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Autophagy is a process leading to the degradation of cellular material, in organelles called lysosomes, to supply energy or generate building blocks for the synthesis of new materials. Over the past decades, its role has been evidenced in several indications, notably in neurodegenerative disorders and orphan diseases called lysosomal storage disorders and its modulation is largely envisioned as a therapeutic avenue to alleviate the symptoms and reverse the clinical courses of these indications. Identifying new chemical classes and drugs is, hence, of huge importance. In this study, we developed automated assays to assess the potential efficacy of chemical compounds on different steps of autophagy, notably its induction through the localization of a largely involved transcription factor, transcription factor EB (TFEB). These assays were then used to screen a collection of 1,520 approved drugs. This study led to the identification of five candidate hits modulating autophagy and TFEB subcellular localization. Our results suggest the repurposing potential of already approved drugs in central nervous system disorders with lysosomal storage impairments.

In vertebrates, intercellular communication is largely mediated by connexins (Cx), a family of structurally related transmembrane proteins that assemble to form hemichannels (HCs) at the plasma membrane. HCs are upregulated in different brain disorders and represent innovative therapeutic targets. Identifying modulators of Cx-based HCs is of great interest to better understand their function and define new treatments. In this study, we developed automated versions of two different cell-based assays to identify new pharmacological modulators of Cx43-HCs. As HCs remain mostly closed under physiological conditions in cell culture, depletion of extracellular Ca 2+ was used to increase the probability of opening of HCs. The first assay follows the incorporation of a fluorescent dye, Yo-Pro, by real-time imaging, while the second is based on the quenching of a fluorescent protein, YFP QL , by iodide after iodide uptake. These assays were then used to screen a collection of 2242 approved drugs and compounds under development. This study led to the identification of 11 candidate hits blocking Cx43-HC, active in the two assays, with 5 drugs active on HC but not on gap junction (GJ) activities. To our knowledge, this is the first screening on HC activity and our results suggest the potential of a new use of already approved drugs in central nervous system disorders with HC impairments.

Donepezil (DPZ) is an acetylcholinesterase inhibitor used in Alzheimer's disease to restore cognitive functions but is endowed with limited efficacy. Recent studies pointed out the implication of astroglial networks in cognitive processes, notably via astrocyte connexins (Cxs), proteins involved in gap junction intercellular communications. Hence, we investigated the impact on cognition of pharmacological or genetic modulations of those astrocyte Cxs during DPZ challenge in two rodent models of Alzheimer's disease-like memory deficits. We demonstrated that the Cx modulator mefloquine (MEF) significantly enhanced the procognitive effect of DPZ in both models. In parallel, we determined that MEF potentiated DPZ-induced release of acetylcholine in hippocampus. Finally, local genetic silencing of astrocyte Cxs in the hippocampus was also found to enhance the procognitive effect of DPZ, pointing out the importance of Cx-dependent astrocyte networks in memory processes., (Copyright © 2020 Droguerre, Duchêne, Picoli, Portal, Lejards, Guiard, Meunier, Villard, Déglon, Hamon, Mouthon and Charvériat.)

Gap junctions (GJs) are dynamic structures composed of hexamers of connexins (Cxs), a class of transmembrane proteins enabling channel-mediated direct intercellular communication through cell-cell diffusion of ions and small metabolites. In defined conditions, Cxs also work as hemichannels allowing exchanges between the cytoplasm and the extracellular medium. The most common GJ channel is formed by connexin 43 (Cx43) and plays an important role in physiological and pathological processes in excitable tissues, such as heart and brain. Hence, Cx43 has been largely envisioned as a new therapeutic target in cancer, neurological and psychiatric indications, or cardiovascular diseases. Identifying new pharmacological inhibitors of Cx43 GJs with different mechanisms of action and from diverse chemical classes is thus highly challenging. We present here a high-content screening method, based on the evaluation of fluorescent dye transfer rates between adjacent cells to monitor the function of GJs in U251 glioblastoma cells expressing high levels of Cx43. This assay was validated using well-described pharmacological GJ inhibitors such as mefloquine. The method was adapted to screen a library of 1,280 Food and Drug Administration- and European Medicines Agency-approved drugs that led to the selection of both known and new inhibitors of GJ channel function. We further focused on a specific class of microtubule-targeting agents, confirming that a proper tubulin network is required for functional Cx43 GJ channels.

Objectives: The phytohormone methyl jasmonate (MeJA) has been identified as a vital cell regulator in plants. This substance is analogous to eicosanoids and similar to that of anti-inflammatory prostaglandins. In animals and in animal cells, it displayed an efficient neuroprotective, anti-inflammatory and antioxidant action; while in tumoral strains, it demonstrates a potentially highly attractive mechanism of apoptosis induction through various cellular and molecular mechanisms. The aim of the present review was to explore two new hypotheses that explain the action of MeJA, a lipid phytohormone and its potentially anti-apoptotic mechanism for use as a therapeutic target for future treatment of Inflammatory bowel diseases (IBDs)., Key Findings: Methyl jasmonate is a new candidate for the treatment of IBDs, modulating the expression of the major classes of caspase-type protease families that selectively act on the extrinsic and intrinsic pathways of the apoptotic process. Its action is based on the reduction of the expression in tumour necrosis factor tissue levels and the modulating action of reactive oxygen species production, acting only on the destruction of cells that express the diseased phenotype, and preserving cells that are not transformed., Conclusions: Methyl jasmonate may represent an alternative for the transduction processes of important signals in the cellular renewal of the intestinal mucosa., (© 2017 Royal Pharmaceutical Society.)

Antidepressants, prescribed as first line treatment of neuropathic pain, have a limited efficacy and poorly tolerated side effects. Because recent studies pointed out the implication of astroglial connexins (Cx) in both neuropathic pain and antidepressive treatment, we investigated whether their blockade by mefloquine could modulate the action of the tricyclic antidepressant amitriptyline. Using primary cultures, we found that both mefloquine and amitriptyline inhibited Cx43-containing gap junctions, and that the drug combination acted synergically. We then investigated whether mefloquine could enhance amitriptyline efficacy in a preclinical model of neuropathic pain. Sprague-Dawley rats that underwent chronic unilateral constriction injury (CCI) to the sciatic nerve (SN) were treated with either amitriptyline, mefloquine or the combination of both drugs. Whereas acute treatments were ineffective, chronic administration of amitriptyline reduced CCI-SN-induced hyperalgesia-like behavior, and this effect was markedly enhanced by co-administration of mefloquine, which was inactive on its own. No pharmacokinetic interactions between both drugs were observed and CCI-SN-induced neuroinflammatory and glial activation markers remained unaffected by these treatments in dorsal root ganglia and spinal cord. Mechanisms downstream of CCI-SN-induced neuroinflammation and glial activation might therefore be targeted. Connexin inhibition in astroglia could represent a promising approach towards improving neuropathic pain therapy by antidepressants., Competing Interests: This work was supported by the Commissariat à l’Energie Atomique and Theranexus Company. TJ, AD, CP, FM and MC are employees of Theranexus. The other authors declare no potential conflict of interest.

Study Objectives: Modafinil is a non-amphetaminic wake-promoting compound used as therapy against sleepiness and narcolepsy. Its mode of action is complex, but modafinil has been recently proposed to act as a cellular-coupling enhancer in glial cells, through modulation of gap junctions constituted by connexins. The present study investigated in mice the impact of connexins on the effects of modafinil using connexin inhibitors., Methods: Modafinil was administered alone or combined with inhibitors of astrocyte connexin, meclofenamic acid, or flecainide, respectively, acting on Cx30 and Cx43. Sleep-wake states were monitored in wild-type and narcoleptic orexin knockout mice. A spontaneous alternation task was used to evaluate working memory in wild-type mice. The effects of the compounds on astroglial intercellular coupling were determined using dye transfer in acute cortical slices., Results: Meclofenamic acid had little modulation on the effects of modafinil, but flecainide enhanced the wake-promoting and pro-cognitive effects of modafinil. Co-administration of modafinil/flecainide resulted in a marked decrease in the number and duration of direct transitions to rapid eye movement sleep, which are characteristic of narcoleptic episodes in orexin knockout mice. Furthermore, modafinil enhanced the connexin-mediated astroglial cell coupling, whereas flecainide reduced it. Finally, this modafinil-induced effect was reversed by co-administration with flecainide., Conclusions: Our study indicates that flecainide impacts the pharmacological effects of modafinil, likely through the normalization of Cx30-dependent gap junctional coupling in astroglial networks. The enhancement of the wake-promoting, behavioral, and cognitive outcomes of modafinil demonstrated here with flecainide would open new perspectives in the management of sleep disorders such as narcolepsy., Commentary: A commentary on this article appears in this issue on page 1175., (© 2016 Associated Professional Sleep Societies, LLC.)

Connexins are transmembrane proteins involved in gap junction intercellular communication. They present cell- and tissue-specific expression, with own electric and metabolic coupling specificities. These proteins are involved in numerous physiological processes in the brain and among them neuronal synchronization and trafficking of glucose. Such proteins are also described as being misregulated in various pathologies in the central nervous system. Thus, connexin blockers have been proposed as pharmacological tools to dissect these implications. However, such approaches lack accurate characterization of known inhibitors toward gap junction isoform specificity. In addition, those compounds are limited to few chemical classes and exhibit other activities, for example, an anti-inflammatory effect. The aims of this study were to evaluate the selectivity of described inhibitors and to enrich this pharmacopeia by new chemical classes. In this study, we present the specificity of published inhibitors toward several connexin isoforms expressed in the brain. Furthermore, after a screening of compounds using cellular models, we identified seven new inhibitors, with high functional reversibility and different relative selectivity toward isoforms. They constitute new chemical classes of connexin modulators completing those previously described. These new inhibitors might also provide new insights in understanding numerous pathophysiological processes involving gap junctions.

At present, there is no effective therapy for any of the neurodegenerative amyloidoses, despite renewed efforts to identify compounds active against the various implicated pathogenetic molecules. We have screened a library of 2960 natural and synthetic compounds in two cell lines chronically infected with mouse prions, and have identified eight new inhibitors of prion replication in vitro. They belong to two distinct chemical families that have not previously been recognised as effective in the field of transmissible spongiform encephalopathies: seven are 3-aminosteroids and one is a derivative of erythromycin A with an oxime functionality. Our results suggest that these aminosteroids inhibit prion replication by triggering a common target, possibly implicated in the regulatory pathways of cellular prion protein metabolism. Furthermore, using a quantitative approach for the study of protein stability, it was shown that the erythromycin A derivative altered prion protein stability by direct interaction. Such direct targeting of this amyloid precursor might provide new clues for the understanding of prion diseases and, more importantly, help to define new molecules that are active against prion diseases.

In the present study, we compared the effect of 5% oxygen tension (hypoxia) with a normal tension of 21% oxygen (normoxia) on macrophage infection by the protozoan parasite Leishmania amazonensis. Macrophages from different sources (human cell line U937, murine cell line J774, and murine peritoneal macrophages) exposed to hypoxia showed a reduction of the percentage of infected cells and the number of intracellular parasites per cell. Observations on the kinetics of infection indicated that hypoxia did not depress L. amazonensis phagocytosis but induced macrophages to reduce intracellular parasitism. Furthermore, hypoxia did not act synergistically with gamma-interferon and bacterial lipopolysaccharides in macrophages to induce killing of parasites. Experiments also indicated no correlation between nitric oxide production and control of infection in macrophages under hypoxic condition. Thus, we have provided the first evidence that hypoxia, which occurs in various pathological conditions, can alter macrophage susceptibility to a parasitic infection.