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Diverse studies have suggested that cytoplasmic inclusions of misfolded α-synuclein in neuronal and glial cells are main pathological features of different α-synucleinopathies, including Parkinson's disease and dementia with Lewy bodies. Up to now, most studies have focused on the effects of α-synuclein on neurons, whereas the possible alterations of astrocyte functions and neuron-glia crosstalk have received minor attention. Recent evidence indicates that cellular signaling mediated by hemichannels and pannexons is critical for astroglial function and dysfunction. These channels constitute a diffusional route of communication between the cytosol and the extracellular space and during pathological scenarios they may lead to homeostatic disturbances linked to the pathogenesis and progression of different diseases. Here, we found that α-synuclein enhances the opening of connexin 43 (Cx43) hemichannels and pannexin-1 (Panx1) channels in mouse cortical astrocytes. This response was linked to the activation of cytokines, the p38 MAP kinase, the inducible nitric oxide synthase, cyclooxygenase 2, intracellular free Ca 2+ concentration ([Ca 2+ ] i ), and purinergic and glutamatergic signaling. Relevantly, the α-synuclein-induced opening of hemichannels and pannexons resulted in alterations in [Ca 2+ ] i dynamics, nitric oxide (NO) production, gliotransmitter release, mitochondrial morphology, and astrocyte survival. We propose that α-synuclein-mediated opening of astroglial Cx43 hemichannels and Panx1 channels might constitute a novel mechanism involved in the pathogenesis and progression of α-synucleinopathies., (© 2019 Wiley Periodicals, Inc.)

The mechanisms involved in Alzheimer's disease are not completely understood and how astrocytes and their gliotransmission contribute to this neurodegenerative disease remains to be fully elucidated. Previous studies have shown that amyloid-β peptide (Aβ) induces neuronal death by a mechanism that involves the excitotoxic release of ATP and glutamate associated to astroglial hemichannel opening. We have demonstrated that synthetic and endogenous cannabinoids (CBs) reduce the opening of astrocyte Cx43 hemichannels evoked by activated microglia or inflammatory mediators. Nevertheless, whether CBs could prevent the astroglial hemichannel-dependent death of neurons evoked by Aβ is unknown. Astrocytes as well as acute hippocampal slices were treated with the active fragment of Aβ alone or in combination with the following CBs: WIN, 2-AG, or methanandamide (Meth). Hemichannel activity was monitored by single channel recordings and by time-lapse ethidium uptake while neuronal death was assessed by Fluoro-Jade C staining. We report that CBs fully prevented the hemichannel activity and inflammatory profile evoked by Aβ in astrocytes. Moreover, CBs fully abolished the Aβ-induced release of excitotoxic glutamate and ATP associated to astrocyte Cx43 hemichannel activity, as well as neuronal damage in hippocampal slices exposed to Aβ. Consequently, this work opens novel avenues for alternative treatments that target astrocytes to maintain neuronal function and survival during AD. GLIA 2016 GLIA 2017;65:122-137., (© 2016 Wiley Periodicals, Inc.)

Several epidemiological studies indicate that children born from mothers exposed to infections during gestation, have an increased risk to develop neurological disorders, including schizophrenia, autism and cerebral palsy. Given that it is unknown if astrocytes and their crosstalk with neurons participate in the above mentioned brain pathologies, the aim of this work was to address if astroglial paracrine signaling mediated by Cx43 and Panx1 unopposed channels could be affected in the offspring of LPS-exposed dams during pregnancy. Ethidium uptake experiments showed that prenatal LPS-exposure increases the activity of astroglial Cx43 and Panx1 unopposed channels in the offspring. Induction of unopposed channel opening by prenatal LPS exposure depended on intracellular Ca 2+ levels, cytokine production and activation of p38 MAP kinase/iNOS pathway. Biochemical assays and Fura-2AM/DAF-FM time-lapse fluorescence images revealed that astrocytes from the offspring of LPS-exposed dams displayed increased spontaneous Ca 2+ dynamics and NO production, whereas iNOS levels and release of IL-1β/TNF-α were also increased. Interestingly, we found that prenatal LPS exposure enhanced the release of ATP through astroglial Cx43 and Panx1 unopposed channels in the offspring, resulting in an increased neuronal death mediated by the activation of neuronal P2X 7 receptors and Panx1 channels. Altogether, this evidence suggests that astroglial Cx43 and Panx1 unopposed channel opening induced by prenatal LPS exposure depended on the inflammatory activation profile and the activation pattern of astrocytes. The understanding of the mechanism underlying astrocyte-neuron crosstalk could contribute to the development of new strategies to ameliorate the brain abnormalities induced in the offspring by prenatal inflammation. GLIA 2015;63:2058-2072., (© 2015 Wiley Periodicals, Inc.)

Under inflammatory conditions, microglia exhibit increased levels of free intracellular Ca(2+) and produce high amounts of nitric oxide (NO). However, whether NO, Ca(2+) dynamics, and gliotransmitter release are reciprocally modulated is not fully understood. More importantly, the effect of astrocytes in the potentiation or suppression of such signaling is unknown. Our aim was to address if astrocytes could regulate NO-dependent Ca(2+) dynamics and ATP release in LPS-stimulated microglia. Griess assays and Fura-2AM time-lapse fluorescence images of microglia revealed that LPS produced an increased basal [Ca(2+) ]i that depended on the sequential activation of iNOS, COXs, and EP1 receptor. TGFβ1 released by astrocytes inhibited the abovementioned responses and also abolished LPS-induced ATP release by microglia. Luciferin/luciferase assays and dye uptake experiments showed that release of ATP from LPS-stimulated microglia occurred via pannexin 1 (Panx1) channels, but not connexin 43 hemichannels. Moreover, in LPS-stimulated microglia, exogenous ATP triggered activation of purinergic P2Y1 receptors resulting in Ca(2+) release from intracellular stores. Interestingly, TGFβ1 released by astrocytes inhibited ATP-induced Ca(2+) response in LPS-stimulated microglia to that observed in control microglia. Finally, COX/EP1 receptor signaling and activation of P2 receptors via ATP released through Panx1 channels were critical for the increased NO production in LPS-stimulated microglia. Thus, Ca(2+) dynamics depended on the inflammatory profile of microglia and could be modulated by astrocytes. The understanding of mechanisms underlying glial cell regulatory crosstalk could contribute to the development of new treatments to reduce inflammatory cytotoxicity in several brain pathologies., (Copyright © 2013 Wiley Periodicals, Inc.)

The ventromedial hypothalamus is involved in regulating feeding and satiety behavior, and its neurons interact with specialized ependymal-glial cells, termed tanycytes. The latter express glucose-sensing proteins, including glucose transporter 2, glucokinase, and ATP-sensitive K(+) (K(ATP) ) channels, suggesting their involvement in hypothalamic glucosensing. Here, the transduction mechanism involved in the glucose-induced rise of intracellular free Ca(2+) concentration ([Ca(2+) ](i) ) in cultured β-tanycytes was examined. Fura-2AM time-lapse fluorescence images revealed that glucose increases the intracellular Ca(2+) signal in a concentration-dependent manner. Glucose transportation, primarily via glucose transporters, and metabolism via anaerobic glycolysis increased connexin 43 (Cx43) hemichannel activity, evaluated by ethidium uptake and whole cell patch clamp recordings, through a K(ATP) channel-dependent pathway. Consequently, ATP export to the extracellular milieu was enhanced, resulting in activation of purinergic P2Y(1) receptors followed by inositol trisphosphate receptor activation and Ca(2+) release from intracellular stores. The present study identifies the mechanism by which glucose increases [Ca(2+) ](i) in tanycytes. It also establishes that Cx43 hemichannels can be rapidly activated under physiological conditions by the sequential activation of glucosensing proteins in normal tanycytes., (Copyright © 2011 Wiley Periodicals, Inc.)

Brain ischemia causes more extensive injury in hyperglycemic than normoglycemic subjects, and the increased damage is to astroglia as well as neurons. In the present work, we found that in cortical astrocytes from rat or mouse, reoxygenation after hypoxia in a medium mimicking interstitial fluid during ischemia increases hemichannel activity and decreases cell-cell communication via gap junctions as indicated by dye uptake and dye coupling, respectively. These effects were potentiated by high glucose during the hypoxia in a concentration-dependent manner (and by zero glucose) and were not observed in connexin 43(-/-) astrocytes. The responses were transient and persistent after short and long periods of hypoxia, respectively. The persistent responses were associated with a progressive reduction in cell viability that was prevented by La(3+) or peptides that block connexin 43 (Cx43) hemichannels or by inhibition of p38 MAP kinase prior to hypoxia-reoxygenation but not by treatments that block pannexin hemichannels. Block of Cx43 hemichannels did not affect the reduction in gap junction mediated dye coupling observed during reoxygenation. Cx43 hemichannels may be a novel therapeutic target to reduce cell death following stroke, particularly in hyperglycemic conditions., ((c) 2009 Wiley-Liss, Inc.)