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Brain metastasis represents a substantial source of morbidity and mortality in various cancers, and is characterized by high resistance to chemotherapy. Here we define the role of the most abundant cell type in the brain, the astrocyte, in promoting brain metastasis. We show that human and mouse breast and lung cancer cells express protocadherin 7 (PCDH7), which promotes the assembly of carcinoma–astrocyte gap junctions composed of connexin 43 (Cx43). Once engaged with the astrocyte gap-junctional network, brain metastatic cancer cells use these channels to transfer the second messenger cGAMP to astrocytes, activating the STING pathway and production of inflammatory cytokines such as interferon-α (IFNα) and tumour necrosis factor (TNF). As paracrine signals, these factors activate the STAT1 and NF-κB pathways in brain metastatic cells, thereby supporting tumour growth and chemoresistance. The orally bioavailable modulators of gap junctions meclofenamate and tonabersat break this paracrine loop, and we provide proof-of-principle that these drugs could be used to treat established brain metastasis. A heterotypic cell interaction between astrocytes and tumour cells colonizing the brain is discovered; by establishing gap junctions, tumour cells trigger the activation of innate immune response signalling in astrocytes, which results in the secretion of factors that support growth and chemoresistance in brain metastatic cells. Metastasis reduced by gap junction inhibitors The development of novel therapeutic approaches to brain metastases has been hampered by a lack of mechanistic insights. These authors report that invasive breast and lung cancer cells engage the normally protective network of brain astrocytes to support metastases. By establishing gap junctions, tumour cells trigger the activation of innate immune response signalling in astrocytes, which then secrete factors that support metastatic growth and chemoresistance. The gap junction inhibitors meclofenamate and tonabersat interfere with this paracrine loop and impair the growth of experimental brain metastases, suggesting possible clinical relevance.

The connexin family of channel-forming proteins is present in every tissue type in the human anatomy. Connexins are best known for forming clustered intercellular channels, structurally known as gap junctions, where they serve to exchange members of the metabolome between adjacent cells. In their single-membrane hemichannel form, connexins can act as conduits for the passage of small molecules in autocrine and paracrine signalling. Here, we review the roles of connexins in health and disease, focusing on the potential of connexins as therapeutic targets in acquired and inherited diseases as well as wound repair, while highlighting the associated clinical challenges.

Collective cell responses to exogenous cues depend on cell–cell interactions. In principle, these can result in enhanced sensitivity to weak and noisy stimuli. However, this has not yet been shown experimentally, and little is known about how multicellular signal processing modulates single-cell sensitivity to extracellular signaling inputs, including those guiding complex changes in the tissue form and function. Here we explored whether cell–cell communication can enhance the ability of cell ensembles to sense and respond to weak gradients of chemotactic cues. Using a combination of experiments with mammary epithelial cells and mathematical modeling, we find that multicellular sensing enables detection of and response to shallow epidermal growth factor (EGF) gradients that are undetectable by single cells. However, the advantage of this type of gradient sensing is limited by the noisiness of the signaling relay, necessary to integrate spatially distributed ligand concentration information. We calculate the fundamental sensory limits imposed by this communication noise and combine them with the experimental data to estimate the effective size of multicellular sensory groups involved in gradient sensing. Functional experiments strongly implicated intercellular communication through gap junctions and calcium release from intracellular stores as mediators of collective gradient sensing. The resulting integrative analysis provides a framework for understanding the advantages and limitations of sensory information processing by relays of chemically coupled cells.

Paclitaxel is a widely used chemotherapy drug, but development of resistance leads to treatment failure. Tumor cells that are treated with a sublethal dose of paclitaxel for a long period of time show the epithelial‐mesenchymal transition (EMT) phenotype, which leads to metastasis and resistance. All‐trans retinoic acid (ATRA) is always used in combination with paclitaxel and can reverse EMT in many types of cancer cells. The ability of ATRA to reverse EMT in chemoresistant cells is still unknown. In the present study, the ability of ATRA to reverse EMT in paclitaxel‐resistant cells was investigated. Three colorectal cancer cell lines, HCT116, LoVo and CT26, were treated with sublethal doses of paclitaxel to create resistant cell lines. Western blotting, immunocytochemistry, and “parachute” dye‐coupling assays showed that ATRA reverses EMT, inhibits nuclear factor kappa B (NF‐κΒ), and upregulates gap junctions in paclitaxel‐resistant cells. Scratch wound‐healing and Transwell assays showed that ATRA decreases the migration and invasion abilities of paclitaxel‐resistant cells. In addition, the CT26 cell line was used in the Balb/c pulmonary metastasis model to show that ATRA reduces metastasis of paclitaxel‐resistant cells in vivo. Given these data, ATRA may reverse EMT by inhibiting NF‐κΒ and upregulating gap junctions in paclitaxel‐resistant cells. In this study, the ability of ATRA to reverse EMT in paclitaxel‐resistant cells was investigated. In vivo and in vitro results showed that ATRA may reverse the EMT by inhibiting NF‐κΒ and upregulating gap junctions in paclitaxel‐resistant cells.

Growing evidence has implicated glial anomalies in the pathophysiology of major depression disorder (MDD). Gap junctional communication is a main determinant of astrocytic function. However, it is unclear whether gap junction dysfunction is involved in MDD development. This study investigates changes in the function of astrocyte gap junction occurring in the rat prefrontal cortex (PFC) after chronic unpredictable stress (CUS), a rodent model of depression. Animals exposed to CUS and showing behavioral deficits in sucrose preference test (SPT) and novelty suppressed feeding test (NSFT) exhibited significant decreases in diffusion of gap junction channel-permeable dye and expression of connexin 43 (Cx43), a major component of astrocyte gap junction, and abnormal gap junctional ultrastructure in the PFC. Furthermore, we analyzed the effects of typical antidepressants fluoxetine and duloxetine and glucocorticoid receptor (GR) antagonist mifepristone on CUS-induced gap junctional dysfunction and depressive-like behaviors. The cellular and behavioral alterations induced by CUS were reversed and/or blocked by treatment with typical antidepressants or mifepristone, indicating that the mechanism of their antidepressant action may involve the amelioration of gap junction dysfunction and the cellular changes may be related to GR activation. We then investigated the effects of pharmacological gap junction blockade in the PFC on depressive-like behaviors. The results demonstrate that carbenoxolone (CBX) infusions induced anhedonia in SPT, and anxiety in NSFT, and Cx43 mimetic peptides Gap27 and Gap26 also induced anhedonia, a core symptom of depression. Together, this study supports the hypothesis that gap junction dysfunction contributes to the pathophysiology of depression.

Small molecules are important tools to measure and modulate intracellular signaling pathways. A longstanding limitation for using chemical compounds in complex tissues has been the inability to target bioactive small molecules to a specific cell class. Here, we describe a generalizable esterase–ester pair capable of targeted delivery of small molecules to living cells and tissue with cellular specificity. We used fluorogenic molecules to rapidly identify a small ester masking motif that is stable to endogenous esterases, but is efficiently removed by an exogenous esterase. This strategy allows facile targeting of dyes and drugs in complex biological environments to label specific cell types, illuminate gap junction connectivity, and pharmacologically perturb distinct subsets of cells. We expect this approach to have general utility for the specific delivery of many small molecules to defined cellular populations.

Gap junctions (GJs) are intercellular junctions that allow the direct transfer of ions and small molecules between neighboring cells, and GJs between astrocytes play an important role in the development of various pathologies of the brain, including regulation of the pathological neuronal synchronization underlying epileptic seizures. Recently, we found that a pathological change is observed in astrocytes during the ictal and interictal phases of 4-aminopyridin (4-AP)-elicited epileptic activity in vitro, which was correlated with neuronal synchronization and extracellular epileptic electrical activity. This finding raises the question: Does this signal depend on GJs between astrocytes? In this study we investigated the effect of the GJ blocker, carbenoxolone (CBX), on epileptic activity in vitro and in vivo. Based on the results obtained, we came to the conclusion that the astrocytic syncytium formed by GJ-associated astrocytes, which is responsible for the regulation of potassium, affects the formation of epileptic activity in astrocytes in vitro and epileptic seizure onset. This effect is probably an important, but not the only, mechanism by which CBX suppresses epileptic activity. It is likely that the mechanisms of selective inhibition of GJs between astrocytes will show important translational benefits in anti-epileptic therapies.

In the current study, we investigated the effects of two legacy per- and polyfluoroalkyl substances (PFASs) namely perfluorooctanoic acid (PFOA) and perfluorooctane sulfonate (PFOS) on osteogenesis. The alterations of connexin 43 (Cx43)-mediated gap junctions (GJs) were further explored as a potential mechanism. The two cell models (C3H10T1/2 and MC3T3-E1 cells) differentiated into osteoblasts (OBs) were utilized, and treated with PFOA and PFOS at the doses of 0.25, 2.5, 25, and 75 μM. Real-time PCR and Western blot were applied to assess the mRNA and protein expression of osteogenic-specific markers and Cx43. ALP staining and ARS staining were used to evaluate the osteogenesis process. The scrape-loading dye transfer assay was performed to assess the GJ-mediated intercellular coupling. To investigate the role of gap-junctional intercellular communication (GJIC) in the PFAS-induced osteogenic inhibition, the Cx43-specific GJIC enhancer, rotigaptide (ZP123), was added into the differentiation medium of C3H10T1/2 cells. After the exposure of PFOA and PFOS, the osteogenic molecules were down-regulated and the calcium deposition was reduced in the two cell models, indicating the inhibitory effects of the legacy PFASs. The Cx43 expression and GJIC activity were significantly suppressed, and the usage of ZP123 rescued the adverse impact on osteogenesis, suggesting the remarkable role of GJIC herein.

Our understanding of the active role that astrocytes play in modulating neuronal function and behavior is rapidly expanding, but little is known about the role that astrocytes may play in drug-seeking behavior for commonly abused substances. Given that the nucleus accumbens is critically involved in substance abuse and motivation, we sought to determine whether nucleus accumbens astrocytes influence the motivation to self-administer ethanol following abstinence. We found that the packing density of astrocytes that were expressing glial fibrillary acidic protein increased in the nucleus accumbens core (NAcore) during abstinence from EtOH self-administration. No change was observed in the nucleus accumbens shell. This increased NAcore astrocyte density positively correlated with the motivation for ethanol. Astrocytes can communicate with one another and influence neuronal activity through gap-junction hemichannels. Because of this, the effect of blocking gap-junction hemichannels on the motivation for ethanol was examined. The motivation to self-administer ethanol after 3 weeks abstinence was increased following microinjection of gap-junction hemichannel blockers into the NAcore at doses that block both neuronal and astrocytic channels. In contrast, no effect was observed following microinjection of doses that are not thought to block astrocytic channels or following microinjection of either dose into the nucleus accumbens shell. Additionally, the motivation for sucrose after 3 weeks abstinence was unaffected by NAcore gap-junction hemichannel blockers. Next, Designer Receptors Exclusively Activated by Designer Drugs (DREADDs) were selectively expressed in NAcore astrocytes to test the effect of astrocyte stimulation. DREADD activation increased cytosolic calcium in primary astrocytes, facilitated responding for rewarding brain stimulation, and reduced the motivation for ethanol after 3 weeks abstinence. This is the first work to modulate drug-seeking behavior with astrocyte-specific DREADDs. Taken together, our findings demonstrate that NAcore astrocytes can shape the motivation to self-administer ethanol; suggesting that the development of ligands which selectively stimulate astrocytes may be a successful strategy to abate ethanol-seeking behavior.

There is increasing evidence that exposure to environmental toxicants may impact fertility, especially during critical windows of reproductive axis development. Hypothalamic gonadotropin-releasing hormone (GnRH) neurons, essential for puberty onset and fertility, originate from the olfactory placode and migrate toward the hypothalamus during development, making them particularly vulnerable to environmental insults. Cadmium (Cd), a widespread heavy metal, is well known for its gonadotoxicity, but its impact on human hypothalamic neuron development remains unclear. Using human fetal GnRH neuroblasts (FNCB4) we investigated the effects of Cd exposure on their morpho-functional and developmental features. Cd induced oxidative stress and COX2 mRNA upregulation, indicative of inflammatory pathway activation, which was accompanied by reduced cell migration and downregulation of motility-related genes. These effects were associated with F-actin disassembly and altered expression of adhesion molecules. Electrophysiological analyses showed that Cd altered membrane potential, increased capacitance and permeability, and disrupted gap junctional communication, as also confirmed by connexin-43 delocalization. Moreover, Cd significantly reduced the expression of specific GnRH neuronal markers, suggesting impaired functional maturation. Overall, our findings provide the first evidence that Cd may interfere with mechanisms crucially involved in human GnRH neuron development, adding new mechanistic insights into the comprehension of how early-life exposure to Cd may contribute to fertility concerns.