MINOCYCLINE PREVENTS GENTAMICIN-INDUCED OTOTOXICITY BY INHIBITING p38 MAP KINASE PHOSPHORYLATION AND CASPASE 3 ACTIVATION X. WEI, a L. ZHAO, a J. LIU, b1 R. C. DODEL, c M. R. FARLOW a AND Y. DU a,d * a Department of Neurology, School of Medicine, Indiana University, 975 West Walnut Street IB 457, Indianapolis, IN 46202, USA b School of Pharmaceutical Education, Peking University, Beijing, China c Department of Neurology, Friedrich-Wilhelms-University, Bonn, Ger- many d Indiana University Center for Aging Research, Indiana University, India- napolis, IN 46202, USA Abstract—Aminoglycosides are commonly used antibiotics that often induce ototoxicity leading to permanent hair cell loss and hearing impairment. We hereby examined whether minocycline protects hair cells from gentamicin-induced hair cell damage. Two millimolar gentamicin significantly induced outer hair cell damage and the addition of minocycline to gentamicin-treated explants significantly increased hair cell survival in a dose-dependent manner. Additionally, we dem- onstrated that gentamicin induced p38 MAPK phosphoryla- tion, cytochrome c release, and caspase 3 activation in these cells and these remarkable changes were blocked by mino- cycline treatments. Furthermore, we showed that the inhibi- tor of p38 MAPK or the inhibitor of caspase 3 only partially blocked gentamicin-induced hair cell damage, and the pre- treatment of explants with the inhibitor of p38 MAPK and the inhibitor of caspase 3 together exerted a synergic protective effect against gentamicin-induced hair cell damage. Our re- sults suggest that minocycline blocks gentamicin-induced hair cell loss possibly by inhibition of three mechanisms: p38 MAPK phosphorylation, cytochrome c release, and caspase 3 activation. This finding may explain why minocycline has protective activity in a variety of apoptotic models. Therapeu- tic intervention by using minocycline or related drugs may be a novel means for preventing inner ear injury following the use of aminoglycoside. © 2005 Published by Elsevier Ltd on behalf of IBRO. Key words: minocycline, apoptosis, caspase 3, p38 MAP ki- nase, ototoxicity, hair cell. Significant hearing loss occurs in approximately 10% of the population. Decline in hearing can be caused by a variety of etiologies, including aging and therapeutic agents, such as aminoglycoside antibiotics (Arslan et al., 1999). The incidence of ototoxicity from aminoglycoside antibiotics is between 10 and 63% (Arslan et al., 1999). Recently, it has been suggested that aminoglycoside-induced hearing im- pairment may be caused by mutations in the mitochondrial DNA (Arslan et al., 1999). Since hair cells do not regener- ate in the mammalian cochlea, their losses are irreversible and cumulative. Development of otoprotective drugs to protect these cells has been relatively elusive. Currently, there is no medical therapy to protect against hair cell loss, largely because the molecular mechanisms responsible for ototoxin- or noise-induced hair cell death remain undeter- mined. Recently, evidence has suggested that C-Jun N- terminal kinase (Pirvola et al., 2000; Ulla et al., 2000; Wang et al., 2003), caspase 3 (Mangiardi et al., 2004; Liu et al.,1998), excitotoxicity (Darlington and Smith, 2003), as well as oxidative stress (Evans and Halliwell, 1999) are involved in ototoxin- and noise-induced hair cell death. Minocycline is a semisynthetic second-generation tet- racycline which exerts anti-inflammatory effects that are completely separate and distinct from its antimicrobial ac- tions (Ryan and Ashley, 1998). Clinical studies have sug- gested that minocycline and related tetracyclines have beneficial anti-inflammatory activities which may be useful for treating both rheumatoid arthritis as well as osteoarthri- tis (Ryan et al., 1996). Recently, the neuroprotective prop- erties of minocycline in models of ischemic injury (global and focal ischemia; Yrjanheikki et al., 1998, 1999) have been shown to be due, in part, to indirect effects in inhib- iting glial (astrocytic/microglial) caspase 1, iNOS activity, as well as p38 MAP kinase (p38 MAPK) phosphorylation (Yrjanheikki et al., 1998, 1999; Tikka et al., 2001). We have also demonstrated that minocycline is able to block 1-methyl- 4-phenyl-1,2,3,6-tetrahydropyridine-induced neurotoxicity by inhibiting neuronal p38 MAPK phosphorylation (Du et al., 2001). Additionally, Chen et al. (2000); Zhu et al. (2002) demonstrated that minocycline treatment delayed mortality or progression in a mouse model of Huntington disease and amyotrophic lateral sclerosis, presumably by inhibiting caspase 3 expression and cytochrome c release. They suggest that the primary target of minocycline is to block the release of cytochrome c. However, since considerable evidence suggests that phosphorylation of p38 MAPK reg- ulates the release of cytochrome c (Zhuang et al., 2000; Cheng et al., 2001), minocycline may be able to block cytochrome c release by inhibition of p38 MAPK. Further- more, since in almost all of these neuroprotective models, using specific inhibitors of these proteins alone provided much less neuroprotection than minocycline, we suggest that minocycline may target additional neuroprotective mechanisms than originally suggested. Most recently, it 1 Present address: School of Pharmaceutical Education, Peking Uni- versity, Beijing, China. *Correspondence to: Y. Du, Department of Neurology, School of Med- icine, Indiana University, 975 West Walnut Street IB 457, Indianapolis, IN 46202, USA. Tel: +1-317-278-0220; fax: +1-317-274-3587. E-mail address: ydu@iupui.edu (Y. Du). Abbreviations: NO, nitric oxide; p38 MAPK, p38 MAP kinase; PBS, phosphate-buffered saline; p-p38, phosphorylated p38 MAPK. Neuroscience 131 (2005) 513–521 0306-4522/05$30.00+0.00 © 2005 Published by Elsevier Ltd on behalf of IBRO. doi:10.1016/j.neuroscience.2004.11.014 513