Microglial PHOX and Mac-1 are Essential to the Enhanced Dopaminergic Neurodegeneration Elicited by A30P and A53T Mutant Alpha-Synuclein WEI ZHANG, 1,2 SHANNON DALLAS, 3 DAN ZHANG, 1 JIAN-PING GUO, 4 HAO PANG, 1 BELINDA WILSON, 1 DAVID S. MILLER, 3 BIAO CHEN, 5 WANQIN ZHANG, 6 PATRICK L. MCGEER, 4 JAU-SHYONG HONG, 1 AND JING ZHANG 7 * 1 Neuropharmacology Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, North Carolina 2 Department of Neurology, Beijing Tian Tan Hospital, Capital Medical University, Beijing, China 3 Intracellular Regulation Section, Laboratory of Pharmacology and Chemistry, National Institute of Environmental Health Sciences/National Institutes of Health, Research Triangle Park, North Carolina 4 Kinsmen Laboratory of Neurological Research, University of British Columbia, Vancouver, BC, Canada 5 Department of Neurology, Xuanwu Hospital, Capital University of Medical Sciences, Beijing, China 6 Department of Physiology, Dalian Medical University, Dalian, China 7 Department of Pathology, University of Washington, Seattle, Washington KEY WORDS Parkinson’s disease; microglia; synuclein ABSTRACT a-Synuclein, a gene whose mutations, duplication, and trip- lication has been linked to autosomal dominant familial Parkinson’s disease (fPD), appears to play a central role in the pathogenesis of sporadic PD (sPD) as well. Enhance- ment of neurodegeneration induced by mutant a-synuclein has been attributed to date largely to faster formation of a- synuclein aggregates in neurons. Recently, we reported that microglial activation enhances wild type (WT) a-synuclein- elicited dopaminergic neurodegeneration. In the present study, using a primary mesencephalic culture system, we tested whether mutated a-synuclein could activate micro- glia more powerfully than WT a -synuclein, thereby contrib- uting to the accelerated neurodegeneration observed in fPD. The results showed that a-synuclein with the A30P or A53T mutations caused greater microglial activation than WT a -synuclein. Furthermore, the extent of microglial acti- vation paralleled the degree of dopaminergic neurotoxicity induced by WT and mutant a-synuclein. Mutant a-synu- clein also induced greater production of reactive oxygen species than WT a -synuclein by NADPH oxidase (PHOX), and PHOX activation was linked to direct activation of macrophage antigen-1 (Mac-1) receptor, rather than a-synu- clein internalization via scavenger receptors. These results have, for the first time, demonstrated that microglia are also critical in enhanced neurotoxicity induced by mutant a-synuclein. V V C 2007 Wiley-Liss, Inc. INTRODUCTION The pathogenesis of Parkinson’s disease (PD), though largely unknown, appears to be related, at least par- tially, to increased oxidative stress, mitochondrial dys- function, abnormal protein aggregation, as well as fail- ure of the ubiquitin–proteasome (UPS) and lysosomal systems (Bahr and Bendiske, 2002; Dawson and Daw- son, 2003b; Greenamyre et al., 2001; Jellinger, 2001; Lansbury and Brice, 2002; Lee et al., 2004; Przedborski and Vila, 2003). More recently, neuroinflammation, char- acterized by microglial activation, has also been impli- cated as a contributor to neurodegeneration in PD. It was generally believed that the microglial activation is a consequence of neurodegeneration. However, recent reports indicate that an inflammatory reaction because of the over-activation of microglia is capable of initiating neurodegeneration directly in animal PD models. For example, lipopolysaccharide, a proinflammatory agent, though incapable of killing neurons directly, potently activates microglia with subsequent dopaminergic (DAergic) neurodegeneration in vitro and in vivo (Gao et al., 2002b; Le et al., 2001; Ling et al., 2004). Further- more, it was reported that in PD, the extent of neuronal degeneration matches up with the degree of microglial activation in the substantia nigra pars compacta (SNpc) (McGeer et al., 1988; Ouchi et al., 2005; Shavali et al., 2006). Factors other than lipopolysaccharide, i.e., rotenone (Gao et al., 2002a), maneb (Zhou et al., 2005), neurome- lanin (Wilms et al., 2003), and aggregated a-synuclein (Zhang et al., 2005), have also been shown to activate microglia and promote DAergic neurodegeneration. In an earlier study, we demonstrated that exogenous aggre- gated a-synuclein triggers microglial activation with re- sultant morphological alterations, as well as production of reactive oxygen species (ROS), including extracellular superoxide (O 2 2 ) and intracellular ROS (iROS) (Zhang et al., 2005). a-Synuclein is a key protein found in Lewy Grant sponsors: Intramural grants from NIH/NIEHS; Extramural grants: R01AG025327, R01ES012703. *Correspondence to: Jing Zhang, Division of Neuropathology, Harborview Medical Center, University of Washington School of Medicine, Box 359635, 325 9th Avenue, Seattle, WA 98104-2499, USA. E-mail: zhangj@u.washington.edu Received 12 November 2006; Revised 17 May 2007; Accepted 23 May 2007 DOI 10.1002/glia.20532 Published online 28 June 2007 in Wiley InterScience (www.interscience. wiley.com). GLIA 55:1178–1188 (2007) V V C 2007 Wiley-Liss, Inc.