Membranes and Synaptosomes Used to Investigate Synaptic GABAergic Currents in Epileptic Patients.

Among the most prevalent neurological disorders, epilepsy affects about 1% of the population worldwide. We previously found, using human epileptic tissues, that GABAergic neurotransmission impairment is a key mechanism that drives the pathological phenomena that ultimately lead to generation and recurrence of seizures. Using both a "microtransplantation technique" and synaptosomes preparations from drug-resistant temporal lobe epilepsies (TLEs), we used the technique of two-electrode voltage clamp to record GABA-evoked currents, focusing selectively on the synaptic "fast inhibition" mediated by low-affinity GABAA receptors. Here, we report that the use-dependent GABA current desensitization (i.e., GABA rundown, which is evoked by applying to the cells consecutive pulses of GABA, at high concentration), which is a distinguishing mark of TLE, is mainly dependent on a dysfunction that affects synaptic GABAA receptors. In addition, using the same approaches, we recorded a depolarized GABA reversal potential in synaptosomes samples from the human epileptic subicula of TLE patients. These results, which confirm previous experiments using total membranes, suggest an altered chloride homeostasis in the synaptic area. Finally, the lack of a Zn2+ block of GABA-evoked currents using the synaptosomes supports the enrichment of "synaptic fast inhibitory" GABAA receptors in this preparation. Altogether, our findings suggest a pathophysiological role of low-affinity GABAA receptors at the synapse, especially during the fast and repetitive GABA release underlying recurrent seizures.

Immune-mediated myogenesis and acetylcholine receptor clustering promote a slow disease progression in ALS mouse models.

BACKGROUND: Amyotrophic lateral sclerosis (ALS) is a heterogeneous disease in terms of onset and progression rate. This may account for therapeutic clinical trial failure. Transgenic SOD1G93A mice on C57 or 129Sv background have a slow and fast disease progression rate, mimicking the variability observed in patients. Based on evidence inferring the active influence of skeletal muscle on ALS pathogenesis, we explored whether dysregulation in hindlimb skeletal muscle reflects the phenotypic difference between the two mouse models. METHODS: Ex vivo immunohistochemical, biochemical, and biomolecular methodologies, together with in vivo electrophysiology and in vitro approaches on primary cells, were used to afford a comparative and longitudinal analysis of gastrocnemius medialis between fast- and slow-progressing ALS mice. RESULTS: We reported that slow-progressing mice counteracted muscle denervation atrophy by increasing acetylcholine receptor clustering, enhancing evoked currents, and preserving compound muscle action potential. This matched with prompt and sustained myogenesis, likely triggered by an early inflammatory response switching the infiltrated macrophages towards a M2 pro-regenerative phenotype. Conversely, upon denervation, fast-progressing mice failed to promptly activate a compensatory muscle response, exhibiting a rapidly progressive deterioration of muscle force. CONCLUSIONS: Our findings further pinpoint the pivotal role of skeletal muscle in ALS, providing new insights into underestimated disease mechanisms occurring at the periphery and providing useful (diagnostic, prognostic, and mechanistic) information to facilitate the translation of cost-effective therapeutic strategies from the laboratory to the clinic.

GABAA receptor function is enhanced by Interleukin-10 in human epileptogenic gangliogliomas and its effect is counteracted by Interleukin-1ß.

Gangliogliomas (GGs) are low-grade brain tumours that cause intractable focal epilepsy in children and adults. In GG, as in epileptogenic focal malformations (i.e., tuberous sclerosis complex, TSC), there is evidence of sustained neuroinflammation with involvement of the pro-inflammatory cytokine IL-1ß. On the other hand, anti-inflammatory mediators are less studied but bear relevance for understanding seizure mechanisms. Therefore, we investigated the effect of the key anti-inflammatory cytokine IL-10 on GABAergic neurotransmission in GG. We assessed the IL-10 dependent signaling by transcriptomic analysis, immunohistochemistry and performed voltage-clamp recordings on Xenopus oocytes microtransplanted with cell membranes from brain specimens, to overcome the limited availability of acute GG slices. We report that IL-10-related mRNAs were up-regulated in GG and slightly in TSC. Moreover, we found IL-10 receptors are expressed by neurons and astroglia. Furthermore, GABA currents were potentiated significantly by IL-10 in GG. This effect was time and dose-dependent and inhibited by blockade of IL-10 signaling. Notably, in the same tissue, IL-1ß reduced GABA current amplitude and prevented the IL-10 effect. These results suggest that in epileptogenic tissue, pro-inflammatory mechanisms of hyperexcitability prevail over key anti-inflammatory pathways enhancing GABAergic inhibition. Hence, boosting the effects of specific anti-inflammatory molecules could resolve inflammation and reduce intractable seizures.

Unexpected Effect of IL-1ß on the Function of GABAA Receptors in Pediatric Focal Cortical Dysplasia.

Focal cortical dysplasia (FCD) type II is an epileptogenic malformation of the neocortex, as well as a leading cause of drug-resistant focal epilepsy in children and young adults. The synaptic dysfunctions leading to intractable seizures in this disease appear to have a tight relationship with the immaturity of GABAergic neurotransmission. The likely outcome would include hyperpolarizing responses upon activation of GABAARs. In addition, it is well-established that neuroinflammation plays a relevant role in the pathogenesis of FCD type II. Here, we investigated whether IL-1ß, a prototypical pro-inflammatory cytokine, can influence GABAergic neurotransmission in FCD brain tissues. To this purpose, we carried out electrophysiological recordings on Xenopus oocytes transplanted with human tissues and performed a transcriptomics analysis. We found that IL-1ß decreases the GABA currents amplitude in tissue samples from adult individuals, while it potentiates GABA responses in samples from pediatric cases. Interestingly, these cases of pediatric FCD were characterized by a more depolarized EGABA and an altered transcriptomics profile, that revealed an up-regulation of chloride cotransporter NKCC1 and IL-1ß. Altogether, these results suggest that the neuroinflammatory processes and altered chloride homeostasis can contribute together to increase the brain excitability underlying the occurrence of seizures in these children.

Dissecting the Molecular Determinants of GABAA Receptors Current Rundown, a Hallmark of Refractory Human Epilepsy.

GABAA receptors-(Rs) are fundamental for the maintenance of an efficient inhibitory function in the central nervous system (CNS). Their dysfunction is associated with a wide range of CNS disorders, many of which characterized by seizures and epilepsy. Recently, an increased use-dependent desensitization due to a repetitive GABA stimulation (GABAA current rundown) of GABAARs has been associated with drug-resistant temporal lobe epilepsy (TLE). Here, we aimed to investigate the molecular determinants of GABAA current rundown with two different heterologous expression systems (Xenopus oocytes and human embryonic kidney cells; HEK) which allowed us to manipulate receptor stoichiometry and to study the GABAA current rundown on different GABAAR configurations. To this purpose, we performed electrophysiology experiments using two-electrode voltage clamp in oocytes and confirming part of our results in HEK. We found that different degrees of GABAA current rundown can be associated with the expression of different GABAAR ß-subunits reaching the maximum current decrease when functional α1ß2 receptors are expressed. Furthermore, the blockade of phosphatases can prevent the current rundown observed in α1ß2 GABAARs. Since GABAAR represents one important therapeutic target in the treatment of human epilepsy, our results could open new perspectives on the therapeutic management of drug-resistant patients showing a GABAergic impairment.

Phytocannabinoids in Neurological Diseases: Could They Restore a Physiological GABAergic Transmission?

γ-Aminobutyric acid type A receptors (GABAARs) are the main inhibitory mediators in the central nervous system (CNS). GABAARs are pentameric ligand gated ion channels, and the main subunit composition is usually 2α2ßγ, with various isotypes assembled within a set of 19 different subunits. The inhibitory function is mediated by chloride ion movement across the GABAARs, activated by synaptic GABA release, reducing neuronal excitability in the adult CNS. Several studies highlighted the importance of GABA-mediated transmission during neuro-development, and its involvement in different neurological and neurodevelopmental diseases, from anxiety to epilepsy. However, while it is well known how different classes of drugs are able to modulate the GABAARs function (benzodiazepines, barbiturates, neurosteroids, alcohol), up to now little is known about GABAARs and cannabinoids interaction in the CNS. Endocannabinoids and phytocannabinoids are lately emerging as a new class of promising drugs for a wide range of neurological conditions, but their safety as medication, and their mechanisms of action are still to be fully elucidated. In this review, we will focus our attention on two of the most promising molecules (Δ9-tetrahydrocannabinol; Δ9-THC and cannabidiol; CBD) of this new class of drugs and their possible mechanism of action on GABAARs.