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The deletion of Phe508 (ΔF508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ΔF508‐CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ΔF508‐NBD1 and housekeeping proteins prevents ΔF508‐CFTR delivery to the plasma membrane. Based on this assumption we applied structure‐based virtual screening to identify new low‐molecular‐weight compounds that should bind to ΔF508‐NBD1 and act as protein–protein interaction inhibitors. Using different functional assays for CFTR activity, we demonstrated that in silico ‐selected compounds induced functional expression of ΔF508‐CFTR in transfected HeLa cells, human bronchial CF cells in primary culture, and in the nasal epithelium of homozygous ΔF508‐CFTR mice. The proposed compounds disrupt keratin8‐ΔF508‐CFTR interaction in ΔF508‐CFTR HeLa cells. Structural analysis of ΔF508‐NBD1 in the presence of these compounds suggests their binding to NBD1. We conclude that our strategy leads to the discovery of new compounds that are among the most potent correctors of ΔF508‐CFTR trafficking defect known to date. Graphical Abstract A virtual screening strategy led to potent novel compounds (correctors), able to correct the function of mutant (ΔF508‐CFTR) CFTR and based on their ability to inhibit interactions between mutant ΔF508‐CFTR and keratin 8.

The antiinflammatory protein annexin-1 (ANXA1) and the adaptor S100A10 (p11), inhibit cytosolic phospholipase A2 (cPLA2alpha) by direct interaction. Since the latter is responsible for the cleavage of arachidonic acid at membrane phospholipids, all three proteins modulate eicosanoid production. We have previously shown the association of ANXA1 expression with that of CFTR, the multifactorial protein mutated in cystic fibrosis. This could in part account for the abnormal inflammatory status characteristic of this disease. We postulated that CFTR participates in the regulation of eicosanoid release by direct interaction with a complex containing ANXA1, p11 and cPLA2alpha. We first analyzed by plasmon surface resonance the in vitro binding of CFTR to the three proteins. A significant interaction between p11 and the NBD1 domain of CFTR was found. We observed in Calu-3 cells a rapid and partial redistribution of all four proteins in detergent resistant membranes (DRM) induced by TNF-alpha. This was concomitant with increased IL-8 synthesis and cPLA2alpha activation, ultimately resulting in eicosanoid (PGE2 and LTB4) overproduction. DRM destabilizing agent methyl-beta-cyclodextrin induced further cPLA2alpha activation and eicosanoid release, but inhibited IL-8 synthesis. We tested in parallel the effect of short exposure of cells to CFTR inhibitors Inh172 and Gly-101. Both inhibitors induced a rapid increase in eicosanoid production. Longer exposure to Inh172 did not increase further eicosanoid release, but inhibited TNF-alpha-induced relocalization to DRM. These results show that (i) CFTR may form a complex with cPLA2alpha and ANXA1 via interaction with p11, (ii) CFTR inhibition and DRM disruption induce eicosanoid synthesis, and (iii) suggest that the putative cPLA2/ANXA1/p11/CFTR complex may participate in the modulation of the TNF-alpha-induced production of eicosanoids, pointing to the importance of membrane composition and CFTR function in the regulation of inflammation mediator synthesis.

Oxidative stress plays a prominent role in the pathophysiology of cystic fibrosis (CF). Despite the presence of oxidative stress markers and a decreased antioxidant capacity in CF airway lining fluid, few studies have focused on the oxidant/antioxidant balance in CF cells. The aim of the current study was to investigate the cellular levels of reactive oxygen species (ROS), oxidative damage and enzymatic antioxidant defenses in the lung of Cftr-knockout mice in basal conditions and as a response to oxidative insult.The results show that endogenous ROS and lipid peroxidation levels are higher in Cftr(-/-) lung when compared to wild-type (Cftr(+/+)) in basal conditions, despite a strong enzymatic antioxidant response involving superoxide dismutases, glutathione peroxidases and peroxiredoxin 6 (Prdx6). The latter has the unique capacity to directly reduce membrane phospholipid hydroperoxides (PL-OOH). A dramatic increase in PL-OOH levels in Cftr(-/-) lung consecutive to in vivo oxidative challenge by paraquat (PQ) unmasks a susceptibility to phospholipid peroxidation. PQ strongly decreases Prdx6 expression in Cftr(-/-) mice compared to Cftr(+/+). Similar results were obtained after P. aeruginosa LPS challenge. Two-dimensional gel analysis of Prdx6 revealed one main molecular form in basal conditions and a PQ-induced form only detected in Cftr(+/+) lung. Mass spectrometry experiments suggested that, as opposed to the main basal form, the one induced by PQ is devoid of overoxidized catalytic Cys47 and could correspond to a fully active form that is not induced in Cftr(-/-) lung. These results highlight a constitutive redox imbalance and a vulnerability to oxidative insult in Cftr(-/-) lung and present Prdx6 as a key component in CF antioxidant failure. This impaired PL-OOH detoxification mechanism may enhance oxidative damage and stress-related signaling, contributing to an exaggerated inflammatory response in CF lung.

The erythroid Rh-associated glycoprotein (RhAG) is strictly required for the expression of the Rh blood group antigens carried by Rh (D,CE) proteins. A biological function for RhAG in ammonium transport has been suggested by its ability to improve survival of an ammonium-uptake-deficient yeast. We investigated the function of RhAG by studying the entry of NH3/NH4+ in HeLa cells transiently expressing the green fluorescent protein (GFP)-RhAG fusion protein and using a fluorescent proton probe to measure intracellular pH (pHi). Under experimental conditions that reduce the intrinsic Na/H exchanger activity, exposure of control cells to a 10 mM NH4Cl- containing solution induces the classic pHi response profile of cells having a high permeability to NH3 (PNH3) but relatively low permeability to NH4+ (PNH4). In contrast, under the same conditions, the pHi profile of cells expressing RhAG clearly indicated an increased PNH4, as evidenced by secondary reacidification during NH4Cl exposure and a pHi undershoot below the initial resting value upon its removal. Measurements of pHi during methylammonium exposure showed that RhAG expression enhances the influx of both the unprotonated and ionic forms of methylammonium. Using a mathematical model to adjust passive permeabilities for a fit to the pHi profiles, we found that RhAG expression resulted in a threefold increase of PNH4 and a twofold increase of PNH3. Our results are the first evidence that the human erythroid RhAG increases the transport of both NH3 and NH4+.

Nonsense (premature stop codon) mutations in mRNA for the cystic fibrosis transmembrane conductance regulator (CFTR) cause cystic fibrosis (CF) in approximately 10% of patients. Ataluren (PTC124) is an oral drug that permits ribosomes to readthrough premature stop codons in mRNA to produce functional protein. To evaluate ataluren activity, safety, and pharmacokinetics in children with nonsense mutation CF. Patients were assessed in two 28-day cycles, comprising 14 days on and 14 days off ataluren. Patients took ataluren three times per day (morning, midday, and evening) with randomization to the order of receiving a lower dose (4, 4, and 8 mg/kg) and a higher dose (10, 10, and 20 mg/kg) in the two cycles. The study enrolled 30 patients (16 male and 14 female, ages 6 through 18 yr) with a nonsense mutation in at least one allele of the CFTR gene, a classical CF phenotype, and abnormal baseline nasal epithelial chloride transport. Ataluren induced a nasal chloride transport response (at least a -5-mV improvement) or hyperpolarization (value more electrically negative than -5 mV) in 50% and 47% of patients, respectively, with more hyperpolarizations at the higher dose. Improvements were seen in seven of nine nonsense mutation genotypes represented. Ataluren significantly increased the proportion of nasal epithelial cells expressing apical full-length CFTR protein. Adverse events and laboratory abnormalities were infrequent and usually mild. Ataluren pharmacokinetics were similar to those in adults. In children with nonsense mutation CF, ataluren can induce functional CFTR production and is well tolerated.

The deletion of Phe508 (ΔF508) in the first nucleotide binding domain (NBD1) of CFTR is the most common mutation associated with cystic fibrosis. The ΔF508‐CFTR mutant is recognized as improperly folded and targeted for proteasomal degradation. Based on molecular dynamics simulation results, we hypothesized that interaction between ΔF508‐NBD1 and housekeeping proteins prevents ΔF508‐CFTR delivery to the plasma membrane. Based on this assumption we applied structure‐based virtual screening to identify new low‐molecular‐weight compounds that should bind to ΔF508‐NBD1 and act as protein–protein interaction inhibitors. Using different functional assays for CFTR activity, we demonstrated that in silico‐selected compounds induced functional expression of ΔF508‐CFTR in transfected HeLa cells, human bronchial CF cells in primary culture, and in the nasal epithelium of homozygous ΔF508‐CFTR mice. The proposed compounds disrupt keratin8‐ΔF508‐CFTR interaction in ΔF508‐CFTR HeLa cells. Structural analysis of ΔF508‐NBD1 in the presence of these compounds suggests their binding to NBD1. We conclude that our strategy leads to the discovery of new compounds that are among the most potent correctors of ΔF508‐CFTR trafficking defect known to date.

Motor skills can take weeks to months to acquire and can diminish over time in the absence of continued practice. Thus, strategies that enhance skill acquisition or retention are of great scientific and practical interest. Here we investigated the effect of noninvasive cortical stimulation on the extended time course of learning a novel and challenging motor skill task. A skill measure was chosen to reflect shifts in the task's speed-accuracy tradeoff function ( SAF), which prevented us from falsely interpreting variations in position along an unchanged SAF as a change in skill. Subjects practiced over 5 consecutive days while receiving transcranial direct current stimulation (tDCS) over the primary motor cortex (M1). Using the skill measure, we assessed the impact of anodal (relative to sham) tDCS on both within-day (online) and betweenday (offline) effects and on the rate of forgetting during a 3-month follow-up (long-term retention). There was greater total (online plus offline) skill acquisition with anodal tDCS compared to sham, which was mediated through a selective enhancement of offline effects. Anodal tDCS did not change the rate of forgetting relative to sham across the 3-month follow-up period, and consequently the skill measure remained greater with anodal tDCS at 3 months. This prolonged enhancement may hold promise for the rehabilitation of brain injury. Furthermore, these findings support the existence of a consolidation mechanism, susceptible to anodal tDCS, which contributes to offline effects but not to online effects or long-term retention.

Non-invasive electrical brain stimulation by application of direct current (DCS) promotes plasticity in neuronal networks in vitro and in in vivo. This effect has been mainly attributed to the direct modulation of neurons. Glia represents approximately 50% of cells in the brain. Glial cells are electrically active and participate in synaptic plasticity. Despite of that, effects of DCS on glial structures and on interaction with neurons are only sparsely investigated. In this perspectives article we review the current literature, present own dose response data and provide a framework for future research from two points of view: first, the direct effects of DCS on glia and second, the contribution of glia to DCS related neuronal plasticity.

Early rehabilitation in the acute phase of stroke, that bears unique neuroplastic properties, is the current standard to reduce disability. Anodal transcranial direct current stimulation can augment neurorehabilitation in chronic stroke. Studies in the acute phase are sparse and held back by inconclusive preclinical data pointing towards potential negative interaction of the excitability increasing tDCS modality with stroke-induced glutamate toxicity. In this present study, we aimed to evaluate structural and behavioral safety of anodal tDCS applied in the acute phase of stroke. Photothrombotic stroke including the right primary motor cortex was induced in rats. 24 h after stroke anodal tDCS was applied for 20 min ipsilesionally at one of four different current densities in freely moving animals. Effects on the infarct volume and on stroke induced neuroinflammation were assessed. Behavioral consequences were monitored. Infarct volume and the modified Neurological Severity Score were not affected by anodal tDCS. Pasta handling, a more sensitive task for sensorimotor deficits, and microglia reactivity indicated potentially harmful effects at the highest tDCS current density tested (47.8 A/m 2 ), which is more than 60 times higher than intensities commonly used in humans. Compared to published safety limits of anodal tDCS in healthy rats, recent stroke does not increase the sensitivity of the brain to anodal tDCS, as assessed by lesion size and neuroinflammatory response. Behavioral deficits only occurred at the highest intensity, which was associated with increased neuroinflammation. When safety limits of commonly used clinical tDCS are met, augmentation of early neurorehabilitation after stroke by anodal tDCS appears to be feasible.

Schizophrenia is a severe neurodevelopmental psychiatric affliction manifested behaviorally at late adolescence/early adulthood. Current treatments comprise antipsychotics which act solely symptomatic, are limited in their effectiveness and often associated with side-effects. We here report that application of non-invasive transcranial direct current stimulation (tDCS) during adolescence, prior to schizophrenia-relevant behavioral manifestation, prevents the development of positive symptoms and related neurobiological alterations in the maternal immune stimulation (MIS) model of schizophrenia.