Epilepsies are characterized by recurrent seizures, which disrupt normal brain function. Alterations in neuronal excitability and excitation-inhibition balance have been shown to promote seizure generation, yet molecular determinants of such alterations remain to be identified. Pannexin channels are nonselective, large-pore channels mediating extracellular exchange of neuroactive molecules. Recent data suggest that these channels are activated under pathological conditions and regulate neuronal excitability. However, whether pannexin channels sustain or counteract chronic epilepsy in human patients remains unknown. We studied the impact of pannexin-1 channel activation in postoperative human tissue samples from patients with epilepsy displaying epileptic activity ex vivo. These samples were obtained from surgical resection of epileptogenic zones in patients suffering from lesional or drug-resistant epilepsy. We found that pannexin-1 channel activation promoted seizure generation and maintenance through adenosine triphosphate signaling via purinergic 2 receptors. Pharmacological inhibition of pannexin-1 channels with probenecid or mefloquine-two medications currently used for treating gout and malaria, respectively-blocked ictal discharges in human cortical brain tissue slices. Genetic deletion of pannexin-1 channels in mice had anticonvulsant effects when the mice were exposed to kainic acid, a model of temporal lobe epilepsy. Our data suggest a proepileptic role of pannexin-1 channels in chronic epilepsy in human patients and that pannexin-1 channel inhibition might represent an alternative therapeutic strategy for treating lesional and drug-resistant epilepsies.
Summary Local translation is a conserved mechanism conferring cells the ability to quickly respond to local stimuli. In the brain, it has been recently reported in astrocytes, whose fine processes contact blood vessels and synapses. Yet the specificity and regulation of astrocyte local translation remain unknown. We study hippocampal perisynaptic astrocytic processes (PAPs) and show that they contain the machinery for translation. Using a refined immunoprecipitation technique, we characterize the entire pool of ribosome-bound mRNAs in PAPs and compare it with the one expressed in the whole astrocyte. We find that a specific pool of mRNAs is highly polarized at the synaptic interface. These transcripts encode an unexpected molecular repertoire, composed of proteins involved in iron homeostasis, translation, cell cycle, and cytoskeleton. Remarkably, we observe alterations in global RNA distribution and ribosome-bound status of some PAP-enriched transcripts after fear conditioning, indicating the role of astrocytic local translation in memory and learning.
Brain postnatal development is characterized by critical periods of experience-dependent remodeling of neuronal circuits. Failure to end these periods results in neurodevelopmental disorders. The cellular processes defining critical-period timing remain unclear. Here, we show that in the mouse visual cortex, astrocytes control critical-period closure. We uncover the underlying pathway, which involves astrocytic regulation of the extracellular matrix, allowing interneuron maturation. Unconventional astrocyte connexin signaling hinders expression of extracellular matrix–degrading enzyme matrix metalloproteinase 9 (MMP9) through RhoA–guanosine triphosphatase activation. Thus, astrocytes not only influence the activity of single synapses but also are key elements in the experience-dependent wiring of brain circuits.
Presynaptic glutamate replenishment is fundamental to brain function. In high activity regimes, such as epileptic episodes, this process is thought to rely on the glutamate-glutamine cycle between neurons and astrocytes. However the presence of an astroglial glutamine supply, as well as its functional relevance in vivo in the healthy brain remain controversial, partly due to a lack of tools that can directly examine glutamine transfer. Here, we generated a fluorescent probe that tracks glutamine in live cells, which provides direct visual evidence of an activity-dependent glutamine supply from astroglial networks to presynaptic structures under physiological conditions. This mobilization is mediated by connexin43, an astroglial protein with both gap-junction and hemichannel functions, and is essential for synaptic transmission and object recognition memory. Our findings uncover an indispensable recruitment of astroglial glutamine in physiological synaptic activity and memory via an unconventional pathway, thus providing an astrocyte basis for cognitive processes.
Epilepsy is a neurological condition that affects 1% of the world population. Conventional treatments of epilepsy use drugs targeting neuronal excitability, inhibitory or excitatory transmission. Yet, one third of patients presents an intractable form of epilepsy and fails to respond to pharmacological anti-epileptic strategies. The ketogenic diet is a well-established non-pharmacological treatment that has been proven to be effective in reducing seizure frequency in the pharmaco-resistant patients. This dietary solution is however extremely restrictive and can be associated with complications caused by the high [fat]:[carbohydrate + protein] ratio. Recent advances suggest that the traditional 4:1 ratio of the ketogenic diet is not a requisite for its therapeutic effect. We show here that combining nutritional strategies targeting specific amino-acids, carbohydrates and fatty acids with a low [fat]:[proteins + carbohydrates] ratio also reduces excitatory drive and protects against seizures to the same extent as the ketogenic diet. Similarly, the morphological and molecular correlates of temporal lobe seizures were reduced in animals fed with the combined diet. These results provide evidence that low-fat dietary strategies more palatable than the ketogenic diet could be useful in epilepsy.
Brain postnatal development is characterized by critical periods of experience-dependent remodeling. Termination of these periods of intense plasticity is associated with settling of neuronal circuits, allowing for efficient information processing. Failure to end critical periods thus results in neurodevelopmental disorders. Yet, the cellular processes defining the timing of these developmental periods remain unclear. Here we show in the mouse visual cortex that astrocytes control the closure of the critical period. We uncover a novel underlying pathway involving regulation of the extracellular matrix that allows interneurons maturation via an unconventional astroglial connexin signaling. We find that timing of the critical period closure is controlled by a marked developmental upregulation of the astroglial protein connexin 30 that inhibits expression of the matrix degrading enzyme MMP9 through the RhoA-GTPase signaling pathway. Our results thus demonstrate that astrocytes not only influence activity and plasticity of single synapses, but are also key elements in the experience-dependent wiring of brain developing circuits. This work, by revealing that astrocytes promote the maturation of inhibitory circuits, hence provide a new cellular target to alleviate malfunctions associated to impaired closure of critical periods.
Local translation is a conserved molecular mechanism conferring cells the ability to quickly respond to local stimuli. It not only permits cells with complex morphology to bypass somatic protein synthesis and transport, but also contributes locally to the establishment of molecular and functional polarity. In the brain, local translation has been extensively studied in neurons and has only been recently reported in astrocytes, whose fine processes contact both blood vessels and synapses. Yet the specificity and regulation of astrocyte local translation remain unknown. Here, we studied hippocampal perisynaptic astrocytic processes (PAPs) and show that they contain all the machinery for translation. Using our recently refined polysome immunoprecipitation technique, we then characterized the pool of polysomal mRNAs in PAPs, referred to as the PAPome, and compared it to the one found in the whole astrocyte. We found that the PAPome encoded an unexpected molecular repertoire, mostly composed of cytoplasmic proteins and of proteins involved in iron homeostasis, translation, cell cycle and cytoskeleton. Among them, ezrin (Ezr), ferritin heavy chain 1 (Fth1) and 60S acidic ribosomal protein1 (Rplp1) were enriched in PAPs compared to perivascular astrocytic processes, indicating that local translation differs at these two interfaces. Remarkably, PAPs were also enriched in transcripts coding for proteins involved in learning and memory, such as ferritin (Ftl1 and Fth1), G1/S-specific cyclin-D2 (Ccnd2), E3 ubiquitin-protein ligase (Mdm2) , Receptor of activated protein C kinase 1 (Gnb2l1) and Elongation factor 1-alpha 1 (Eef1a1).To address their regulation in a physiological context, we assessed their local translation after fear conditioning. We found alterations in their density and/or distribution in astrocytes as well as a drop in their translation specifically in PAPs. In all, our results reveal an unexpected molecular repertoire of hippocampal PAPs, which is regulated by local translation during learning and memory processes.
Astroglial release of molecules is thought to actively modulate neuronal activity, but the nature, release pathway, and cellular targets of these neuroactive molecules are still unclear. Pannexin 1, expressed by neurons and astrocytes, form nonselective large pore channels that mediate extracellular exchange of molecules. The functional relevance of these channels has been mostly studied in brain tissues, without considering their specific role in different cell types, or in neurons. Thus, our knowledge of astroglial pannexin 1 regulation and its control of neuronal activity remains very limited, largely due to the lack of tools targeting these channels in a cell-specific way. We here show that astroglial pannexin 1 expression in mice is developmentally regulated and that its activation is activity-dependent. Using astrocyte-specific molecular tools, we found that astroglial-specific pannexin 1 channel activation, in contrast to pannexin 1 activation in all cell types, selectively and negatively regulates hippocampal networks, with their disruption inducing a drastic switch from bursts to paroxysmal activity. This decrease in neuronal excitability occurs via an unconventional astroglial mechanism whereby pannexin 1 channel activity drives purinergic signaling-mediated regulation of hyperpolarisation-activated cyclic nucleotide (HCN)-gated channels. Our findings suggest that astroglial pannexin 1 channel activation serves as a negative feedback mechanism crucial for the inhibition of hippocampal neuronal networks.
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