Amyloid ß-induced death in neurons involves glial and neuronal hemichannels.

The mechanisms involved in Alzheimer's disease are not completely understood and how glial cells contribute to this neurodegenerative disease remains to be elucidated. Because inflammatory treatments and products released from activated microglia increase glial hemichannel activity, we investigated whether amyloid-ß peptide (Aß) could regulate these channels in glial cells and affect neuronal viability. Microglia, astrocytes, or neuronal cultures as well as acute hippocampal slices made from GFAP-eGFP transgenic mice were treated with the active fragment of Aß. Hemichannel activity was monitored by single-channel recordings and by time-lapse ethidium uptake, whereas neuronal death was assessed by Fluoro-Jade C staining. We report that low concentrations of Aß(25-35) increased hemichannel activity in all three cell types and microglia initiate these effects triggered by Aß. Finally, neuronal damage occurs by activation of neuronal hemichannels induced by ATP and glutamate released from Aß(25-35)-activated glia. These responses were observed in the presence of external calcium and were differently inhibited by hemichannel blockers, whereas the Aß(25-35)-induced neuronal damage was importantly reduced in acute slices made from Cx43 knock-out mice. Thus, Aß leads to a cascade of hemichannel activation in which microglia promote the release of glutamate and ATP through glial (microglia and astrocytes) hemichannels that induces neuronal death by triggering hemichannels in neurons. Consequently, this work opens novel avenues for alternative treatments that target glial cells and neurons to maintain neuronal survival in the presence of Aß.

Inhibition of cytokine-induced connexin43 hemichannel activity in astrocytes is neuroprotective.

Astrocytes express high levels of connexin43, a protein that forms two types of channels: gap junction channels for direct intercellular communication, and hemichannels for exchanges with the extracellular space. Inflammation induces connexin43 hemichannel activation, which has been proposed to be involved in neuroglial interactions. Here, we investigated the contribution of connexin43 to NMDA-induced excitotoxicity in neuron/astrocyte co-cultures, after treatment with a pro-inflammatory cytokine mixture, containing TNF-alpha and IL1-beta (Mix), that stimulated astroglial connexin43 hemichannel activity. Interestingly, NMDA treatment induced a higher amount of neurotoxicity in Mix-treated co-cultures than in untreated ones, whereas this extent of neurotoxicity was absent in enriched neuron cultures or in co-cultures with connexin43 knock-out astrocytes. Furthermore, application of connexin43 hemichannel blockers or a synthetic cannabinoid prevented the Mix-induced potentiated NMDA neurotoxicity. Altogether, these data demonstrate that inflammation-induced astroglial hemichannel activation plays a critical role in neuronal death and suggest a neuroprotective role of connexin43 hemichannel blockade.

Cannabinoids prevent the opposite regulation of astroglial connexin43 hemichannels and gap junction channels induced by pro-inflammatory treatments.

Brain injuries as well as neurodegenerative diseases, are associated with neuro-inflammation characterized by astroglial and microglial activation and/or proliferation. Recently, we reported that lipopolysaccharide (LPS)-activation of microglia inhibits junctional channels and promotes hemichannels, two connexin43 functions in astrocytes. This opposite regulation is mediated by two pro-inflammatory cytokines, interleukin-1 beta and tumor necrosis factor-alpha, released from activated microglia. Because cannabinoids (CBs) have anti-inflammatory properties and their receptors are expressed by glial cells, we investigated on primary cortical cultures the effects of CB agonists, methanandamide and synthetic CBs on (i) cytokines released from LPS-activated microglia and (ii) connexin43 functions in astrocytes subjected to pro-inflammatory treatments. We observed that CBs inhibited the LPS-induced release of interleukin-1 beta and tumor necrosis factor-alpha from microglia. Moreover, the connexin43 dual regulation evoked by the pro-inflammatory treatments, was prevented by CB treatments. Pharmacological characterizations of CB actions on astrocytic connexin43 channels revealed that these effects were mainly mediated through CB1 receptors activation, although non-CB1/CB2 receptors seemed to mediate the action of the methanandamide. Altogether these data demonstrate that in inflammatory situations CBs exert, through the activation of different sub-types of glial CB receptors, a regulation on two functions of connexin43 channels in astrocytes known to be involved in neuron survival.

Proinflammatory cytokines released from microglia inhibit gap junctions in astrocytes: potentiation by beta-amyloid.

Brain inflammation is characterized by a reactive gliosis involving the activation of astrocytes and microglia. This process, common to many brain injuries and diseases, underlies important phenotypic changes in these two glial cell types. One characteristic feature of astrocytes is their high level of intercellular communication mediated by gap junctions. Previously, we have reported that astrocyte gap junctional communication (AGJC) and the expression of connexin 43 (Cx43), the main constitutive protein of gap junctions, are inhibited in microglia (MG)-astrocyte cocultures. Here, we report that bacterial lipopolysaccharide activation of microglia increases their inhibitory effect on Cx43 expression and AGJC. This inhibition is mimicked by treating astrocyte cultures with conditioned medium harvested from activated microglia. Interleukin-1beta (IL-1beta) and tumor necrosis factor-alpha (TNF-alpha) were identified as being the main factors responsible for this conditioned medium-mediated activity. Interestingly, an inflammatory response characterized by MG activation and reactive astrocytes occurs in Alzheimer's disease, at sites of beta-amyloid (Abeta) deposits. We found that this peptide potentiates the inhibitory effect of a conditioned medium diluted at a concentration that is not effective per se. This potentiation is prevented by treating astrocytes with specific blockers of IL-1beta and TNF-alpha activities. Thus, the suppression of communication between astrocytes, induced by activated MG could contribute to the proposed role of reactive gliosis in this neurodegenerative disease.

A pro- and an anti-inflammatory cytokine are synthesised in distinct brain macrophage cells during innate activation.

Brain macrophages are known to exert dual and opposing functions on neuronal survival, which can be either beneficial or detrimental. The rationale of our study is that this duality could arise from an exclusive secretion of either pro- or anti-inflammatory cytokine by distinct cell subsets, cytokines that could respectively mediate neurotoxic or neurotrophic effects. Innate immune response was induced in macrophage cultures prepared from embryonic-day-16 to postnatal-day-8 mouse brains. By immunofluorescent detection of intracellular cytokines, we have assessed the occurrence of TNFalpha or IL10 synthesis at single cell level and observed distinct secretory patterns that include cells producing exclusively TNFalpha or IL10, cells producing both cytokines and non-producer cells. These secretory patterns are differentially regulated by MAP-kinase inhibitors. Altogether, these results demonstrate that synthesis of either a pro- or an anti-inflammatory cytokine can segregate distinct brain macrophages and suggests a functional cell-subset-specialisation.

Increased interaction of connexin43 with zonula occludens-1 during inhibition of gap junctions by G protein-coupled receptor agonists.

Astrocytes are extensively coupled through gap junctions (GJs) that are composed of channels mostly constituted by connexin43 (Cx43). This astroglial gap junctional intercellular communication (GJIC) allows propagation of ions and signaling molecules critical for neuronal activity and survival. It is drastically inhibited by a short-term exposure to endothelin-1 (ET-1) or to sphingosine-1-phosphate (S1P), both compounds being inflammatory mediators acting through activation of GTP-binding protein-coupled receptors (GPCRs). Previously, we have identified the GTPases G(i/o) and Rho as key actors in the process of S1P-induced inhibition. Here, we asked whether similar mechanisms underlied the effects of ET-1 and S1P by investigating changes in the phosphorylation status of Cx43 and in the molecular associations of Cx43 with zonula occludens (ZO) proteins and occludin. We showed that the inhibitory effect of ET-1 on GJIC was entirely dependent on the activation of G(i/o) but not on Rho and Rho-associated kinase. Both ET-1 and S1P induced dephosphorylation of Cx43 located at GJs through a process mediated by G(i/o) and calcineurin. Thanks to co-immunoprecipitation approaches, we found that a population of Cx43 (likely junctional Cx43) was associated to ZO-1-ZO-2-occludin multiprotein complexes and that acute treatments of astrocytes with ET-1 or S1P induced a G(i/o)-dependent increase in the amount of Cx43 linked to these complexes. As a whole, this study identifies a new mechanism of GJIC regulation in which two GPCR agonists dynamically alter interactions of Cx43 with its molecular partners.

Bmcc1s, a novel brain-isoform of Bmcc1, affects cell morphology by regulating MAP6/STOP functions.

The BCH (BNIP2 and Cdc42GAP Homology) domain-containing protein Bmcc1/Prune2 is highly enriched in the brain and is involved in the regulation of cytoskeleton dynamics and cell survival. However, the molecular mechanisms accounting for these functions are poorly defined. Here, we have identified Bmcc1s, a novel isoform of Bmcc1 predominantly expressed in the mouse brain. In primary cultures of astrocytes and neurons, Bmcc1s localized on intermediate filaments and microtubules and interacted directly with MAP6/STOP, a microtubule-binding protein responsible for microtubule cold stability. Bmcc1s overexpression inhibited MAP6-induced microtubule cold stability by displacing MAP6 away from microtubules. It also resulted in the formation of membrane protrusions for which MAP6 was a necessary cofactor of Bmcc1s. This study identifies Bmcc1s as a new MAP6 interacting protein able to modulate MAP6-induced microtubule cold stability. Moreover, it illustrates a novel mechanism by which Bmcc1 regulates cell morphology.

Albumin stimulates monocyte chemotactic protein-1 expression in rat embryonic mixed brain cells.

Albumin, a blood protein absent from the adult brain in physiological situations, can be brought into contact with brain cells during development or, in adult, following breakdown of the blood-brain barrier occurring as a result of local inflammation. In the present study, we show that ovalbumin and albumin induce the release of monocyte chemotactic protein 1 (MCP-1/CCL2) from rat embryonic mixed brain cells. A short-term exposure to ovalbumin during the cell dissociation procedure is sufficient to generate MCP-1 mRNA. A comparable effect is observed when the cells are incubated for 4 hr with ovalbumin or rat albumin, while MCP-1 messengers are barely detectable following bovine albumin exposure. The amount of MCP-1 protein measured in 4 hr-supernatants of albumin-treated cells followed the same albumin-inducing pattern as that of MCP-1 mRNA, while all albumins tested induced MCP-1 protein after a 17 hr-incubation period. The albumin-induced MCP-1 production is significantly inhibited in calphostin C-treated cells, suggesting the implication of a protein kinase C-dependent signaling pathway. This MCP-1-inducing activity is maintained after a lipid extraction procedure but abolished by proteinase K or trypsin treatments of albumin. The MCP-1 secretion following albumin contact with nervous cells could thus interfere, by chemotactic gradient formation, with the brain infiltration program of blood-derived cells during development or brain injury.