CHLORIDE INTRACELLULAR ION CHANNEL 1 AS A CUTTING EDGE BETWEEN NEUROGENESIS AND NEURODEGENERATION.

Chloride intracellular ion channel 1 (CLIC1) has peculiar properties compared to most known ion channels, in that it exists both as a soluble protein and as a membrane-integrated protein. Previous studies have shown that CLIC1 insertion into the plasma membrane is driven by different stimuli such as oxidation, cytoplasmic acidification, increased calcium concentration. Once inserted into the plasma membrane CLIC1 forms a chloride selective ion channel. The best characterized mechanism leading CLIC1 into the plasma membrane is cell oxidation. Our laboratory has previously shown that in microglia cells stimulation by amyloid beta peptides, one of the most common hallmarks of Alzheimer disease (AD), determines the increase of CLIC1-mediated current. Many studies have demonstrated that many AD patients show at first visual impairment rather than other cognitive deficits. AD patients show severe abnormalities in the structure and function of the retina, including retinal ganglion cells (RGCs) loss. Several cellular mechanisms precede RGCs degeneration, including oxidative stress. Therefore, in view of the oxidation-driven behavior of CLIC1, during my PhD, I analyzed CLIC1 expression in mouse retina. We found out that CLIC1 expression is increased in retinas of two different AD mouse models. Interestingly, CLIC1 increased expression is prior to the manifestation of the first cognitive symptoms, thus proposing CLIC1 as an early marker of neurodegeneration in AD. Although CLIC1 expression is largely increased in AD mouse retinas, CLIC1 is also expressed in wild type retinas, mainly in the gangliar layer, where the nuclei of RGCs, whose axons make up the optic nerve, are located. Thus, we wondered about the role of CLIC1 in the normal RGCs physiology. Our studies unraveled the crucial role of CLIC1-mediated current in axon outgrowth of RGCs, showing that the inhibition of the chloride current causes a reduced axon outgrowth that is parallel to an altered morphology of the growth cones and a reduced co-localization of CLIC1 with the actin cytoskeleton. Our results also suggest that CLIC1 involvement in axon outgrowth might be due to CLIC1 modulation by cAMP. Taken together our data propose cAMP, or cAMP homologues, as a putative tool to modulate CLIC1 transient expression at the plasma membrane and consequently axon outgrowth

Proteome from patients with metabolic syndrome is regulated by quantity and quality of dietary lipids

Background: Metabolic syndrome is a multi-component disorder associated to a high risk of cardiovascular disease. Its etiology is the result of a complex interaction between genetic and environmental factors, including dietary habits. We aimed to identify the target proteins modulated by the long-term consumption of four diets differing in the quality and quantity of lipids in the whole proteome of peripheral blood mononuclear cells (PBMC). Results: A randomized, controlled trial conducted within the LIPGENE study assigned 24 MetS patients for 12 weeks each to 1 of 4 diets: a) high-saturated fatty acid (HSFA), b) high-monounsaturated fatty acid (HMUFA), c) low-fat, high-complex carbohydrate diets supplemented with placebo (LFHCC) and d) low-fat, high-complex carbohydrate diets supplemented with long chain (LC) n-3 polyunsaturated fatty acids (PUFA) (LFHCC n-3). We analyzed the changes induced in the proteome of both nuclear and cytoplasmic fractions of PBMC using 2-D proteomic analysis. Sixty-seven proteins were differentially expressed after the long-term consumption of the four diets. The HSFA diet induced the expression of proteins responding to oxidative stress, degradation of ubiquitinated proteins and DNA repair. However, HMUFA, LFHCC and LFHCC n-3 diets down-regulated pro-inflammatory and oxidative stress-related proteins and DNA repairing proteins. Conclusion: The long-term consumption of HSFA, compared to HMUFA, LFHCC and LFHCC n-3, seems to increase the cardiovascular disease (CVD) risk factors associated with metabolic syndrome, such as inflammation and oxidative stress, and seem lead to DNA damage as a consequence of high oxidative stress

Chloride intracellular channel 1 (CLIC1): Sensor and effector during oxidative stress

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

Chloride intracellular channel 1 (CLIC1): Sensor and effector during oxidative stress

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

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

Toxic effects of expanded ataxin-1 involve mechanical instability of the nuclear membrane

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

Biallelic PDE2A variants: a new cause of syndromic paroxysmal dyskinesia

International audienceCause of complex dyskinesia remains elusive in some patients. A homozygous missense variant leading to drastic decrease of PDE2A enzymatic activity was reported in one patient with childhood-onset choreodystonia preceded by paroxysmal dyskinesia and associated with cognitive impairment and interictal EEG abnormalities. Here, we report three new cases with biallelic PDE2A variants identified by trio whole-exome sequencing. Mitochondria network was analyzed after Mitotracker™ Red staining in control and mutated primary fibroblasts. Analysis of retrospective video of patients' movement disorder and refinement of phenotype was carried out. We identified a homozygous gain of stop codon variant c.1180C>T; p.(Gln394*) in PDE2A in siblings and compound heterozygous variants in young adult: a missense c.446C>T; p.(Pro149Leu) and splice-site variant c.1922+5G>A predicted and shown to produce an out of frame transcript lacking exon 22. All three patients had cognitive impairment or developmental delay. The phenotype of the two oldest patients, aged 9 and 26, was characterized by childhood-onset refractory paroxysmal dyskinesia initially misdiagnosed as epilepsy due to interictal EEG abnormalities. The youngest patient showed a proven epilepsy at the age of 4 months and no paroxysmal dyskinesia at 15 months. Interestingly, analysis of the fibroblasts with the biallelic variants in PDE2A variants revealed mitochondria network morphology changes. Together with previously reported case, our three patients confirm that biallelic PDE2A variants are a cause of childhood-onset refractory paroxysmal dyskinesia with cognitive impairment, sometimes associated with choreodystonia and interictal baseline EEG abnormalities or epilepsy