Downregulation of the 18-kDa Translocator Protein: Effects on the Ammonia-Induced Mitochondrial Permeability Transition and Cell Swelling in Cultured Astrocytes K. S. PANICKAR, 1,2 A. R. JAYAKUMAR, 1 K. V. RAMA RAO, 1 AND M. D. NORENBERG 1,3,4 * 1 Department of Pathology, University of Miami School of Medicine, Miami, Florida 2 Diet, Genomics, and Immunology Laboratory, Beltsville Human Nutrition Research Center, US Department of Agriculture, Beltsville, Maryland 3 Veterans Affairs Medical Center 4 Department of Biochemistry and Molecular Biology, University of Miami School of Medicine, Miami, Florida KEY WORDS ammonia toxicity; astrocytes; cell swelling; hepatic ence- phalopathy; mitochondrial permeability transition; periphe- ral benzodiazepine receptor; 18-kDa translocator protein ABSTRACT Hepatic encephalopathy (HE) is a major neurological com- plication in patients with severe liver disease. While the pathogenesis of HE is unclear, elevated blood and brain ammonia levels are believed to be major etiological factors, and astrocytes appear to be the primary target of its toxic- ity. A notable feature of ammonia neurotoxicity is an upre- gulation of the 18-kDa translocator protein (TSPO) (for- merly referred to as the peripheral benzodiazepine receptor or PBR), which is found on the outer mitochondrial mem- brane. However, the precise significance of this upregulation is unclear. To examine its potential role in ammonia-induced astrocyte dysfunction, we downregulated the TSPO using antisense oligonucleotides, and examined whether such downregulation could alter two prominent features of am- monia gliotoxicity, namely, the mitochondrial permeability transition (MPT) and astrocyte swelling. Nontransfected cultures treated with NH 4 Cl (5 mM; 48 h) showed a signifi- cant increase in astrocyte cell volume (37.5%). In cultured astrocytes transfected with TSPO antisense oligonucleo- tides, such cell swelling was reduced to 17%, but this change was not significantly different from control cell volume. Simi- larly, nontransfected cultures treated with NH 4 Cl (5 mM; 24 h) exhibited a 40% decline in the cyclosporin A-sensitive mitochondrial inner membrane potential (DW m )(P < 0.01) (a measure of the MPT). By contrast, cells transfected with TSPO antisense oligonucleotides did not display a signifi- cant loss of the DW m following ammonia exposure. Our find- ings highlight the important role of the TSPO in the mech- anism of ammonia neurotoxicity. V V C 2007 Wiley-Liss, Inc. INTRODUCTION Hepatic encephalopathy (HE) is a major neurological disorder in patients with severe liver failure. Acute HE [fulminant hepatic failure (FHF)] results from massive liver necrosis following viral hepatitis, acetaminophen toxicity or exposure to other hepatotoxins. It is charac- terized by an abrupt onset of delirium, seizures, and coma, and it has an extremely poor prognosis with an 80–90% mortality rate (Capocaccia and Angelico, 1991). Chronic HE (portal-systemic encephalopathy) is charac- terized by a change in personality, altered mood, dimin- ished intellectual capacity, abnormal muscle tone and tremor (asterixis), and when severe, stupor and coma (for review, see Jones and Weissenborn, 1997). While the pathogenesis of HE is still not fully known, elevated levels of brain ammonia due to failure of the liver to detoxify this substance has been strongly impli- cated as an etiological factor (for reviews, see Albrecht and Jones, 1999; Hazell and Butterworth, 1999). Addi- tionally, astrocytes appear to be the main target of ammo- nia central nervous system toxicity (Norenberg, 1987). Exposure of cultured astrocytes to ammonia mimics many of the changes observed in HE in vivo (Norenberg, 1996, 1998). A prominent feature of HE is the upregulation of the peripheral benzodiazepine receptor (Butterworth, 2000; Norenberg et al., 1997), which has been recently renamed as the 18-kDa translocator protein (TSPO) (Papadopoulos et al., 2006). Increased number of TSPOs have been reported in portacaval-shunted rats (model of chronic HE) (Giguere et al., 1992), in an acute in vivo model of HE (Itzhak et al., 1995), in acute hyperammo- nemic mice (Itzhak et al., 1995), as well as in postmor- tem brain specimens from patients with HE (Lavoie et al., 1990). Furthermore, a significant increase of TSPOs has been identified in cultured astrocytes after treatment with ammonia (Itzhak and Norenberg, 1994). The significance of this upregulation relative to the Grant sponsors: Department of Veterans Affairs (Merit Review Grant), American Association for the Study of Liver Disease/American Liver Foundation. Grant spon- sor: NIH; Grant number: DK063311. *Correspondence to: Michael D. Norenberg, Department of Pathology (D-33), P.O. Box 016960, University of Miami School of Medicine, Miami, FL 33101, USA. E-mail: mnorenbe@med.miami.edu Received 3 May 2007; Revised 20 August 2007; Accepted 21 August 2007 DOI 10.1002/glia.20584 Published online 24 September 2007 in Wiley InterScience (www.interscience. wiley.com). GLIA 55:1720–1727 (2007) V V C 2007 Wiley-Liss, Inc.