Fluorochromes were imaged separately and merged with Leica Power Scan software. and ROS generation were prevented by pharmacological inhibition of CLIC1, by replacement of chloride with impermeant anions, by an anti-CLIC1 antibody and by suppression of CLIC1 expression using siRNA. Thus, the CLIC1-mediated Cl? conductance is required for A-induced generation of neurotoxic ROS by microglia. Amazingly, CLIC1 activation is usually itself dependent on oxidation by ROS derived from the activated NADPH oxidase. We therefore propose that CLIC1 translocation from your cytosol to the plasma membrane, in response to redox modulation by NADPH oxidase-derived ROS, provides a feedforward mechanism that facilitates sustained microglial ROS generation by the NAPDH oxidase. Keywords: Rabbit Polyclonal to OR10Z1 microglia, -amyloid, ROS, CLIC1, NADPH oxidase, neurodegeneration Introduction A major feature of Alzheimer’s disease (AD) is the accumulation of extracellular -amyloid (A) into plaques infiltrated with activated microglia. Exposure of microglia to A increases the expression of the chloride intracellular channel 1 (CLIC1). We have previously exhibited that blocking CLIC1 reduces A-induced microglial-mediated neurotoxicity (Novarino et al., 2004). In the present work, we identify a novel mechanism through which CLIC1 plays a pivotal role in the microglial response to A, which could have profound implications for the pathophysiology of AD. The CLIC family consists of seven proteins (Shanks et al., 2002), with CLICs 1, 4, and 5 known to possess chloride channel activity (Tonini et al., 2000; Tulk et al., 2002; Berryman et al., 2004; Singh and Ashley, 2006). CLIC1 was originally recognized in monocytes (Valenzuela et al., 1997) and is able to place into membranes from your aqueous phase (Tulk et al., 2002; Warton et al., 2002). CLIC1 channel activity is increased by oxidation, probably through the formation of an intrachain disulfide bond that promotes dimerization (Harrop et al., PROTAC MDM2 Degrader-4 2001; Littler et al., 2004). Although CLIC1 is the most highly expressed transcript of a range of chloride channels encoded by mammalian microglial mRNA (Ducharme et al., 2007), its functional role remains obscure. In response to A, microglia secrete a range of proinflammatory molecules including cytokines (Meda et al., 1999) and reactive oxygen species (ROS) (Bianca et al., 1999). Oxidative damage is a feature of the AD brain (Lyras et al., 1997), and considerable evidence suggests oxidative stress induced by microglial-derived ROS is usually a major contributor to neurodegeneration (Wilkinson and Landreth, 2006; Block et al., 2007). Exposure of microglia to A induces PROTAC MDM2 Degrader-4 ROS generation by the NADPH oxidase (McDonald et al., 1997; Bianca et al., 1999), specifically by NOX2 (Sankarapandi et al., 1998). Neurons are guarded by pharmacological inhibition (Qin et al., 2002; Abramov et al., 2003) or genetic modification of the NADPH oxidase (Block et al., 2006), supporting a neurotoxic role for NADPH oxidase-derived ROS (Abramov and Duchen, 2005). Because NADPH oxidase-derived ROS and resultant oxidative stress are strongly implicated in the pathogenesis of AD (Shimohama et al., 2000; Wilkinson and Landreth, 2006; Park et al., 2008), these processes and their mechanisms clearly represent attractive therapeutic targets. Both NADPH PROTAC MDM2 Degrader-4 oxidase and CLIC1 are upregulated in the AD brain (Shimohama et al., 2000; Parachikova et al., 2007) and expression of both increases in microglia in response to A (Bianca et al., 1999; Novarino et al., 2004). CLIC1 blockade limits A-induced microglial-mediated neurotoxicity after 24 h (Novarino et al., 2004). Here, we describe a primary role for CLIC1 in microglial activation by A. Using electrophysiological and live cell imaging methods, we show that A promotes the acute translocation of CLIC1 from your cytoplasm to the microglia cell membrane, resulting in the appearance of an anion conductance within minutes. This conductance is usually shown to be essential for ROS generation by the NADPH oxidase, and is itself regulated by it, thus defining a fundamental role for CLIC1 in A-induced oxidative stress. Materials and Methods Cell culture and manipulation Experiments have been performed using main cultures of microglia from rat cortex and cells of the murine microglial immortalized cell collection BV2 (Blasi et al., 1990; Bocchini et al., 1992). We have used the BV2 microglial cell collection for most experiments that require manipulation of gene expression, because transfection of main microglial is problematic with a very low transfection efficiency. The BV2 cell collection was managed in DMEM supplemented with 10% fetal bovine serum and 2 mm l-glutamine, without antibiotic. Purified main microglial cultures were obtained from mixed glial cultures as explained previously (Novarino et al., 2004). Mixed cultures were obtained from.