Molecular basis for substrate recognition and transport of human GABA transporter GAT1.

γ-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in the central nervous system, is recycled through specific GABA transporters (GATs). GAT1, which is mainly expressed in the presynaptic terminals of axons, is a potential drug target of neurological disorders due to its essential role in GABA transport. Here we report four cryogenic electron microscopy structures of human GAT1, at resolutions of 2.2-3.2 Å. GAT1 in substrate-free form or in complex with the antiepileptic drug tiagabine exhibits an inward-open conformation. In the presence of GABA or nipecotic acid, inward-occluded structures are captured. The GABA-bound structure reveals an interaction network bridged by hydrogen bonds and ion coordination for GABA recognition. The substrate-free structure unwinds the last helical turn of transmembrane helix TM1a to release sodium ions and substrate. Complemented by structure-guided biochemical analyses, our studies reveal detailed mechanism of GABA recognition and transport, and elucidate mode of action of the inhibitors, nipecotic acid and tiagabine.

Tiagabine suppresses pentylenetetrazole-induced seizures in mice and improves behavioral and cognitive parameters by modulating BDNF/TrkB expression and neuroinflammatory markers.

Tiagabine (Tia), a new-generation antiseizure drug that mimics the GABAergic signaling by inhibiting GABA transporter type-1, is the least studied molecule in chronic epilepsy models with comorbid neurobehavioral and neuroinflammatory parameters. Therefore, the current study investigated the effects of Tia in a real-time manner on electroencephalographic (EEG) activity, behavioral manifestations and mRNA expression in pentylenetetrazole (PTZ)-kindled mice. Male BALB/c mice were treated with tiagabine (0.5, 1 and 2 mg/kg) for 21 days with simultaneous PTZ (40 mg/kg) injection every other day for a total of 11 injections and monitored for seizure progression with synchronized validation through EEG recordings from cortical electrodes. The post-kindling protection from anxiety and memory deficit was verified by a battery of behavioral experiments. Isolated brains were evaluated for oxidative alterations and real-time changes in mRNA expression for BDNF/TrkB, GAT-1 and GAT-3 as well as neuroinflammatory markers. Experimental results revealed that Tia at the dose of 2 mg/kg maximally inhibited the development of full bloom seizure and reduced epileptic spike discharges from the cortex. Furthermore, Tia dose-dependently exerted the anxiolytic effects and protected from PTZ-evoked cognitive impairment. Tia reduced lipid peroxidation and increased superoxide dismutase and glutathione levels in the brain via augmentation of GABAergic modulation. PTZ-induced upregulated BDNF/TrkB signaling and pro-inflammatory cytokines were mitigated by Tia with upregulation of GAT-1 and GAT-3 transporters in whole brains. In conclusion, the observed effects of Tia might have resulted from reduced oxidative stress, BDNF/TrkB modulation and mitigated neuroinflammatory markers expression leading to reduced epileptogenesis and improved epilepsy-related neuropsychiatric effects.

GABA transport goes structural.

The γ-aminobutyric acid transporter 1 (GAT1) is a transporter which clears the inhibitory neurotransmitter γ-aminobutyric acid (GABA) from the synaptic cleft. The paper by Motiwala et al. documents a structure of GAT1 in complex with the antiepileptic drug tiagabine. This study will enable structure-based docking of large chemical libraries for the discovery of novel antiepileptics.

Analysis of Different Binding Modes for Tiagabine within the GAT-1 Transporter.

The recently obtained cryo-electron microscopy structure (PDB code: 7SK2) of the human γ-aminobutyric acid transporter type 1 (hGAT-1) in complex with the antiepileptic drug, tiagabine, revealed a rather unexpected binding mode for this inhibitor in an inward-open state of the transporter. The simultaneously released crystal structures of the modified dopamine transporter with mutations mimicking hGAT-1 indicated an alternative binding mode for the tiagabine analogues that were found to block the transporter in an outward-open state, which is more consistent with the results of previous biological and molecular modeling studies. In view of the above discrepancies, our study compares different hypothetical tiagabine binding modes using classical and accelerated molecular dynamics simulations, as well as MM-GBSA free binding energy (dG) calculations. The results indicate that the most stable and energetically favorable binding mode of tiagabine is the one where the nipecotic acid fragment is located in the main binding site (S1) and the aromatic rings are arranged within the S2 site of the hGAT-1 transporter in an outward-open state, confirming the previous molecular modelling findings. The position of tiagabine bound to hGAT-1 in an inward-open state, partially within the intracellular release pathway, was significantly less stable and the dG values calculated for this complex were higher. Furthermore, analysis of the cryo-electron map for the 7SK2 structure shows that the model does not appear to fit into the map optimally at the ligand binding site. These findings suggest that the position of tiagabine found in the 7SK2 structure is rather ambiguous and requires further experimental verification. The identification of the main, high-affinity binding site for tiagabine and its analogues is crucial for the future rational design of the GABA transporter inhibitors.

Breastfeeding while on treatment with antiseizure medications: a systematic review from the ILAE Women Task Force

We carried out a systematic review of published information on transfer of antiseizure medications (ASMs) into breastmilk, ASM serum concentrations in breastfed infants, and the wellbeing of infants breastfed by mothers on ASM treatment. Information was extracted from 85 relevant articles. No data on ASM levels in breastmilk or in breastfed infants was identified for cannabidiol, cenobamate, clobazam, eslicarbazepine-acetate, everolimus, felbamate, fenfluramine, retigabine, rufinamide, stiripentol, tiagabine, and vigabatrin. For ASMs, with available information on levels in breastfed infants, very low concentrations (in the order of 10% or less of maternal serum concentrations) were reported for carbamazepine, gabapentin, levetiracetam, oxcarbazepine, phenytoin, valproate, and clonazepam. Slightly higher levels (up to approximately 30% of maternal serum concentrations) have been observed with lamotrigine and topiramate, and in single case reports for brivaracetam, lacosamide, and perampanel. High infant levels (30% up to 100% of maternal serum concentrations) have been reported with ethosuximide, phenobarbital and zonisamide. Adverse infant effects during breastfeeding by mothers on ASMs appear to be rare regardless of the type of ASM, but systematic study is limited. Prospective long-term follow-up studies of developmental outcomes among children who have been breastfed by mothers taking ASMs are sparse and have mainly involved children whose mothers were taking carbamazepine, lamotrigine, levetiracetam, phenytoin or valproate as monotherapy while breastfeeding. Although these studies have not indicated poorer outcome among breastfed children compared with those who were not breastfed, further data on long-term outcomes are needed to draw firm conclusions. It is concluded that breastfeeding should in general be encouraged in women taking ASMs, given the well-established benefits of breastfeeding with regard to both short- and long-term infant health in the general population. Counselling needs to be individualized including information on the current knowledge regarding the woman's specific ASM treatment.

Structural basis of GABA reuptake inhibition.

γ-Aminobutyric acid (GABA) transporter 1 (GAT1)1 regulates neuronal excitation of the central nervous system by clearing the synaptic cleft of the inhibitory neurotransmitter GABA upon its release from synaptic vesicles. Elevating the levels of GABA in the synaptic cleft, by inhibiting GABA reuptake transporters, is an established strategy to treat neurological disorders, such as epilepsy2. Here we determined the cryo-electron microscopy structure of full-length, wild-type human GAT1 in complex with its clinically used inhibitor tiagabine3, with an ordered part of only 60 kDa. Our structure reveals that tiagabine locks GAT1 in the inward-open conformation, by blocking the intracellular gate of the GABA release pathway, and thus suppresses neurotransmitter uptake. Our results provide insights into the mixed-type inhibition of GAT1 by tiagabine, which is an important anticonvulsant medication. Its pharmacodynamic profile, confirmed by our experimental data, suggests initial binding of tiagabine to the substrate-binding site in the outward-open conformation, whereas our structure presents the drug stalling the transporter in the inward-open conformation, consistent with a two-step mechanism of inhibition4. The presented structure of GAT1 gives crucial insights into the biology and pharmacology of this important neurotransmitter transporter and provides blueprints for the rational design of neuromodulators, as well as moving the boundaries of what is considered possible in single-particle cryo-electron microscopy of challenging membrane proteins.

Non-convulsive status epilepticus in the setting of cannabidiol adjunctive therapy

Anti-seizure medications (ASMs) can cause non-convulsive status epilepticus (NCSE), but account for less than 5% of all NCSE cases. We present a 63-year-old, right-handed male with a history of intractable focal epilepsy since age seven years old, whose bouts of NCSE were triggered by cannabidiol (CBD) adjunctive therapy. His most common seizure types included focal myoclonic or tonic seizures with vocalization, usually with awakening, which occurred on a monthly basis despite the combination of tiagabine, perampanel, levetiracetam, lacosamide and clonazepam. After CBD was initiated, he began to exhibit episodes of prolonged confusion, at times with myoclonic or tonic seizures. Increasing CBD doses led to more frequent and prolonged episodes. The confusional episodes occurred predominantly in the morning, with spontaneous resolution by the afternoon. During one of these episodes, he was hospitalized, and NCSE was confirmed by video-EEG monitoring. CBD was withdrawn and the patient had no further episodes of NCSE. While CBD can cause NCSE, the medication interaction between CBD and tiagabine also needs to be considered.

Nipecotic acid as potential lead molecule for the development of GABA uptake inhibitors; structural insights and design strategies.

Gamma-Aminobutyric Acid (GABA) inhibitory neurotransmitter departs an energetic role in brain signalling system. Levels of GABA in the brain influence human behaviour, diminishes in the degree of GABA can cause seizures, depression, Parkinson's. To put it plainly, it plays a basic part in the significant issues of mind. It is exceptionally important to cure the issues linked to GABA. Writing overview proposed that nipecotic acid is an intense GABA reuptake inhibitor. This scaffold is likewise present in one of the promoted anticonvulsant drugs 'Tiagabine'. Tiagabine is only drug in the market which works through this mechanism however the medication is regulated with one more prescription for the synergistic impact. Nipecotic acid has several disadvantages such as it can't cross the blood-brain barrier because of its hydrophilic and zwitterionic nature. To avoid this problem nipecotic acid scaffold hybrids with the different aromatic groups can enhance the physical (lipophilicity) as well as biological properties of the resultant compound. So, there is a dire requirement for compounds that work through this mechanism. Several medicinal chemists and researchers are already working in this field and developed outstanding newer molecules. This review article compiles these developed new hybrids along with design strategies, structure-activity relationship, and biological activity as well as in silico studies. This review also demonstrates the synthesis of nipecotic acid and the core mechanism through which nipecotic acid acts as a GABA reuptake inhibitor.

Antiepileptic Drug Tiagabine Does Not Directly Target Key Cardiac Ion Channels Kv11.1, Nav1.5 and Cav1.2.

Tiagabine is an antiepileptic drug used for the treatment of partial seizures in humans. Recently, this drug has been found useful in several non-epileptic conditions, including anxiety, chronic pain and sleep disorders. Since tachycardia-an impairment of cardiac rhythm due to cardiac ion channel dysfunction-is one of the most commonly reported non-neurological adverse effects of this drug, in the present paper we have undertaken pharmacological and numerical studies to assess a potential cardiovascular risk associated with the use of tiagabine. A chemical interaction of tiagabine with a model of human voltage-gated ion channels (VGICs) is described using the molecular docking method. The obtained in silico results imply that the adverse effects reported so far in the clinical cardiological of tiagabine could not be directly attributed to its interactions with VGICs. This is also confirmed by the results from the isolated organ studies (i.e., calcium entry blocking properties test) and in vivo (electrocardiogram study) assays of the present research. It was found that tachycardia and other tiagabine-induced cardiac complications are not due to a direct effect of this drug on ventricular depolarization and repolarization.

Tiagabine induced modulation of oscillatory connectivity and activity match PET-derived, canonical GABA-A receptor distributions.

As the most abundant inhibitory neurotransmitter in the mammalian brain, γ-aminobutyric acid (GABA) plays a crucial role in shaping the frequency and amplitude of oscillations, which suggests a role for GABA in shaping the topography of functional connectivity and activity. This study explored the effects of pharmacologically blocking the reuptake of GABA (increasing local concentrations) using the GABA transporter 1 (GAT1) blocker, tiagabine (15 mg). In a placebo-controlled crossover design, we collected resting magnetoencephalography (MEG) recordings from 15 healthy individuals prior to, and at 1-, 3- and 5- hours post, administration of tiagabine and placebo. We quantified whole brain activity and functional connectivity in discrete frequency bands. Drug-by-session (2 × 4) analysis of variance in connectivity revealed interaction and main effects. Post-hoc permutation testing of each post-drug recording vs. respective pre-drug baseline revealed consistent reductions of a bilateral occipital network spanning theta, alpha and beta frequencies, across 1- 3- and 5- hour recordings following tiagabine only. The same analysis applied to activity revealed significant increases across frontal regions, coupled with reductions in posterior regions, across delta, theta, alpha and beta frequencies. Crucially, the spatial distribution of tiagabine-induced changes overlap with group-averaged maps of the distribution of GABAA receptors, from flumazenil (FMZ-VT) PET, demonstrating a link between GABA availability, GABAA receptor distribution, and low-frequency network oscillations. Our results indicate that the relationship between PET receptor distributions and MEG effects warrants further exploration, since elucidating the nature of this relationship may uncover electrophysiologically-derived maps of oscillatory activity as sensitive, time-resolved, and targeted receptor-mapping tools for pharmacological imaging.

GABAergic cortical network physiology in frontotemporal lobar degeneration.

The clinical syndromes caused by frontotemporal lobar degeneration are heterogeneous, including the behavioural variant frontotemporal dementia (bvFTD) and progressive supranuclear palsy. Although pathologically distinct, they share many behavioural, cognitive and physiological features, which may in part arise from common deficits of major neurotransmitters such as γ-aminobutyric acid (GABA). Here, we quantify the GABAergic impairment and its restoration with dynamic causal modelling of a double-blind placebo-controlled crossover pharmaco-magnetoencephalography study. We analysed 17 patients with bvFTD, 15 patients with progressive supranuclear palsy, and 20 healthy age- and gender-matched controls. In addition to neuropsychological assessment and structural MRI, participants undertook two magnetoencephalography sessions using a roving auditory oddball paradigm: once on placebo and once on 10 mg of the oral GABA reuptake inhibitor tiagabine. A subgroup underwent ultrahigh-field magnetic resonance spectroscopy measurement of GABA concentration, which was reduced among patients. We identified deficits in frontotemporal processing using conductance-based biophysical models of local and global neuronal networks. The clinical relevance of this physiological deficit is indicated by the correlation between top-down connectivity from frontal to temporal cortex and clinical measures of cognitive and behavioural change. A critical validation of the biophysical modelling approach was evidence from parametric empirical Bayes analysis that GABA levels in patients, measured by spectroscopy, were related to posterior estimates of patients' GABAergic synaptic connectivity. Further evidence for the role of GABA in frontotemporal lobar degeneration came from confirmation that the effects of tiagabine on local circuits depended not only on participant group, but also on individual baseline GABA levels. Specifically, the phasic inhibition of deep cortico-cortical pyramidal neurons following tiagabine, but not placebo, was a function of GABA concentration. The study provides proof-of-concept for the potential of dynamic causal modelling to elucidate mechanisms of human neurodegenerative disease, and explains the variation in response to candidate therapies among patients. The laminar- and neurotransmitter-specific features of the modelling framework, can be used to study other treatment approaches and disorders. In the context of frontotemporal lobar degeneration, we suggest that neurophysiological restoration in selected patients, by targeting neurotransmitter deficits, could be used to bridge between clinical and preclinical models of disease, and inform the personalized selection of drugs and stratification of patients for future clinical trials.

Molecular Dynamic Simulations to Probe Stereoselectivity of Tiagabine Binding with Human GAT1.

The human gamma aminobutyric acid transporter subtype 1 (hGAT1) located in the nerve terminals is known to catalyze the neuronal function by the electrogenic reuptake of γ-aminobutyric acid (GABA) with the co-transport of Na+ and Cl- ions. In the past, there has been a major research drive focused on the dysfunction of hGAT1 in several neurological disorders. Thus, hGAT1 of the GABAergic system has been well established as an attractive target for such diseased conditions. Till date, there are various reports about stereo selectivity of -COOH group of tiagabine, a Food and Drug Administration (FDA)-approved hGAT1-selective antiepileptic drug. However, the effect of the stereochemistry of the protonated -NH group of tiagabine has never been scrutinized. Therefore, in this study, tiagabine has been used to explore the binding hypothesis of different enantiomers of tiagabine. In addition, the impact of axial and equatorial configuration of the-COOH group attached at the meta position of the piperidine ring of tiagabine enantiomers was also investigated. Further, the stability of the finally selected four hGAT1-tiagabine enantiomers namely entries 3, 4, 6, and 9 was evaluated through 100 ns molecular dynamics (MD) simulations for the selection of the best probable tiagabine enantiomer. The results indicate that the protonated -NH group in the R-conformation and the -COOH group of Tiagabine in the equatorial configuration of entry 4 provide maximum strength in terms of interaction within the hGAT1 binding pocket to prevent the change in hGAT1 conformational state, i.e., from open-to-out to open-to-in as compared to other selected tiagabine enantiomers 3, 6, and 9.

Tiagabine-related status epilepticus: a case report and systematic literature review.

Tiagabine-related status epilepticus (SE) is an uncommon complication of tiagabine use. We aimed to detail the features and outcomes in a patient with tiagabine poisoning and review the relevant literature. We describe a case of tiagabine-related SE and literature review based on a 1995-2019 PubMed search. We report the case of a 30-year-old man with super-refractory SE after tiagabine poisoning. He fully recovered after 72 h of general anesthesia and was discharged from the ICU on day 16. A literature review showed distinct features among patients with tiagabine-related SE. Tiagabine side effects were characterized by non-convulsive SE after a slight increase in tiagabine dose and a rapid favorable evolution after benzodiazepine and early tiagabine withdrawal. Generalized convulsive SE was a complication of voluntary or involuntary tiagabine poisoning and was particularly refractory. Both presentations are characterized by a return to baseline after prompt and adequate management. Tiagabine-related SE electroclinical features vary according to the underlying pathophysiological mechanism and can be life threatening. Recovery is the rule after tiagabine withdrawal and SE management with progressive therapeutic escalation guided by response to prior anticonvulsant treatments.

Various Aspects of Tiagabine Effectiveness as Add-on Therapy in Patients with Refractory Epilepsy.

The aim of the present study was to investigate various aspects of tiagabine (TGB) effectiveness in Bulgarian patients with drug-resistant epilepsy. This open, prospective study recruited the patients with epilepsy attending the Clinic of Neurology at the University Hospital of Plovdiv, Bulgaria. The patients completed diaries about the seizure frequency, severity, and adverse events. There were regular documented visits at 3 or 6 months during the first year of treatment with TGB and at 6 months or 1 year afterwards, with dynamic assessment of seizure frequency, severity, adverse events, and EEG recordings. TGB was applied as an add-on treatment in 43 patients (24 males, mean age 39 years). There was relatively mild and transient dynamic improvement of seizure severity, satisfactory seizure frequency reduction in 32.6% of participants, stable mean seizure frequency reduction (40-50%) from month 6 to month 24 and a stable response rate (52.3-50%) during the same period. New seizure types (myoclonic, myoclonic-atonic) occurred in 2 patients. The final clinical efficacy was higher in patients with initial monotherapy. There were adverse events (dizziness/vertigo, sedation, memory impairment, loss of appetite and weight, confusion, psychosis, insomnia, transient diplopia, lymphadenomegaly, rash, nausea, depression, anxiety, tremor of hands, unstable gait, legs edema, thrombocytopenia, cervical muscles tightening) in 26.19% of patients. In conclusion, TGB treatment is associated with low and transient improvement of seizure severity, good and stable improvement of seizure frequency, possible worsening of seizure control, possible appearance of new seizure types, and acceptable safety and tolerability.

Human Serum Albumin-Occupying-Based Fluorescence Turn-On Analysis of Antiepileptic Drug Tiagabine Hydrochloride.

Tiagabine hydrochloride (TGB) is a clinically frequently used drug for anticonvulsion and reducing epileptic frequency. Over administration of TGB could bring about adverse effects, such as speech disorder, depression, and even suicidal tendencies. Therefore, accessible and sensitive assay for analysis of TGB becomes an urgent need toward guiding clinical medication. Here, we present the first report on fluorescence turn-on detection of TGB in urine testing. In this protocol, a fluorescent dye, perylene tetracarboxylic acid imide derivative (PTAI), is found specifically occupying the Sudlow site II of human serum albumin (HSA) and displays a new phenomenon of binding-induced quenching (BIQ). In presence of TGB, competitive binding of the TGB to the site II of HSA will trigger release of PTAI, thus successfully lighting up the fluorescence of PTAI. This label-free assay enjoys a broader working range (1-350 µM) and lower detection limit (0.218 µM) than the traditional liquid chromatography method and is uninterfered by the miscellaneous in the artificial urine. The BIQ probe highlights the merits of HSA as a quencher and a molecular recognition unit, and it opens up a way for studying drug-HSA interaction mechanism and noninvasive pharmaceutical testing.

GABA-ergic Dynamics in Human Frontotemporal Networks Confirmed by Pharmaco-Magnetoencephalography.

To bridge the gap between preclinical cellular models of disease and in vivo imaging of human cognitive network dynamics, there is a pressing need for informative biophysical models. Here we assess dynamic causal models (DCM) of cortical network responses, as generative models of magnetoencephalographic observations during an auditory oddball roving paradigm in healthy adults. This paradigm induces robust perturbations that permeate frontotemporal networks, including an evoked 'mismatch negativity' response and transiently induced oscillations. Here, we probe GABAergic influences in the networks using double-blind placebo-controlled randomized-crossover administration of the GABA reuptake inhibitor, tiagabine (oral, 10 mg) in healthy older adults. We demonstrate the facility of conductance-based neural mass mean-field models, incorporating local synaptic connectivity, to investigate laminar-specific and GABAergic mechanisms of the auditory response. The neuronal model accurately recapitulated the observed magnetoencephalographic data. Using parametric empirical Bayes for optimal model inversion across both drug sessions, we identify the effect of tiagabine on GABAergic modulation of deep pyramidal and interneuronal cell populations. We found a transition of the main GABAergic drug effects from auditory cortex in standard trials to prefrontal cortex in deviant trials. The successful integration of pharmaco- magnetoencephalography with dynamic causal models of frontotemporal networks provides a potential platform on which to evaluate the effects of disease and pharmacological interventions.SIGNIFICANCE STATEMENT Understanding human brain function and developing new treatments require good models of brain function. We tested a detailed generative model of cortical microcircuits that accurately reproduced human magnetoencephalography, to quantify network dynamics and connectivity in frontotemporal cortex. This approach identified the effect of a test drug (GABA-reuptake inhibitor, tiagabine) on neuronal function (GABA-ergic dynamics), opening the way for psychopharmacological studies in health and disease with the mechanistic precision afforded by generative models of the brain.

Isobolographic Analysis of Antiseizure Activity of the GABA Type A Receptor-Modulating Synthetic Neurosteroids Brexanolone and Ganaxolone with Tiagabine and Midazolam.

Epilepsy is often treated with a combination of antiepileptic drugs. Although neurosteroids are potent anticonvulsants, little is known about their combination potential for the treatment of refractory epilepsy. Here, we investigated the combination efficacy of neurosteroids allopregnanolone (AP, brexanolone) and ganaxolone (GX) with the GABA-reuptake inhibitor tiagabine (TG) or the benzodiazepine midazolam (MDZ) on tonic inhibition in dentate gyrus granule cells and seizure protection in the hippocampus kindling and 6-Hz seizure models. Isobolographic analysis indicated that combinations of GX and TG or AP and TG at three standard ratios (1:1, 3:1, and 1:3) displayed significant synergism in augmenting tonic inhibition. In pharmacological studies, GX, AP, and TG produced dose-dependent antiseizure effects in mice (ED50 = 1.46, 4.20, and 0.20 mg/kg, respectively). The combination of GX and TG at the fixed ratio of 1:1 exerted the greatest combination index (CI = 0.53), indicating strong synergistic interaction in seizure protection. In addition, combination regimens of AP and TG showed robust synergism for seizure protection (CI = 0.4). Finally, combination regimens of GX and MDZ elicited synergistic (CI = 0.6) responses for seizure protection. These results demonstrate striking synergism of neurosteroids and TG combination for seizure protection, likely because of their effects at extrasynaptic GABA type A (GABA-A) receptors from TG-induced elevation in GABA levels. Superadditive antiseizure activity of neurosteroid-MDZ combinations may stem from their actions at both synaptic and extrasynaptic GABA-A receptors. Together, these findings provide a potential mechanistic basis for combination potential of neurosteroids with TG or benzodiazepines for the management of refractory epilepsy, status epilepticus, and seizure disorders. SIGNIFICANCE STATEMENT: This paper investigates for the first time the potential synergistic interactions between two neurosteroids with anticonvulsant properties, allopregnanolone (brexanolone) and the very similar synthetic analog, ganaxolone, and two conventional antiepileptic drugs active at GABA type A receptors: the GABA-reuptake inhibitor tiagabine and a benzodiazepine, midazolam. The results demonstrate a synergistic protective effect of neurosteroid-tiagabine combinations, as well as neurosteroid-midazolam regimens in seizure models.

Decreased directed functional connectivity in the psychedelic state.

Neuroimaging studies of the psychedelic state offer a unique window onto the neural basis of conscious perception and selfhood. Despite well understood pharmacological mechanisms of action, the large-scale changes in neural dynamics induced by psychedelic compounds remain poorly understood. Using source-localised, steady-state MEG recordings, we describe changes in functional connectivity following the controlled administration of LSD, psilocybin and low-dose ketamine, as well as, for comparison, the (non-psychedelic) anticonvulsant drug tiagabine. We compare both undirected and directed measures of functional connectivity between placebo and drug conditions. We observe a general decrease in directed functional connectivity for all three psychedelics, as measured by Granger causality, throughout the brain. These data support the view that the psychedelic state involves a breakdown in patterns of functional organisation or information flow in the brain. In the case of LSD, the decrease in directed functional connectivity is coupled with an increase in undirected functional connectivity, which we measure using correlation and coherence. This surprising opposite movement of directed and undirected measures is of more general interest for functional connectivity analyses, which we interpret using analytical modelling. Overall, our results uncover the neural dynamics of information flow in the psychedelic state, and highlight the importance of comparing multiple measures of functional connectivity when analysing time-resolved neuroimaging data.

Characterization of HemY-type protoporphyrinogen IX oxidase genes from cyanobacteria and their functioning in transgenic Arabidopsis.

KEY MESSAGE: We investigated the functions of two cyanobacterial HemY protoporphyrinogen IX oxidase (PPO) genes with in vitro and in vivo assays and evaluated their applicability as resistance traits to PPO-inhibiting herbicides. We isolated HemY-type protoporphyrinogen IX oxidase (PPO) genes from cyanobacteria, OnPPO gene from Oscillatoria nigro-viridis PCC7112 and HaPPO gene from Halothece sp. PCC7418. The alignment of amino acid sequences as well as phylogenetic analyses conducted showed that OnPPO and HaPPO are classified as HemY-type PPO and are more closely related to plastidic PPOs than to mitochondrial PPOs. The PPO-deficient Escherichia coli BT3 strain, which requires heme supplementation, could obtain normal growth in the absence of heme supplementation when complemented with OnPPO and HaPPO. The enzyme assays of OnPPO, HaPPO, and Arabidopsis thaliana PPO1 (AtPPO1) proteins each revealed different kinetic properties in terms of catalytic efficiency, substrate affinity, and the degree of inhibition by PPO inhibitors. In particular, the catalytic efficiencies (kcat/Km) of OnPPO and HaPPO were approximately twofold higher than that of AtPPO1. The elution profiles of all three PPOs, acquired by size-exclusion chromatography, showed only a single peak with a molecular weight of approximately 52-54 kDa, which corresponds to a monomeric form. Moreover, functional complementation with OnPPO and HaPPO in AtPPO1-silenced Arabidopsis resulted in restored growth, whereas AtPPO1-silenced wild type Arabidopsis suffered necrotic death. In addition, we observed that overexpression of OnPPO and HaPPO in Arabidopsis conferred resistance to the PPO-inhibiting herbicides tiafenacil and saflufenacil. These results suggest that two HemY-type PPOs of cyanobacteria can functionally substitute for plastidic PPO activity in Arabidopsis and can enhance resistance to tiafenacil and saflufenacil.

Acute effect of cannabidiol on the activity of various novel antiepileptic drugs in the maximal electroshock- and 6 Hz-induced seizures in mice: Pharmacodynamic and pharmacokinetic studies.

Cannabidiol and cannabidiol-enriched products have recently attracted much attention as an add-on therapy for epilepsy, especially drug-resistant seizures. It should be, however, remembered that concomitant use of cannabidiol and antiepileptic drugs may pose a risk of interactions between them. For this reason, the aim of our study was to assess the effect of cannabidiol on the activity of selected new antiepileptic drugs in the electrically-induced seizure models in mice. We studied the effect of cannabidiol on the anticonvulsant action of topiramate, oxcarbazepine, lamotrigine, and pregabalin in the maximal electroshock-induced seizure test as well as on the activity of levetiracetam, tiagabine, lacosamide, and gabapentin in the 6 Hz seizure test in mice. We showed that cannabidiol increased the activity of topiramate, oxcarbazepine, pregabalin, tiagabine, and gabapentin. It did not affect the anticonvulsant effect of lamotrigine and lacosamide. Interestingly, cannabidiol attenuated the anticonvulsant activity of levetiracetam. Co-administration of antiepileptic drugs with cannabidiol did not cause adverse effects such as impairment of motor coordination, changes in neuromuscular strength or potentiation of the cannabidiol-induced hypolocomotion. Serum and brain levels of antiepileptic drugs and cannabidiol were determined by using HPLC in order to ascertain any pharmacokinetic contribution to the observed behavioral effects. Only interaction with levetiracetam was purely pharmacodynamic in nature because no changes in serum and brain concentration of either levetiracetam or cannabidiol were observed. Increased anticonvulsant activity of topiramate, oxcarbazepine, pregabalin, tiagabine, and gabapentin could be, at least in part, related to pharmacokinetic interactions with cannabidiol because there were changes in serum and/or brain concentrations of antiepileptic drugs and/or cannabidiol. Pharmacokinetic interactions cannot be also excluded between lacosamide and cannabidiol because cannabidiol increased brain concentration of lacosamide and lacosamide increased brain concentration of cannabidiol. Further pharmacokinetic studies are required to evaluate the type of interactions between cannabidiol and novel antiepileptic drugs.