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

AbstractA 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 β 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

Glucocorticoid receptor in astrocytes regulates midbrain dopamine neurodegeneration through connexin hemichannel activity

The precise contribution of astrocytes in neuroinflammatory process occurring in Parkinson's disease (PD) is not well characterized. In this study, using GR(Cx30CreERT2) mice that are conditionally inactivated for glucocorticoid receptor (GR) in astrocytes, we have examined the actions of astrocytic GR during dopamine neuron (DN) degeneration triggered by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). The results show significantly augmented DN loss in GR(Cx30CreERT2) mutant mice in substantia nigra (SN) compared to controls. Hypertrophy of microglia but not of astrocytes was greatly enhanced in SN of these astrocytic GR mutants intoxicated with MPTP, indicating heightened microglial reactivity compared to similarly-treated control mice. In the SN of GR astrocyte mutants, specific inflammation-associated transcripts ICAM-1, TNF-alpha and Il-1 beta as well as TNF-alpha protein levels were significantly elevated after MPTP neurotoxicity compared to controls. Interestingly, this paralleled increased connexin hemichannel activity and elevated intracellular calcium levels in astrocytes examined in acute midbrain slices from control and mutant mice treated with MPP+. The increased connexin-43 hemichannel activity was found in vivo in MPTP-intoxicated mice. Importantly, treatment of MPTP-injected GR(Cx30CreERT2) mutant mice with TAT-Gap19 peptide, a specific connexin-43 hemichannel blocker, reverted both DN loss and microglial activation; in wild-type mice there was partial but significant survival effect. In the SN of postmortem PD patients, a significant decrease in the number of astrocytes expressing nuclear GR was observed, suggesting the participation of astrocytic GR deregulation of inflammatory process in PD. Overall, these data provide mechanistic insights into GR-modulated processes in vivo, specifically in astrocytes, that contribute to a pro-inflammatory state and dopamine neurodegeneration in PD pathology

Monitoring gap junctional communication in astrocytes from acute adult mouse brain slices using the gap-FRAP technique

International audienceIntercellular communication through gap junction channels plays a key role in cellular homeostasis and in synchronizing physiological functions, a feature that is modified in number of pathological situations. In the brain, astrocytes are the cell population that expresses the highest amount of gap junction proteins, named connexins. Several techniques have been used to assess the level of gap junctional communication in astrocytes, but so far they remain very difficult to apply in adult brain tissue. Here, using specific loading of astrocytes with sulforhodamine 101, we adapted the gap-FRAP (Fluorescence Recovery After Photobleaching) to acute hippocampal slices from 9 month-old adult mice. We show that gap junctional communication monitored in astrocytes with this technique was inhibited either by pharmacological treatment with a gap junctional blocker or in mice lacking the two main astroglial connexins, while a partial inhibition was measured when only one connexin was knocked-out. We validate this approach using a mathematical model of sulforhodamine 101 diffusion in an elementary astroglial network and a quantitative analysis of the exponential fits to the fluorescence recovery curves. Consequently, we consider that the adaptation of the gap-FRAP technique to acute brain slices from adult mice provides an easy going and valuable approach that allows overpassing this age-dependent obstacle and will facilitate the investigation of gap junctional communication in adult healthy or pathological brain

Neurons and brain macrophages regulate connexin expression in cultured astrocytes.

International audienceNeurons and brain macrophages (BM), respectively, increase and inhibit gap junctional communication (GJC) and connexin expression in cultured astrocytes. Thus, in brain diseases and injuries, neuronal death associated with the BM activation may decrease GJC in astrocytes and therefore have a physiopathological relevance

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

International audienceBrain 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

Contribution of Astroglial Cx43 Hemichannels to the Modulation of Glutamatergic Currents by D-Serine in the Mouse Prefrontal Cortex

International audienceAstrocytes interact dynamically with neurons by modifying synaptic activity and plasticity. This interplay occurs through a process named gliotransmission, meaning that neuroactive molecules are released by astrocytes. Acting as a gliotransmitter, D-serine, a co-agonist of the NMDA receptor at the glycine-binding site, can be released by astrocytes in a calcium [Ca2+]i-dependent manner. A typical feature of astrocytes is their high expression level of connexin43 (Cx43), a protein forming gap junction channels and hemichannels associated with dynamic neuroglial interactions. Pharmacological and genetic inhibition of Cx43 hemichannel activity reduced the amplitude of NMDA EPSCs in mouse layer 5 prefrontal cortex pyramidal neurons without affecting AMPA EPSC currents. This reduction of NMDA EPSCs was rescued by addition of D-serine in the extracellular medium. LTP of NMDA and AMPA EPSCs after high-frequency stimulation was reduced by prior inhibition of Cx43 hemichannel activity. Inactivation of D-serine synthesis within the astroglial network resulted in the reduction of NMDA EPSCs, which was rescued by adding extracellular D-serine. We showed that the activity of Cx43 hemichannels recorded in cultured astrocytes was [Ca2+]I dependent. Accordingly, in acute cortical slices, clamping [Ca2+]i at a low level in astroglial network resulted in an inhibition of NMDA EPSC potentiation that was rescued by adding extracellular D-serine. This work demonstrates that astroglial Cx43 hemichannel activity is associated with D-serine release. This process, occurring by direct permeation of D-serine through hemichannels or indirectly by Ca2+ entry and activation of other [Ca2+]i-dependent mechanisms results in the modulation of synaptic activity and plasticity.SIGNIFICANCE STATEMENT We recorded neuronal glutamatergic (NMDA and AMPA) responses in prefrontal cortex (PFC) neurons and used pharmacological and genetic interventions to block connexin-mediated hemichannel activity specifically in a glial cell population. For the first time in astrocytes, we demonstrated that hemichannel activity depends on the intracellular calcium concentration and is associated with D-serine release. Blocking hemichannel activity reduced the LTP of these excitatory synaptic currents triggered by high-frequency stimulation. These observations may be particularly relevant in the PFC, where D-serine and its converting enzyme are highly expressed

A c-Src Inhibitor Peptide Based on Connexin43 Exerts Neuroprotective Effects through the Inhibition of Glial Hemichannel Activity

The non-receptor tyrosine kinase c-Src is an important mediator in several signaling pathways related to neuroinflammation. Our previous study showed that cortical injection of kainic acid (KA) promoted a transient increase in c-Src activity in reactive astrocytes surrounding the neuronal lesion. As a cell-penetrating peptide based on connexin43 (Cx43), specifically TAT-Cx43266–283, inhibits Src activity, we investigated the effect of TAT-Cx43266–283 on neuronal death promoted by cortical KA injections in adult mice. As expected, KA promoted neuronal death, estimated by the reduction in NeuN-positive cells and reactive gliosis, characterized by the increase in glial fibrillary acidic protein (GFAP) expression. Interestingly, TAT-Cx43266–283 injected with KA diminished neuronal death and reactive gliosis compared to KA or KA+TAT injections. In order to gain insight into the neuroprotective mechanism, we used in vitro models. In primary cultured neurons, TAT-Cx43266–283 did not prevent neuronal death promoted by KA, but when neurons were grown on top of astrocytes, TAT-Cx43266–283 prevented neuronal death promoted by KA. These observations demonstrate the participation of astrocytes in the neuroprotective effect of TAT-Cx43266–283. Furthermore, the neuroprotective effect was also present in non-contact co-cultures, suggesting the contribution of soluble factors released by astrocytes. As glial hemichannel activity is associated with the release of several factors, such as ATP and glutamate, that cause neuronal death, we explored the participation of these channels on the neuroprotective effect of TAT-Cx43266–283. Our results confirmed that inhibitors of ATP and NMDA receptors prevented neuronal death in co-cultures treated with KA, suggesting the participation of astrocyte hemichannels in neurotoxicity. Furthermore, TAT-Cx43266–283 reduced hemichannel activity promoted by KA in neuron-astrocyte co-cultures as assessed by ethidium bromide (EtBr) uptake assay. In fact, TAT-Cx43266–283 and dasatinib, a potent c-Src inhibitor, strongly reduced the activation of astrocyte hemichannels. In conclusion, our results suggest that TAT-Cx43266–283 exerts a neuroprotective effect through the reduction of hemichannel activity likely mediated by c-Src in astrocytes. These data unveil a new role of c-Src in the regulation of Cx43-hemichannel activity that could be part of the mechanism by which astroglial c-Src participates in neuroinflammation