CLIC1 function is required for beta-amyloid-induced generation of reactive oxygen species by microglia

The Alzheimer's disease (AD) brain is characterized by plaques containing beta-amyloid (Abeta) protein surrounded by astrocytes and reactive microglia. Activation of microglia by Abeta initiates production of reactive oxygen species (ROS) by the plasmalemmal NADPH oxidase; the resultant oxidative stress is thought to contribute to neurodegeneration in AD. We have previously shown that Abeta upregulates a chloride current mediated by the chloride intracellular channel 1 (CLIC1) protein in microglia. We now demonstrate that Abeta promotes the acute translocation of CLIC1 from the cytosol to the plasma membrane of microglia, where it mediates a chloride conductance. Both the Abeta induced Cl(-) conductance 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 Abeta-induced generation of neurotoxic ROS by microglia. Remarkably, CLIC1 activation is itself dependent on oxidation by ROS derived from the activated NADPH oxidase. We therefore propose that CLIC1 translocation from the 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

Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimer's disease, for example, is characterized by an accumulation of \u3b2-amyloid protein (A\u3b2) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although A\u3b2 and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that A\u3b2 stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with A\u3b2, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-\u3b1 (TNF-\u3b1). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with A\u3b2-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-\u3b1 release induced by A\u3b2 stimulation. These results provide a direct link between A\u3b2-induced microglial activation and CLIC1 functional expression

New Proteins from the Mobile Genome: Structural Definitions of Function

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The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24-Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity

Involvement of the intracellular ion channel CLIC1 in microglia-mediated beta-amyloid-induced neurotoxicity.

It is widely believed that the inflammatory events mediated by microglial activation contribute to several neurodegenerative processes. Alzheimer's disease, for example, is characterized by an accumulation of β-amyloid protein (Aβ) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although Aβ and its fragment 25-35 exert a direct toxic effect on neurons, they also activate microglia. Microglial activation is accompanied by morphological changes, cell proliferation, and release of various cytokines and growth factors. A number of scientific reports suggest that the increased proliferation of microglial cells is dependent on ionic membrane currents and in particular on chloride conductances. An unusual chloride ion channel known to be associated with macrophage activation is the chloride intracellular channel-1 (CLIC1). Here we show that Aβ stimulation of neonatal rat microglia specifically leads to the increase in CLIC1 protein and to the functional expression of CLIC1 chloride conductance, both barely detectable on the plasma membrane of quiescent cells. CLIC1 protein expression in microglia increases after 24 hr of incubation with Aβ, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-α (TNF-α). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy] acetic acid)] prevents neuronal apoptosis in neurons cocultured with Aβ-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-α release induced by Aβ stimulation. These results provide a direct link between Aβ-induced microglial activation and CLIC1 functional expression

Involvement of the intracellular ion channel CLIC1 in microglia-mediatedbeta-amyloid-induced neurotoxicity.

It is widely believed that the inflammatory events mediated by microglialactivation contribute to several neurodegenerative processes. Alzheimer'sdisease, for example, is characterized by an accumulation of beta-amyloidprotein (Abeta) in neuritic plaques that are infiltrated by reactive microgliaand astrocytes. Although Abeta and its fragment 25-35 exert a direct toxiceffect on neurons, they also activate microglia. Microglial activation isaccompanied by morphological changes, cell proliferation, and release of variouscytokines and growth factors. A number of scientific reports suggest that theincreased proliferation of microglial cells is dependent on ionic membranecurrents and in particular on chloride conductances. An unusual chloride ionchannel known to be associated with macrophage activation is the chlorideintracellular channel-1 (CLIC1). Here we show that Abeta stimulation of neonatalrat microglia specifically leads to the increase in CLIC1 protein and to thefunctional expression of CLIC1 chloride conductance, both barely detectable onthe plasma membrane of quiescent cells. CLIC1 protein expression in microgliaincreases after 24 hr of incubation with Abeta, simultaneously with theproduction of reactive nitrogen intermediates and of tumor necrosis factor-alpha(TNF-alpha). We demonstrate that reducing CLIC1 chloride conductance by aspecific blocker [IAA-94(R(+)-[(6,7-dichloro-2-cyclopentyl-2,3-dihydro-2-methyl-1-oxo-1H-inden-5yl)-oxy]acetic acid)] prevents neuronal apoptosis in neurons cocultured withAbeta-treated microglia. Furthermore, we show that small interfering RNAs usedto knock down CLIC1 expression prevent TNF-alpha release induced by Abetastimulation. These results provide a direct link between Abeta-inducedmicroglial activation and CLIC1 functional expression