The connexin43 mimetic peptide Gap19 inhibits hemichannels without altering gap junctional communication in astrocytes

In the brain, astrocytes represent the cellular population that expresses the highest amount of connexins (Cxs). This family of membrane proteins is the molecular constituent of gap junction channels and hemichannels that provide pathways for direct cytoplasm-to-cytoplasm and inside-out exchange, respectively. Both types of Cx channels are permeable to ions and small signaling molecules allowing astrocytes to establish dynamic interactions with neurons. So far, most pharmacological approaches currently available do not distinguish between these two channel functions, stressing the need to develop new specific molecular tools. In astrocytes two major Cxs are expressed, Cx43 and Cx30, and there is now evidence indicating that at least Cx43 operates as a gap junction channel as well as a hemichannel in these cells. Based on studies in primary cultures as well as in acute hippocampal slices, we report here that Gap 19, a nonapeptide derived from the cytoplasmic loop of Cx43, inhibits astroglial Cx43 hemichannels in a dose-dependent manner, without affecting gap junction channels. This peptide, which not only selectively inhibits hemichannels but is also specific for Cx43, can be delivered in vivo in mice as TAT-Gap19, and displays penetration into the brain parenchyma. As a result, Gap 19 combined with other tools opens up new avenues to decipher the role of Cx43 hemichannels in interactions between astrocytes and neurons in physiological as well as pathological situations

Connexin43 hemichannels in satellite glial cells, can they influence sensory neuron activity?

Indexación: Scopus.In this review article, we summarize the current insight on the role of Connexin- and Pannexin-based channels as modulators of sensory neurons. The somas of sensory neurons are located in sensory ganglia (i.e., trigeminal and nodose ganglia). It is well known that within sensory ganglia, sensory neurons do not form neither electrical nor chemical synapses. One of the reasons for this is that each soma is surrounded by glial cells, known as satellite glial cells (SGCs). Recent evidence shows that connexin43 (Cx43) hemichannels and probably pannexons located at SGCs have an important role in paracrine communication between glial cells and sensory neurons. This communication may be exerted via the release of bioactive molecules from SGCs and their subsequent action on receptors located at the soma of sensory neurons. The glio-neuronal communication seems to be relevant for the establishment of chronic pain, hyperalgesia and pathologies associated with tissue inflammation. Based on the current literature, it is possible to propose that Cx43 hemichannels expressed in SGCs could be a novel pharmacological target for treating chronic pain, which need to be directly evaluated in future studies.https://www.frontiersin.org/articles/10.3389/fnmol.2017.00374/ful

Role of Astrocyte Network in Edema after Juvenile Traumatic Brain Injury

Juvenile traumatic brain injury (jTBI) is the leading cause of death and disability in young children and adolescents. Despite its lasting detrimental effects on the developing brain, no pharmacological treatment exists. One of the pathological hallmarks of jTBI is edema. Astrocytes play a key role in the edema process, and have been hypothesized that numerous astrocyte networks allow communication and propagation of edema and secondary injury spread. Two key astrocyte proteins are hypothesized to have a central role in the edema process: Aquaporin 4 (AQP4) and Connexin 43 (Cx43). AQP4 is expressed extensively in astrocyte endfeet, which surrounds the blood vessels as part of the blood brain barrier (BBB). Cx43 is central in astrocyte to astrocyte connection and communication. We hypothesized that AQP4 acted as one of the potential passageway of water into the astrocyte, whereas Cx43 acted as the bridge between astrocytes once inside the brain. By blocking these strategically located pathways, we hypothesized that edema would decrease post-jTBI. In order to achieve specific inhibitions of APQ4 or Cx43, we utilized small interference RNA (siRNA), which is also an endogenous mechanism. We observed that after jTBI both AQP4 and Cx43 was significantly upregulated, edema was prominent, and reactive astrogliosis occurred. When siAQP4 was administered after jTBI, there was functional improvement, decreased edema, and decreased reactive astrogliosis. When siCx43 was administered, there was functional improvement and decreased reactive astrogliosis, but the level of edema did not change. From these findings, it can be seen that (1) AQP4 and Cx43 are upregulated acutely after jTBI, (2) both siAQP4 and siCx43 have therapeutic potentials after jTBI leading to functional recovery, (3) although both target astrocyte endfeet proteins, the mechanism of action seem to be different and AQP4 may play a more direct role in the edema process than Cx43. Future studies could focus on (1) a more clinically relevant delivery of siRNA for jTBI, (2) elucidating the mechanism behind functional improvement of siCx43, and (3) the relationship between AQP4 and Cx43 regarding astrocyte pathology after jTBI

Inhibition of Connexin43 hemichannels impairs spatial short-term memory without affecting spatial working memory

Astrocytes are active players in higher brain function as they can release gliotransmitters, which are essential for synaptic plasticity. Various mechanisms have been proposed for gliotransmission, including vesicular mechanisms as well as non-vesicular ones, for example by passive diffusion via connexin hemichannels (HCs). We here investigated whether interfering with connexin43 (Cx43) HCs influenced hippocampal spatial memory. We made use of the peptide Gap19 that blocks HCs but not gap junction channels and is specific for Cx43. To this end, we microinfused transactivator of transcription linked Gap19 (TAT-Gap19) into the brain ventricle of male NMRI mice and assessed spatial memory in a Y maze. We found that the in vivo blockade of Cx43 HCs did not affect the locomotor activity or spatial working memory in a spontaneous alternation Y maze task. Cx43 blockade did however significantly impair the spatial short-term memory in a delayed spontaneous alternation Y maze task. These results indicate that Cx43 HCs play a role in spatial short-term memory

Aktiini-välitteisten liikkuvuuden rooli ääreis-astrosyyttejen kehityskulkussa synaptisessa toiminnassa

Among other glial cell types such as microglia, oligodendrocytes and radial glia, astrocytes are known to be involved in brain function; metabolically supporting neurons, regulating blood flow dynamics, participating in the development of pathological states, sensing and modulating synaptic activity. At the same time the complex astrocytic morphology, with a number of highly ramified peripheral processes located near the synaptic terminals, suggests them as a possible source for morpho-functional plasticity in the brain. This thesis summarizes the work on the in vitro development and further in vivo implementation, using a gene delivery system, of a tool for suppressing activity-dependent astrocytic motility. Calciuminduced astrocyte process outgrowth and its dependence on Profilin-1, novel in vivo gene delivery approaches, a demonstration of astrocytic motility in vivo and the independence of visual processing from astrocytic motility rates are the main findings of the project. The results described in this work increase our understanding of the interactions occurring between astrocytes and neurons as well as the consequences for brain function.Glia-soluihin kuuluvat astrosyytit ovat tiedettävästi osa aivojen toimintaa. Astrosyytit tukevat metabolisesti neuroneita, säätelevät verenkierron dynamiikkaa, ovat hyvin läsnä patoloogisten tilojen kehittymisessä, mukana aistimassa ja muuntamassa synaptista aktiivisuutta. Samanaikaisesti astrosyyttejen kompleksi morfologia, läheinen sijainti synaptisien terminalejen kanssa viittaavat mahdolliseen morfo-funtionaaliseen muovautumiseen aivoissa. Tämä väitöskirja keskittyy töihin jotka kehittävät in vitro ja in vivo tekniikoita jolla voidaan käyttää geeni jakelu menetelmää joka vaimentaa astrosyyttejen liikkuvuutta. Keskeiset havainnot tässä väitöskirjassa ovat astrosyyttejen kasliumin indusoima haarottuminen joka on riippuvainen Profiliini-1:stä ja havainnollistaminen astrosyyttejen liikkuvuudesta in vivo menetelmillä ja astrosyyttejen liikkuvuus aste joka on itsenäinen visuaalisesta prosessoinnista. Kuvaillut tulokset tässä väitöskirjassa kohentavat meidän ymmärrystä astrosyytetjen ja neuronejen välisestä kanssakäymisestä ja niiden seuraamus aivojen toiminnassa

Astrocytic Connexin43 channels as candidate targets in epilepsy treatment

In epilepsy research, emphasis is put on exploring non-neuronal targets such as astrocytic proteins, since many patients remain pharmacoresistant to current treatments, which almost all target neuronal mechanisms. This paper reviews available data on astrocytic connexin43 (Cx43) signaling in seizures and epilepsy. Cx43 is a widely expressed transmembrane protein and the constituent of gap junctions (GJs) and hemichannels (HCs), allowing intercellular and extracellular communication, respectively. A plethora of research papers show altered Cx43 mRNA levels, protein expression, phosphorylation state, distribution and/or functional coupling in human epileptic tissue and experimental models. Human Cx43 mutations are linked to seizures as well, as 30% of patients with oculodentodigital dysplasia (ODDD), a rare genetic condition caused by mutations in the GJA1 gene coding for Cx43 protein, exhibit neurological symptoms including seizures. Cx30/Cx43 double knock-out mice show increased susceptibility to evoked epileptiform events in brain slices due to impaired GJ-mediated redistribution of K+ and glutamate and display a higher frequency of spontaneous generalized chronic seizures in an epilepsy model. Contradictory, Cx30/Cx43 GJs can traffic nutrients to high-energy demanding neurons and initiate astrocytic Ca2+ waves and hyper synchronization, thereby supporting proconvulsant effects. The general connexin channel blocker carbenoxolone and blockers from the fenamate family diminish epileptiform activity in vitro and improve seizure outcome in vivo. In addition, interventions with more selective peptide inhibitors of HCs display anticonvulsant actions. To conclude, further studies aiming to disentangle distinct roles of HCs and GJs are necessary and tools specifically targeting Cx43 HCs may facilitate the search for novel epilepsy treatments

Characterization of panglial gap junction networks in the thalamus and hippocampus reveals glial heterogeneity

Increasing evidence over the past decades revealed the importance of glia in the brain. In this study, astrocytes in the ventrobasal thalamus were characterized in detail for the first time. Glial heterogeneity was investigated by comparing their properties with those of astrocytes in the hippocampus, a brain region in which glial cells have been widely studied. During development, astrocytes establish gap junction channels with each other. In this study, the networks in the ventrobasal thalamus increased in size until the end of the second postnatal week. Cx30 expression was strongly upregulated during postnatal development and had a predominating role in glial network formation in the mature thalamus. Cx30 and Cx43, but not Cx26, mediated gap junction coupling in the hippocampus and thalamus. Strikingly, some astrocytes were devoid of Cx43. Expression of Cx43 in RG-like cells influences proliferation. The effect of two Cx43 mutations on network sizes were studied to identify the key function, gap junction coupling or adhesion, for proliferation. The function remained elusive. In summary, connexin expression differs among brain regions revealing glial heterogeneity throughout the brain. Tracer diffusion from astrocytes into the myelin sheath was observed in the ventrobasal thalamus. Such functional gap junction channels between astrocytes and oligodendrocytes were so far unknown. In this study, a panglial network formed by oligodendrocytes and astrocytes was discovered in the hippocampus and the thalamus. Employing Cx30/47 DKO mice, a major impact of Cx30 on panglial network formation was observed in the thalamus. Cx30 and Cx47 do not form functional channelsin vitro. This leads to the conclusion that panglial coupling is mainly mediated by Cx30:Cx32 channels. Immunohistochemical analysis in PLP-GFP and Cx43-ECFP mice identified overlapping protein expression of "classical" markers in thalamic astrocytes and oligodendrocytes in contrast to hippocampal glia. S100β was the most reliable marker to distinguish astrocytes and oligodendrocytes in the ventrobasal thalamus. Unexpectedly, an "intermediate" cell-type was identified co-expressing Cx43 and Olig2. It is a mature cell-type which is part of thalamic glial networks and has been described for the first time in this study. The functional role of panglial networks in metabolite supply to neurons was analysed in the present study. Glucose diffusion from astrocytes to oligodendrocytes was demonstrated in the thalamus employing the fluorescent glucose analogue 2-NBDG in PLP-GFP mice with SR101 labelled astrocytes. For further investigation of glia-neuron interactions, a method to analyse neuronal field potentials in the ventrobasal thalamus was established and characterized. Extracellular glucose deprivation abolished neuronal postsynaptic field potentials, thereby confirming the neuronal requirement of glial energy supply. In addition to connexin mediated gap junction channels, the expression of ionotropic AMPA and GABAA receptors was studied in thalamic astrocytes. In the juvenile thalamus, two astrocyte populations were distinguished by the presence or absence of AMPA receptor expression. The GluA2 subunit was abundantly expressed when AMPA receptors were expressed. All astrocytes expressed GABAA receptors. They were devoid of the α3 subunit and rarely expressed the γ2 subunit required for synaptic GABAA receptor localization. Instead, all cells expressed the γ1 subunit. These data indicate that glial heterogeneity occurs even within a given brain regions. The present study describes panglial networks in grey matter and their distinct properties among brain regions for the first time. In addition, glial heterogeneity was observed between and within brain regions and enhanced our understanding of glial specializations. In addition, a new "intermediate" cell-type was discovered which is abundantly present in the ventrobasal thalamus

Targeting MAPK phosphorylation of Connexin43 provides neuroprotection in stroke

Connexin43 (Cx43) function is influenced by kinases that phosphorylate specific serine sites located near its C-terminus. Stroke is a powerful inducer of kinase activity, but its effect on Cx43 is unknown. We investigated the impact of wild-type (WT) and knock-in Cx43 with serine to alanine mutations at the protein kinase C (PKC) site Cx43(S368A), the casein kinase 1 (CK1) sites Cx43(S325A/328Y/330A), and the mitogen-activated protein kinase (MAPK) sites Cx43(S255/262/279/282A) (MK4) on a permanent middle cerebral artery occlusion (pMCAO) stroke model. We demonstrate that MK4 transgenic animals exhibit a significant decrease in infarct volume that was associated with improvement in behavioral performance. An increase in astrocyte reactivity with a concomitant decrease in microglial reactivity was observed in MK4 mice. In contrast to WT, MK4 astrocytes displayed reduced Cx43 hemichannel activity. Pharmacological blockade of Cx43 hemichannels with TAT-Gap19 also significantly decreased infarct volume in WT animals. This study provides novel molecular insights and charts new avenues for therapeutic intervention associated with Cx43 function

Small Interference RNA Targeting Connexin-43 Improves Motor Function and Limits Astrogliosis After Juvenile Traumatic Brain Injury

International audienceJuvenile traumatic brain injury (jTBI) is the leading cause of death and disability for children and adolescents worldwide, but there are no pharmacological treatments available. Aquaporin 4 (AQP4), an astrocytic perivascular protein, is increased after jTBI, and inhibition of its expression with small interference RNA mitigates edema formation and reduces the number of reactive astrocytes after jTBI. Due to the physical proximity of AQP4 and gap junctions, coregulation of AQP4 and connexin 43 (Cx43) expressions, and the possibility of water diffusion via gap junctions, we decided to address the potential role of astrocytic gap junctions in jTBI pathophysiology. We evaluated the role of Cx43 in the spread of the secondary injuries via the astrocyte network, such as edema formation associated with blood-brain barrier dysfunctions, astrogliosis, and behavioral outcome. We observed that Cx43 was altered after jTBI with increased expression in the perilesional cortex and in the hippocampus at several days post injury. In a second set of experiments, cortical injection of small interference RNA against Cx43 decreased Cx43 protein expression, improved motor function recovery, and decreased astrogliosis but did not result in differences in edema formation as measured via T2-weighted imaging or diffusion-weighted imaging at 1 day or 3 days. Based on our findings, we can speculate that while decreasing Cx43 has beneficial roles, it likely does not contribute to the spread of edema early after jTBI