The Alzheimer's disease (AD) brain is characterized by plaques containing ␤-amyloid (A␤) protein surrounded by astrocytes and reactive microglia. Activation of microglia by A␤ 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 A␤ upregulates a chloride current mediated by the chloride intracellular channel 1 (CLIC1) protein in microglia. We now demonstrate that A␤ promotes the acute translocation of CLIC1 from the cytosol to the plasma membrane of microglia, where it mediates a chloride conductance. Both the A␤ induced Cl Ϫ 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 Ϫ conductance is required for A␤-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.

The development of neuronal circuits is controlled by guidance molecules that are hypothesized to interact with the cholesterol-enriched domains of the plasma membrane termed lipid rafts. Whether such domains enable local intracellular signalling at the submicrometre scale in developing neurons and are required for shaping the nervous system connectivity in vivo remains controversial. Here, we report a role for lipid rafts in generating domains of local cAMP signalling in axonal growth cones downstream of ephrin-A repulsive guidance cues. Ephrin-A-dependent retraction of retinal ganglion cell axons involves cAMP signalling restricted to the vicinity of lipid rafts and is independent of cAMP modulation outside of this microdomain. cAMP modulation near lipid rafts controls the pruning of ectopic axonal branches of retinal ganglion cells in vivo, a process requiring intact ephrin-A signalling. Together, our findings indicate that lipid rafts structure the subcellular organization of intracellular cAMP signalling shaping axonal arbors during the nervous system development.

Edited by Adam SzewczykKeywords: Intracellular chloride channel 1 Microglia Reactive oxygen species Nicotinamide adenine dinucleotide phosphate oxidase Alzheimer disease Charge compensation a b s t r a c t Oxidative stress, characterized by overproduction of reactive oxygen species (ROS), is a major feature of several pathological states. Indeed, many cancers and neurodegenerative diseases are accompanied by altered redox balance, which results from dysregulation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. In this review, we consider the role of the intracellular chloride channel 1 (CLIC1) in microglial cells during oxidative stress. Following microglial activation, CLIC1 translocates from the cytosol to the plasma membrane where it promotes a chloride conductance. The resultant anionic current balances the excess charge extruded by the active NADPH oxidase, supporting the generation of superoxide by the enzyme. In this scenario, CLIC1 could be considered to act as both a second messenger and an executor. Ó 2010 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved. CLIC1 and oxidative stressReactive oxygen species (ROS), may act as compounds that impair cell and protein function, but they may also act as second messengers in cellular processes that involve changes in the cellular redox state, including migration, differentiation, and cell replication. Indeed, many proteins have redox-sensitive motifs, such as cystein residues and metal co-factors, that are altered by redox state. Kinases such as mitogen-activated protein kinase (MAPK), protein kinase C (PKC) and protein kinase B (PKB), are also activated by cell oxidation [1]. Cell homeostatic mechanisms establish a balance between ROS production and their removal by antioxidant systems. The overwhelming of antioxidant defences by ROS generation results in a condition of oxidative stress. Several pathological conditions are characterized by changes in cellular redox state, in particular chronic inflammatory states, oncologic conditions [2,3] and degenerative process [4][5][6].Alzheimer's disease (AD) is a progressive neurodegenerative disease that affects millions of people every year and is the main cause of dementia in the elderly for which an effective therapy is yet to be found. The AD brain is characterized by the presence of intraneuronal neurofibrillary tangles constituted by the hyperphosphorilated form of the cytoskeleton protein Tau and deposits of the amyloid beta (Ab) protein, also known as senile plaques [7]. Ab is the aberrant form of the transmembrane protein APP (amyloid precursor protein), resulting from the cleavage by gamma and beta secretases [8]. Although for decades the presence of amyloid plaques in the central nervous system (CNS) have been thought to be the main causative factor for neurodegeneration, recent studies propose that soluble oligomers can be more dangerous for neurons than the actual plaques [9]. Indeed, the neurological deficits in AD patients do not always correlat...

cAMP critically modulates the development of neuronal connectivity. It is involved in a wide range of cellular processes that require independent regulation. However, our understanding of how this single second messenger achieves specific modulation of the signaling pathways involved remains incomplete. The subcellular compartmentalization and temporal regulation of cAMP signals have recently been identified as important coding strategies leading to specificity. Dynamic interactions of this cyclic nucleotide with other second messenger including calcium and cGMP are critically involved in the regulation of spatiotemporal control of cAMP. Recent technical improvements of fluorescent sensors facilitate cAMP monitoring, whereas optogenetic tools permit spatial and temporal control of cAMP manipulations, all of which enabled the direct investigation of spatiotemporal characteristics of cAMP modulation in developing neurons. Focusing on neuronal polarization, neurotransmitter specification, axon guidance, and refinement of neuronal connectivity, we summarize herein the recent advances in understanding the features of cAMP signals and their dynamic interactions with calcium and cGMP involved in shaping the nervous system.