Involvement of the Intracellular Ion Channel CLIC1 in Microglia-Mediated  -Amyloid-Induced Neurotoxicity

Abstract

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 ␤-amyloid protein (A␤) in neuritic plaques that are infiltrated by reactive microglia and astrocytes. Although A␤ 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␤ 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␤, simultaneously with the production of reactive nitrogen intermediates and of tumor necrosis factor-␣ (TNF-␣). We demonstrate that reducing CLIC1 chloride conductance by a specific blocker [IAA-94 (R(ϩ)-[ (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␤-treated microglia. Furthermore, we show that small interfering RNAs used to knock down CLIC1 expression prevent TNF-␣ release induced by A␤ stimulation. These results provide a direct link between A␤-induced microglial activation and CLIC1 functional expression.

CLIC1 Function Is Required for β-Amyloid-Induced Generation of Reactive Oxygen Species by Microglia

“…Exposure of microglia to A␤ increases the expression of the chloride intracellular channel 1 (CLIC1). We have previously demonstrated 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.…”

“…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␤.…”

“…Purified primary microglial cultures were obtained from mixed glial cultures as described previously (Novarino et al, 2004). Mixed cultures were obtained from 2-d-old Sprague Dawley rats.…”

“…To knock down CLIC1 protein expression, we manipulated psiUbclic1B as described previously (Novarino et al, 2004) to generate siRNAs targeting a 21-nt region in exon 6 of CLIC1 mRNA (5Ј-GATGATGAAGAGATAGAGCTA-3Ј). The plasmid was produced by annealing corresponding complementary synthetic oligonucleotides which were then cloned into the BglII-XhoI sites of pSiUx.…”

“…Both NADPH 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␤ in vitro (Bianca et al, 1999;Novarino et al, 2004). CLIC1 blockade limits A␤-induced microglial-mediated neurotoxicity after 24 h (Novarino et al, 2004).…”