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.
Chloride intracellular Channel 1 (CLIC1) is a metamorphic protein that changes from a soluble cytoplasmic protein into a transmembrane protein. Once inserted into membranes, CLIC1 multimerises and is able to form chloride selective ion channels. Whilst CLIC1 behaves as an ion channel both in cells and in artificial lipid bilayers, its structure in the soluble form has led to some uncertainty as to whether it really is an ion channel protein.CLIC1 has a single putative transmembrane region that contains only two charged residues: arginine 29 (Arg29) and lysine 37 (Lys37). As charged residues are likely to have a key role in ion channel function, we hypothesized that mutating them to neutral alanine to generate K37A and R29A CLIC1 would alter the electrophysiological characteristics of CLIC1. By using three different electrophysiological approaches: i) single channel Tip-Dip in artificial bilayers using soluble recombinant CLIC1, ii) cell-attached and iii) whole-cell patch clamp recordings in transiently transfected HEK cells, we determined that the K37A mutation altered the single-channel conductance while the R29A mutation affected the single-channel open probability in response to variation in membrane potential.Our results show that mutation of the two charged amino acids (K37 and R29) in the putative transmembrane region of CLIC1 alters the biophysical properties of the ion channel in both artificial bilayers and cells. Hence these charged residues are directly involved in regulating its ion channel activity. This strongly suggests that, despite its unusual structure, CLIC1 itself is able to form a chloride ion channel.
During neuronal differentiation, axonal elongation is regulated by both external and intrinsic stimuli, including neurotropic factors, cytoskeleton dynamics, second messengers such as cyclic adenosine monophosphate (cAMP), and neuronal excitability. Chloride intracellular channel 1 (CLIC1) is a cytoplasmic hydrophilic protein that, upon stimulation, dimerizes and translocates to the plasma membrane, where it contributes to increase the membrane chloride conductance. Here, we investigated the expression of CLIC1 in primary hippocampal neurons and retinal ganglion cells (RGCs) and examined how the functional expression of CLIC1 specifically modulates neurite outgrowth of neonatal murine RGCs. Using a combination of electrophysiology and immunohistochemistry, we found that CLIC1 is expressed in hippocampal neurons and RGCs and that the chloride current mediated by CLIC1 is required for maintaining growth cone morphology and sustaining cAMP-stimulated neurite elongation in dissociated immunopurified RGCs. In cultured RGCs, inhibition of CLIC1 ionic current through the pharmacological blocker IAA94 or a specific anti-CLIC1 antibody directed against its extracellular domain prevents the neurite outgrowth induced by cAMP. CLIC1-mediated chloride current, which results from an increased open probability of the channel, is detected only when cAMP is elevated. Inhibition of protein kinase A prevents such current. These results indicate that CLIC1 functional expression is regulated by cAMP via protein kinase A and is required for neurite outgrowth modulation during neuronal differentiation. Keywords: cAMP, chloride current, CLIC1, neurite growth, PKA, retinal ganglion cells. Several external stimuli and intrinsic factors regulate axonal elongation during neuronal differentiation, including neurotrophic factors, cytoskeleton dynamics, second messengers such as cyclic adenosine monophosphate (cAMP), and neuronal excitability (Goldberg et al. 2002). In particular, neuronal activity plays a key role in regulating axonal outgrowth (Goldberg et al. 2002). Indeed, in cultured retinal ganglion cells (RGCs), depolarizing stimuli (e.g., KCl) and electrical stimulation of RGCs potentiate axonal outgrowth induced by brain-derived neurotrophic factor (BDNF) in a cAMP-dependent manner (Goldberg et al. 2002). Within this context, ion channels may play a crucial role. It has been demonstrated that in cerebellar neurons, the inhibition of tetrodotoxin-sensitive voltage-gated sodium channels impairs axon outgrowth by reducing cell excitability (Brackenbury et al. 2010). Transient receptor potential channels control growth cone (GC) morphology through their specific localization (Greka et al. 2003). In addition, calcium channels have a key role in calcium waves in GC and affect neurite growth in motor neurons (Jablonka et al. 2007). It has also Abbreviations used: AcS, actin-covered surface; BDNF, brain-derived neurotrophic factor; BSA, bovine serum albumin; CLIC1, chloride intracellular channel 1; N2a, neuro-2a; PKA, protein kinase ...
Ataxin 1 (ATXN1) is the protein involved in spinocerebellar ataxia type 1, one of nine dominantly inherited neurodegenerative diseases triggered by polyglutamine expansion. One of the isolated polyglutamine tracts properties is to interact with lipid bilayers. Here we used a multidisciplinary approach to test whether one of the mechanisms responsible for neuronal degeneration involves the destabilization of the nuclear membrane. We thus analyzed the interaction between ATXN1 and lipid membranes, both on cellular models and on artificial lipid bilayers, comparing pathological expanded polyglutamine and histidine interrupted non-harmful polyglutamine tracts of the same length. The toxicity of the different constructs was tested in transiently transfected COS1 cells. Cells expressing pathological ATXN1 presented a significantly higher frequency of anomalous nuclei with respect to those expressing non-harmful ATXN1. Immunofluorescence and electron microscopy showed severe damage in the nuclear membrane of cells expressing the pathological protein. Atomic force microscopy on artificial membranes containing interrupted and non-interrupted partial ATXN1 peptides revealed a different arrangement of the peptides within the lipid bilayer. Force-distance measurements indicated that membrane fragility increases with the lengthening of the uninterrupted glutamine. Transmembrane electrical measurements were performed on artificial bilayers and on the inner nuclear membrane of ATXN1 full length transfected cells. Both artificial lipid bilayers and cellular models demonstrated the dynamic appearance of ionic pathways. Uninterrupted polyglutamines showed not only a larger ionic flow, but also an increase in the single event conductance. Collectively, our results suggest that expanded ATXN1 may induce unregulated ionic pathways in the nuclear membrane, causing severe damage to the cell.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.