Inhibition of astroglial hemichannels prevents synaptic transmission decline during spreading depression.

BACKGROUND: Spreading depression (SD) is an intriguing phenomenon characterized by massive slow brain depolarizations that affect neurons and glial cells. This phenomenon is repetitive and produces a metabolic overload that increases secondary damage. However, the mechanisms associated with the initiation and propagation of SD are unknown. Multiple lines of evidence indicate that persistent and uncontrolled opening of hemichannels could participate in the pathogenesis and progression of several neurological disorders including acute brain injuries. Here, we explored the contribution of astroglial hemichannels composed of connexin-43 (Cx43) or pannexin-1 (Panx1) to SD evoked by high-K+ stimulation in brain slices. RESULTS: Focal high-K+ stimulation rapidly evoked a wave of SD linked to increased activity of the Cx43 and Panx1 hemichannels in the brain cortex, as measured by light transmittance and dye uptake analysis, respectively. The activation of these channels occurs mainly in astrocytes but also in neurons. More importantly, the inhibition of both the Cx43 and Panx1 hemichannels completely prevented high K+-induced SD in the brain cortex. Electrophysiological recordings also revealed that Cx43 and Panx1 hemichannels critically contribute to the SD-induced decrease in synaptic transmission in the brain cortex and hippocampus. CONCLUSIONS: Targeting Cx43 and Panx1 hemichannels could serve as a new therapeutic strategy to prevent the initiation and propagation of SD in several acute brain injuries.

A preclinical mice model of multiple sclerosis based on the toxin-induced double-site demyelination of callosal and cerebellar fibers.

BACKGROUND: Multiple sclerosis (MS) is an irreversible progressive CNS pathology characterized by the loss of myelin (i.e. demyelination). The lack of myelin is followed by a progressive neurodegeneration triggering symptoms as diverse as fatigue, motor, locomotor and sensory impairments and/or bladder, cardiac and respiratory dysfunction. Even though there are more than fourteen approved treatments for reducing MS progression, there are still no cure for the disease. Thus, MS research is a very active field and therefore we count with different experimental animal models for studying mechanisms of demyelination and myelin repair, however, we still lack a preclinical MS model assembling demyelination mechanisms with relevant clinical-like signs. RESULTS: Here, by inducing the simultaneous demyelination of both callosal and cerebellar white matter fibers by the double-site injection of lysolecithin (LPC), we were able to reproduce CNS demyelination, astrocyte recruitment and increases levels of proinflammatory cytokines levels along with motor, locomotor and urinary impairment, as well as cardiac and respiratory dysfunction, in the same animal model. Single site LPC-injections either in corpus callosum or cerebellum only, fails in to reproduce such a complete range of MS-like signs. CONCLUSION: We here report that the double-site LPC injections treatment evoke a complex MS-like mice model. We hope that this experimental approach will help to deepen our knowledge about the mechanisms of demyelinated diseases such as MS.

Cx43 hemichannels and panx1 channels contribute to ethanol-induced astrocyte dysfunction and damage.

BACKGROUND: Alcohol, a widely abused drug, significantly diminishes life quality, causing chronic diseases and psychiatric issues, with severe health, societal, and economic repercussions. Previously, we demonstrated that non-voluntary alcohol consumption increases the opening of Cx43 hemichannels and Panx1 channels in astrocytes from adolescent rats. However, whether ethanol directly affects astroglial hemichannels and, if so, how this impacts the function and survival of astrocytes remains to be elucidated. RESULTS: Clinically relevant concentrations of ethanol boost the opening of Cx43 hemichannels and Panx1 channels in mouse cortical astrocytes, resulting in the release of ATP and glutamate. The activation of these large-pore channels is dependent on Toll-like receptor 4, P2X7 receptors, IL-1ß and TNF-α signaling, p38 mitogen-activated protein kinase, and inducible nitric oxide (NO) synthase. Notably, the ethanol-induced opening of Cx43 hemichannels and Panx1 channels leads to alterations in cytokine secretion, NO production, gliotransmitter release, and astrocyte reactivity, ultimately impacting survival. CONCLUSION: Our study reveals a new mechanism by which ethanol impairs astrocyte function, involving the sequential stimulation of inflammatory pathways that further increase the opening of Cx43 hemichannels and Panx1 channels. We hypothesize that targeting astroglial hemichannels could be a promising pharmacological approach to preserve astrocyte function and synaptic plasticity during the progression of various alcohol use disorders.

Astroglial gliotransmitters released via Cx43 hemichannels regulate NMDAR-dependent transmission and short-term fear memory in the basolateral amygdala.

Astrocytes 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.

SARS-CoV-2 spike protein S1 activates Cx43 hemichannels and disturbs intracellular Ca2+ dynamics.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes the ongoing coronavirus disease 2019 (COVID-19). An aspect of high uncertainty is whether the SARS-CoV-2 per se or the systemic inflammation induced by viral infection directly affects cellular function and survival in different tissues. It has been postulated that tissue dysfunction and damage observed in COVID-19 patients may rely on the direct effects of SARS-CoV-2 viral proteins. Previous evidence indicates that the human immunodeficiency virus and its envelope protein gp120 increase the activity of connexin 43 (Cx43) hemichannels with negative repercussions for cellular function and survival. Here, we evaluated whether the spike protein S1 of SARS-CoV-2 could impact the activity of Cx43 hemichannels. RESULTS: We found that spike S1 time and dose-dependently increased the activity of Cx43 hemichannels in HeLa-Cx43 cells, as measured by dye uptake experiments. These responses were potentiated when the angiotensin-converting enzyme 2 (ACE2) was expressed in HeLa-Cx43 cells. Patch clamp experiments revealed that spike S1 increased unitary current events with conductances compatible with Cx43 hemichannels. In addition, Cx43 hemichannel opening evoked by spike S1 triggered the release of ATP and increased the [Ca2+]i dynamics elicited by ATP. CONCLUSIONS: We hypothesize that Cx43 hemichannels could represent potential pharmacological targets for developing therapies to counteract SARS-CoV-2 infection and their long-term consequences.

Hypertensive Nephropathy: Unveiling the Possible Involvement of Hemichannels and Pannexons.

Hypertension is one of the most common risk factors for developing chronic cardiovascular diseases, including hypertensive nephropathy. Within the glomerulus, hypertension causes damage and activation of mesangial cells (MCs), eliciting the production of large amounts of vasoactive and proinflammatory agents. Accordingly, the activation of AT1 receptors by the vasoactive molecule angiotensin II (AngII) contributes to the pathogenesis of renal damage, which is mediated mostly by the dysfunction of intracellular Ca2+ ([Ca2+]i) signaling. Similarly, inflammation entails complex processes, where [Ca2+]i also play crucial roles. Deregulation of this second messenger increases cell damage and promotes fibrosis, reduces renal blood flow, and impairs the glomerular filtration barrier. In vertebrates, [Ca2+]i signaling depends, in part, on the activity of two families of large-pore channels: hemichannels and pannexons. Interestingly, the opening of these channels depends on [Ca2+]i signaling. In this review, we propose that the opening of channels formed by connexins and/or pannexins mediated by AngII induces the ATP release to the extracellular media, with the subsequent activation of purinergic receptors. This process could elicit Ca2+ overload and constitute a feed-forward mechanism, leading to kidney damage.

TNF-α Plus IL-1ß Induces Opposite Regulation of Cx43 Hemichannels and Gap Junctions in Mesangial Cells through a RhoA/ROCK-Dependent Pathway.

Connexin 43 (Cx43) is expressed in kidney tissue where it forms hemichannels and gap junction channels. However, the possible functional relationship between these membrane channels and their role in damaged renal cells remains unknown. Here, analysis of ethidium uptake and thiobarbituric acid reactive species revealed that treatment with TNF-α plus IL-1ß increases Cx43 hemichannel activity and oxidative stress in MES-13 cells (a cell line derived from mesangial cells), and in primary mesangial cells. The latter was also accompanied by a reduction in gap junctional communication, whereas Western blotting assays showed a progressive increase in phosphorylated MYPT (a target of RhoA/ROCK) and Cx43 upon TNF-α/IL-1ß treatment. Additionally, inhibition of RhoA/ROCK strongly antagonized the TNF-α/IL-1ß-induced activation of Cx43 hemichannels and reduction in gap junctional coupling. We propose that activation of Cx43 hemichannels and inhibition of cell-cell coupling during pro-inflammatory conditions could contribute to oxidative stress and damage of mesangial cells via the RhoA/ROCK pathway.

Astroglial Hemichannels and Pannexons: The Hidden Link between Maternal Inflammation and Neurological Disorders.

Maternal inflammation during pregnancy causes later-in-life alterations of the offspring's brain structure and function. These abnormalities increase the risk of developing several psychiatric and neurological disorders, including schizophrenia, intellectual disability, bipolar disorder, autism spectrum disorder, microcephaly, and cerebral palsy. Here, we discuss how astrocytes might contribute to postnatal brain dysfunction following maternal inflammation, focusing on the signaling mediated by two families of plasma membrane channels: hemi-channels and pannexons. [Ca2+]i imbalance linked to the opening of astrocytic hemichannels and pannexons could disturb essential functions that sustain astrocytic survival and astrocyte-to-neuron support, including energy and redox homeostasis, uptake of K+ and glutamate, and the delivery of neurotrophic factors and energy-rich metabolites. Both phenomena could make neurons more susceptible to the harmful effect of prenatal inflammation and the experience of a second immune challenge during adulthood. On the other hand, maternal inflammation could cause excitotoxicity by producing the release of high amounts of gliotransmitters via astrocytic hemichannels/pannexons, eliciting further neuronal damage. Understanding how hemichannels and pannexons participate in maternal inflammation-induced brain abnormalities could be critical for developing pharmacological therapies against neurological disorders observed in the offspring.

HIV gp120 Protein Increases the Function of Connexin 43 Hemichannels and Pannexin-1 Channels in Astrocytes: Repercussions on Astroglial Function.

At least half of human immunodeficiency virus (HIV)-infected individuals suffer from a wide range of cognitive, behavioral and motor deficits, collectively known as HIV-associated neurocognitive disorders (HAND). The molecular mechanisms that amplify damage within the brain of HIV-infected individuals are unknown. Recently, we described that HIV augments the opening of connexin-43 (Cx43) hemichannels in cultured human astrocytes, which result in the collapse of neuronal processes. Whether HIV soluble viral proteins such as gp120, can regulate hemichannel opening in astrocytes is still ignored. These channels communicate the cytosol with the extracellular space during pathological conditions. We found that gp120 enhances the function of both Cx43 hemichannels and pannexin-1 channels in mouse cortical astrocytes. These effects depended on the activation of IL-1ß/TNF-α, p38 MAP kinase, iNOS, cytoplasmic Ca2+ and purinergic signaling. The gp120-induced channel opening resulted in alterations in Ca2+ dynamics, nitric oxide production and ATP release. Although the channel opening evoked by gp120 in astrocytes was reproduced in ex vivo brain preparations, these responses were heterogeneous depending on the CA1 region analyzed. We speculate that soluble gp120-induced activation of astroglial Cx43 hemichannels and pannexin-1 channels could be crucial for the pathogenesis of HAND.

Permeation of Molecules through Astroglial Connexin 43 Hemichannels Is Modulated by Cytokines with Parameters Depending on the Permeant Species.

Recent studies indicate that connexin hemichannels do not act as freely permeable non-selective pores, but they select permeants in an isoform-specific manner with cooperative, competitive and saturable kinetics. The aim of this study was to investigate whether the treatment with a mixture of IL-1ß plus TNF-α, a well-known pro-inflammatory condition that activates astroglial connexin 43 (Cx43) hemichannels, could alter their permeability to molecules. We found that IL-1ß plus TNF-α left-shifted the dye uptake rate vs. dye concentration relationship for Etd and 2-NBDG, but the opposite took place for DAPI or YO-PRO-1, whereas no alterations were observed for Prd. The latter modifications were accompanied of changes in Kd (Etd, DAPI, YO-PRO-1 or 2-NBDG) and Hill coefficients (Etd and YO-PRO-1), but not in alterations of Vmax. We speculate that IL-1ß plus TNF-α may distinctively affect the binding sites to permeants in astroglial Cx43 hemichannels rather than their number in the cell surface. Alternatively, IL-1ß plus TNF-α could induce the production of endogenous permeants that may favor or compete for in the pore-lining residues of Cx43 hemichannels. Future studies shall elucidate whether the differential ionic/molecule permeation of Cx43 hemichannels in astrocytes could impact their communication with neurons in the normal and inflamed nervous system.

Connexin 43 hemichannels and pannexin-1 channels contribute to the α-synuclein-induced dysfunction and death of astrocytes.

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 Ca2+ concentration ([Ca2+ ]i ), and purinergic and glutamatergic signaling. Relevantly, the α-synuclein-induced opening of hemichannels and pannexons resulted in alterations in [Ca2+ ]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.

Cannabinoids prevent the amyloid ß-induced activation of astroglial hemichannels: A neuroprotective mechanism.

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-ß peptide (Aß) 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 Aß is unknown. Astrocytes as well as acute hippocampal slices were treated with the active fragment of Aß 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 Aß in astrocytes. Moreover, CBs fully abolished the Aß-induced release of excitotoxic glutamate and ATP associated to astrocyte Cx43 hemichannel activity, as well as neuronal damage in hippocampal slices exposed to Aß. 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.

Physiological Functions of Glial Cell Hemichannels.

The brain performs exceptionally complex and dynamic tasks that depend on the coordinated interaction of neurons, glial cells, endothelial cells, pericytes, smooth muscle cells, ependymal cells, and circulating blood cells. Among these cells, glial cells have emerged as crucial protagonists in the regulation of synaptic transmission and neural function. Indeed, these cells express a wide range of receptors that enable them to sense changes in neuronal activity and the microenvironment by responding locally via the release of bioactive molecules known as gliotransmitters. In the central nervous system (CNS), a novel mechanism that allows gliotransmission via the opening of hemichannels has been proposed. These channels are composed of six protein subunits consisting of connexins or pannexins, which are two highly conserved protein families that are encoded by 21 and 3 genes, respectively, in humans. Typically, glial cell hemichannels exhibit low levels of activity, but this activity is sufficient to ensure the release of a broad spectrum of gliotransmitters, including ATP, D-serine, glutamate, adenosine, and glutathione. Here, we briefly review the current findings regarding the effects of the hemichannel-dependent release of gliotransmitters on the physiology of the CNS.

Prenatal exposure to inflammatory conditions increases Cx43 and Panx1 unopposed channel opening and activation of astrocytes in the offspring effect on neuronal survival.

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 Ca2+ 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 Ca2+ dynamics and NO production, whereas iNOS levels and release of IL-1ß/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 P2X7 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.

Activation of Pannexin-1 channels causes cell dysfunction and damage in mesangial cells derived from angiotensin II-exposed mice.

Chronic kidney disease (CKD) is a prevalent health concern associated with various pathological conditions, including hypertensive nephropathy. Mesangial cells are crucial in maintaining glomerular function, yet their involvement in CKD pathogenesis remains poorly understood. Recent evidence indicates that overactivation of Pannexin-1 (Panx1) channels could contribute to the pathogenesis and progression of various diseases. Although Panx1 is expressed in the kidney, its contribution to the dysfunction of renal cells during pathological conditions remains to be elucidated. This study aimed to investigate the impact of Panx1 channels on mesangial cell function in the context of hypertensive nephropathy. Using an Ang II-infused mouse model and primary mesangial cell cultures, we demonstrated that in vivo exposure to Ang II sensitizes cultured mesangial cells to show increased alterations when they are subjected to subsequent in vitro exposure to Ang II. Particularly, mesangial cell cultures treated with Ang II showed elevated activity of Panx1 channels and increased release of ATP. The latter was associated with enhanced basal intracellular Ca2+ ([Ca2+]i) and increased ATP-mediated [Ca2+]i responses. These effects were accompanied by increased lipid peroxidation and reduced cell viability. Crucially, all the adverse impacts evoked by Ang II were prevented by the blockade of Panx1 channels, underscoring their critical role in mediating cellular dysfunction in mesangial cells. By elucidating the mechanisms by which Ang II negatively impacts mesangial cell function, this study provides valuable insights into the pathogenesis of renal damage in hypertensive nephropathy.

Hemichannels contribute to mitochondrial Ca2+ and morphology alterations evoked by ethanol in astrocytes.

Alcohol, a toxic and psychoactive substance with addictive properties, severely impacts life quality, leading to significant health, societal, and economic consequences. Its rapid passage across the blood-brain barrier directly affects different brain cells, including astrocytes. Our recent findings revealed the involvement of pannexin-1 (Panx1) and connexin-43 (Cx43) hemichannels in ethanol-induced astrocyte dysfunction and death. However, whether ethanol influences mitochondrial function and morphology in astrocytes, and the potential role of hemichannels in this process remains poorly understood. Here, we found that ethanol reduced basal mitochondrial Ca2+ but exacerbated thapsigargin-induced mitochondrial Ca2+ dynamics in a concentration-dependent manner, as evidenced by Rhod-2 time-lapse recordings. Similarly, ethanol-treated astrocytes displayed increased mitochondrial superoxide production, as indicated by MitoSox labeling. These effects coincided with reduced mitochondrial membrane potential and increased mitochondrial fragmentation, as determined by MitoRed CMXRos and MitoGreen quantification, respectively. Crucially, inhibiting both Cx43 and Panx1 hemichannels effectively prevented all ethanol-induced mitochondrial abnormalities in astrocytes. We speculate that exacerbated hemichannel activity evoked by ethanol may impair intracellular Ca2+ homeostasis, stressing mitochondrial Ca2+ with potentially damaging consequences for mitochondrial fusion and fission dynamics and astroglial bioenergetics.

Acute activation of hemichannels by ethanol leads to Ca2+-dependent gliotransmitter release in astrocytes.

Multiple studies have demonstrated that acute ethanol consumption alters brain function and cognition. Nevertheless, the mechanisms underlying this phenomenon remain poorly understood. Astrocyte-mediated gliotransmission is crucial for hippocampal plasticity, and recently, the opening of hemichannels has been found to play a relevant role in this process. Hemichannels are plasma membrane channels composed of six connexins or seven pannexins, respectively, that oligomerize around a central pore. They serve as ionic and molecular exchange conduits between the cytoplasm and extracellular milieu, allowing the release of various paracrine substances, such as ATP, D-serine, and glutamate, and the entry of ions and other substances, such as Ca2+ and glucose. The persistent and exacerbated opening of hemichannels has been associated with the pathogenesis and progression of several brain diseases for at least three mechanisms. The uncontrolled activity of these channels could favor the collapse of ionic gradients and osmotic balance, the release of toxic levels of ATP or glutamate, cell swelling and plasma membrane breakdown and intracellular Ca2+ overload. Here, we evaluated whether acute ethanol exposure affects the activity of astrocyte hemichannels and the possible repercussions of this phenomenon on cytoplasmatic Ca2+ signaling and gliotransmitter release. Acute ethanol exposure triggered the rapid activation of connexin43 and pannexin1 hemichannels in astrocytes, as measured by time-lapse recordings of ethidium uptake. This heightened activity derived from a rapid rise in [Ca2+]i linked to extracellular Ca2+ influx and IP3-evoked Ca2+ release from intracellular Ca2+ stores. Relevantly, the acute ethanol-induced activation of hemichannels contributed to a persistent secondary increase in [Ca2+]i. The [Ca2+]i-dependent activation of hemichannels elicited by ethanol caused the increased release of ATP and glutamate in astroglial cultures and brain slices. Our findings offer fresh perspectives on the potential mechanisms behind acute alcohol-induced brain abnormalities and propose targeting connexin43 and pannexin1 hemichannels in astrocytes as a promising avenue to prevent deleterious consequences of alcohol consumption.

Evidence for TGF-ß1/Nrf2 Signaling Crosstalk in a Cuprizone Model of Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic and degenerative disease that impacts central nervous system (CNS) function. One of the major characteristics of the disease is the presence of regions lacking myelin and an oxidative and inflammatory environment. TGF-ß1 and Nrf2 proteins play a fundamental role in different oxidative/inflammatory processes linked to neurodegenerative diseases such as MS. The evidence from different experimental settings has demonstrated a TGF-ß1-Nrf2 signaling crosstalk under pathological conditions. However, this possibility has not been explored in experimental models of MS. Here, by using the cuprizone-induced demyelination model of MS, we report that the in vivo pharmacological blockage of the TGF-ß1 receptor reduced Nrf2, catalase, and TGFß-1 protein levels in the demyelination phase of cuprizone administration. In addition, ATP production, locomotor function and cognitive performance were diminished by the treatment. Altogether, our results provide evidence for a crosstalk between TGF-ß1 and Nrf2 signaling pathways under CNS demyelination, highlighting the importance of the antioxidant cellular response of neurodegenerative diseases such as MS.

Glial connexin expression and function in the context of Alzheimer's disease.

A hallmark of neurodegenerative diseases is the reactive gliosis characterized by a phenotypic change in astrocytes and microglia. This glial response is associated with modifications in the expression and function of connexins (Cxs), the proteins forming gap junction channels and hemichannels. Increased Cx expression is detected in most reactive astrocytes located at amyloid plaques, the histopathological lesions typically present in the brain of Alzheimer's patients and animal models of the disease. The activity of Cx channels analyzed in vivo as well as in vitro after treatment with the amyloid ß peptide is also modified and, in particular, hemichannel activation may contribute to neuronal damage. In this review, we summarize and discuss recent data that suggest glial Cx channels participate in the neurodegenerative process of Alzheimer's disease. This article is part of a Special Issue entitled: The Communicating junctions, composition, structure and characteristics.

Astrocytes inhibit nitric oxide-dependent Ca(2+) dynamics in activated microglia: involvement of ATP released via pannexin 1 channels.

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. TGFß1 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, TGFß1 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.