Browsing by Author "Linsambarth, Sergio"
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Item 4-Hydroxynonenal induces Cx46 hemichannel inhibition through its carbonylation(2020) Retamal, Mauricio; Fiori, Mariana C.; Fernández-Olivares, Ainoa; Linsambarth, Sergio; Peña, Francisca; Quintana, Daisy; Stehberg, Jimmy; Altenberg, Guillermo A.Hemichannels formed by connexins mediate the exchange of ions and signaling molecules between the cytoplasm and the extracellular milieu. Under physiological conditions hemichannels have a low open probability, but in certain pathologies their open probability increases, which can result in cell damage. Pathological conditions are characterized by the production of a number of proinflammatory molecules, including 4-hydroxynonenal (4-HNE), one of the most common lipid peroxides produced in response to inflammation and oxidative stress. The aim of this work was to evaluate whether 4-HNE modulates the activity of Cx46 hemichannels. We found that 4-HNE (100 μM) reduced the rate of 4′,6-diamino-2-fenilindol (DAPI) uptake through hemichannels formed by recombinant human Cx46 fused to green fluorescent protein, an inhibition that was reversed partially by 10 mM dithiothreitol. Immunoblot analysis showed that the recombinant Cx46 expressed in HeLa cells becomes carbonylated after exposure to 4-HNE, and that 10 mM dithiothreitol reduced its carbonylation. We also found that Cx46 was carbonylated by 4-HNE in the lens of a selenite-induced cataract animal model. The exposure to 100 μM 4-HNE decreased hemichannel currents formed by recombinant rat Cx46 in Xenopus laevis oocytes. This inhibition also occurred in a mutant expressing only the extracellular loop cysteines, suggesting that other Cys are not responsible for the hemichannel inhibition by carbonylation. This work demonstrates for the first time that Cx46 is post-translationally modified by a lipid peroxide and that this modification reduces Cx46 hemichannel activity.Item Astroglial gliotransmitters released via Cx43 hemichannels regulate NMDAR-dependent transmission and short-term fear memory in the basolateral amygdala(2021) Linsambarth, Sergio; Carvajal, Francisco; Moraga, Rodrigo; Mendez, Luis; Tamburini, Giovanni; Jimenez, Ivanka; Verdugo, Daniel; Gómez, Gonzalo I.; Jury, Nur; Martínez, Pablo; Van Zundert, Brigitte; Varela, Lorena; Retamal, Mauricio; Martin, Claire; Altenberg, Guillermo; Fiori, Mariana; Cerpa, Waldo; Orellana, Juan; Stehberg, JimmyAstrocytes release gliotransmitters via connexin 43 (Cx43) hemichannels into neighboring synapses, which can modulate synaptic activity and are necessary for fear memory consolidation. However, the gliotransmitters released, and their mechanisms of action remain elusive. Here, we report that fear conditioning training elevated Cx43 hemichannel activity in astrocytes from the basolateral amygdala (BLA). The selective blockade of Cx43 hemichannels by microinfusion of TAT-Cx43L2 peptide into the BLA induced memory deficits 1 and 24 h after training, without affecting learning. The memory impairments were prevented by the co-injection of glutamate and D-serine, but not by the injection of either alone, suggesting a role for NMDA receptors (NMDAR). The incubation with TAT-Cx43L2 decreased NMDAR-mediated currents in BLA slices, effect that was also prevented by the addition of glutamate and D-serine. NMDARs in primary neuronal cultures were unaffected by TAT-Cx43L2, ruling out direct effects of the peptide on NMDARs. Finally, we show that D-serine permeates through purified Cx43 hemichannels reconstituted in liposomes. We propose that the release of glutamate and D-serine from astrocytes through Cx43 hemichannels is necessary for the activation of post-synaptic NMDARs during training, to allow for the formation of short-term and subsequent long-term memory, but not for learning per se.Publication The role of astrocytes in depression, its prevention and treatment by targeting astroglial gliotransmitter release(2024) Duarte, Yorley; Quintana, Daisy; Moraga-Amaro, Rodrigo; Dinamarca, Ivanka; Lemunao, Yordan; Cárdenas, Kevin; Bahamonde, Tamara; Barrientos, Tabita; Olivares, Pedro; Navas, Camila; Carvajal, Francisco J.; Santibáñez, Yessenia; Castro, Raimundo; Meza, María Paz; Jorquera, Ramón; Gómez, Gonzalo I.; Henke, Marina; Alarcón, Rodrigo; Gabriel, Laureen A.; Schiffmann, Susanne; Cerpa, Waldo; Retamal, Mauricio A.; Simon, Felipe; Linsambarth, Sergio; González Nilo, Fernando; Stehberg, JimmyThe role of ventral hippocampus (vHipp) astroglial gliotransmission in depression was studied using chronic restraint stress (CRS) and chronic unpredictable mild stress (CUMS) rodent models. CRS increased Cx43 hemichannel activity and extracellular glutamate levels in the vHipp and blocking astroglial Cx43 hemichannel-dependent gliotransmission during CRS prevented the development of depression and glutamate buildup. Moreover, the acute blockade of Cx43 hemichannels induced antidepressant effects in rats previously subjected to CRS or CUMS. This antidepressant effect was prevented by coinjection of glutamate and D-serine. Furthermore, Cx43 hemichannel blockade decreased postsynaptic NMDAR currents in vHipp slices in a glutamate and D-serine-dependent manner. Notably, chronic microinfusion of glutamate and D-serine, L-serine, or the NMDAR agonist NMDA, into the vHipp induced depressive-like symptoms in nonstressed rats. We also identified a small molecule, cacotheline, which blocks Cx43 hemichannels and its systemic administration induced rapid antidepressant effects, preventing stress-induced increases in astroglial Cx43 hemichannel activity and extracellular glutamate in the vHipp, without sedative or locomotor side effects. In conclusion, chronic stress increases Cx43 hemichannel-dependent release of glutamate and D-/L-serine from astrocytes in the vHipp, overactivating postsynaptic NMDARs and triggering depressive-like symptoms. This study highlights the critical role of astroglial gliotransmitter release in chronic stress-induced depression and suggests it can be used as a target for the prevention and treatment of depression.