Localization of the mitochondrial uncoupling protein family in the rat inner ear Tadashi Kitahara a , Ha-Sheng Li a , Carey D. Balaban a,b,c, * a Department of Otolaryngology, University of Pittsburgh School of Medicine, 107 Eye and Ear Institute, 203 Lothrop Street, Pittsburgh, PA 15123, USA b Department of Neurobiology, University of Pittsburgh School of Medicine, 107 Eye and Ear Institute, 203 Lothrop Street, Pittsburgh, PA 15123, USA c Department of Communication Sciences, University of Pittsburgh School of Medicine, 107 Eye and Ear Institute, 203 Lothrop Street, Pittsburgh, PA 15123, USA Received 2 December 2003; accepted 12 February 2004 Available online 8 July 2004 Abstract Uncoupling proteins (UCPs) are a proton transporter family located in the mitochondrial inner membrane. The molecular expression and activity of UCPs in brown adipose tissue and skeletal muscle are regulated by factors as diverse as chronic overeating and cold exposure, suggesting roles in energy expenditure and heat production. Although UCP2, UCP4 and brain mitochondrial carrier protein-1 (BMCP-1, i.e. UCP5) mRNAs are expressed in the central nervous system, their central function is unknown. This study presents the first evidence on localization and quantitative expression of UCPs in the rat inner ear by real-time PCR and immunohistochemistry. Real-time PCR studies revealed that UCP2 mRNA was expressed in the vestibular and spiral ganglia more abundantly than any other UCP. Neocortex, by contrast, contained UCP2 and UCP4 equally. Notably, UCP3 and UCP4 mRNAs were expressed in inner ear ganglia, but brain UCP3 mRNA expression level was undetectable by simple PCR. Immunohisto- chemical studies confirmed that both UCP2- and UCP3-like immunoreactivities were detected in vestibular and spiral ganglion cells and co-localized with a mitochondrial marker, MitoFluorGreen. According to previous reports, UCP2 and UCP3 are thermogenic in yeast and brain UCP2 has been suggested to modulate pre- and post-synaptic events by axonal thermogenesis. It has also been reported recently that UCP2 and UCP3 responses to superoxide application may be an antioxidant protective mechanism. Therefore, it is suggested that mitochondrial UCPs (UCP2, UCP3, UCP4) may play both a protective role against oxidative damage and a thermal signaling role in the eighth nerve. Ó 2004 Published by Elsevier B.V. Keywords: Uncoupling protein; PGC-1a; PPARc; Vestibular ganglion; Spiral ganglion; Mitochondria; Real-time PCR; Immunohistochemistry 1. Introduction Uncoupling proteins (UCPs) are a proton transporter family located in the mitochondrial inner membrane. Five molecules have been identified as members of the UCP family. UCP1 is the classical UCP form in brown adipose tissue (BAT) (Enerback et al., 1997). UCP2 is distributed more widely in many tissues including the central nervous system (CNS) (Fleury et al., 1997) and UCP3 is expressed preferentially in muscle (Boss et al., 1997). The molecular expression and activity of UCPs in BAT and muscle are regulated by factors as diverse as chronic overeating and cold exposure, so that UCPs play important roles in regulating expenditure of energy and heat production (Argyropoulos and Harper, 2002). In particular, UCP2 and UCP3 appear to be important for regulation of intracellular levels of reactive oxygen species and regulation of fatty acid cycling within * Corresponding author. Tel.: +1-412-647-8050; fax: +1-412-647- 0108. E-mail address: balabancd@pitt.edu (C.D. Balaban). Abbreviations: CT: cycle threshold; Cx: cerebral cortex; LIR: like immunoreactivity; MFG: MitoFluorGreen; MS: skeletal muscle; PCR: polymerase chain reaction; PGC-1: PPAR coactivator-1; PPAR: peroxisome proliferator-activated receptor subunit; SG: spiral gan- glion; UCP: uncoupling protein; VG: vestibular ganglion 0378-5955/$ - see front matter Ó 2004 Published by Elsevier B.V. doi:10.1016/j.heares.2004.02.002 Hearing Research 196 (2004) 39–48 www.elsevier.com/locate/heares