibitor of JNK signalling [Pirvola et al., J. Neurosci. 20 (2000) 43–50]. In the present study, we demonstrate that gentamicin-induced ototoxicity leads to JNK activation and apoptosis in the inner ear hair cells in vivo. We also show that systemic administration of CEP-1347 attenuates gentamicin-induced decrease of auditory sensitivity and cochlear hair cell damage. In addition, CEP-1347 treatment reduces the extent of hair cell loss in the ampullary cristae after gentamicin intoxication. Particularly, the inner hair cells of the cochlea and type I hair cells of the vestibular organs are protected. We have previously shown that also acoustic overstimulation leads to apoptosis of cochlear hair cells and that CEP-1347 can attenuate noise-induced sensory cell loss. These results suggest that activation of the JNK cascade may be a common molecular outcome of cellular stress in the inner ear sensory epithelia, and that attenuation of the lesion can be provided by inhibiting JNK activation.

Introduction

The ototoxic potential of aminoglycoside antibiotics is well known. Therefore, their clinical use has been limited to the treatment of severe infections caused by Gram-negative microorganisms. The ototoxic capacity of aminoglycoside antibiotics has been widely used in experimental research to create inner ear lesions, despite the fact that the molecular mechanisms of ototoxicity have remained obscure. This has further been complicated by the recent demonstration that megalin, a high-affinity receptor for aminoglycosides, is expressed in the secretory, but not in the sensory epithelia of the inner ear (Ylikoski et al., 1997). Further understanding of the ototoxic mechanisms of aminoglycosides has been provided by a series of articles during the last decade indicating that the ototoxic effects of gentamicin (GM) require an ‘activated’ form of the drug (Huang and Schacht, 1990). This ‘activated’ GM leads to the formation of a redox-active iron–GM complex (Priuska and Schacht, 1995) and the generation of reactive oxygen species (ROS) (Clerici et al., 1996, Hirose et al., 1997, Sha and Schacht, 1999). This hypothesis is supported by the ability of certain antioxidants or free radical scavengers and iron chelators to protect from aminoglycoside-induced ototoxicity (Hulka et al., 1993, Garetz et al., 1994, Song and Schacht, 1996, Song et al., 1997). Another step towards understanding the mechanisms of aminoglycoside ototoxicity came from a study showing that concurrent administration of GM and N-methyl-D-aspartate antagonists markedly attenuates both hearing loss and destruction of cochlear hair cells (HCs) in guinea pigs (Basile et al., 1996). From that study it was concluded that aminoglycoside-induced ototoxicity is mediated, in part, through an excitotoxic process.

During recent years, an increasing body of evidence suggests that HC death after aminoglycoside ototoxicity can occur through apoptosis. This possibility was mentioned 15 years ago in GM-intoxicated cochleas (Forge, 1985). Apoptosis and necrosis are two forms of cell death that are defined based on morphological and biochemical criteria. In apoptosis, chromatin condensation, cellular shrinkage and early preservation of plasma membrane integrity contrast with the cytoplasmic disintegration and disorganized clumping of chromatin in necrosis (Kerr et al., 1972, Wyllie et al., 1980). Apoptosis is a gene-directed self-destruction program that mainly results from posttranslational activation of a set of proteins, which are involved in intracellular signalling cascades (Raff, 1992, Weil et al., 1996). In contrast, necrosis is thought to result from more passive mechanisms triggered by extrinsic insults (e.g. trauma, toxins, microbes). The apoptosis versus necrosis classification has been useful in categorizing cell death in numerous settings, but the relationship between these modes of cell death is not always clear. For instance, following excitotoxic or anoxic–ischemic injury, biochemical features of apoptosis and morphological evidence of necrosis have been observed even in the same individual neurons of the adult brain (Portera-Caillau et al., 1997).

Revealing the intracellular signalling pathways that are activated in stressed HCs might offer a possibility to attenuate stress-induced HC death. We have recently demonstrated that the c-Jun N-terminal kinase (JNK) pathway (Derijard et al., 1994, Kyriakis et al., 1994) is activated in stressed cochlear HCs in vitro and that CEP-1347, an indolocarbazole that inhibits JNK signalling (Maroney et al., 1998), protects auditory HCs from neomycin damage in vitro and from noise trauma in vivo (Pirvola et al., 2000). In the present study, we have studied whether aminoglycoside antibiotics cause activation of the JNK pathway and induction of apoptosis in the inner ear HCs in vivo. Further, we have investigated whether CEP-1347 can protect cochlear and vestibular HCs from aminoglycoside-induced death in vivo.