Tiagabine and zonisamide differentially regulate the glial properties in an astrocyte-microglia co-culture model of inflammation.

Due to the role of astrocytes and microglia in the pathophysiology of epilepsy and limited studies of antiseizure medication (ASM) effects on glial cells, we studied tiagabine (TGB) and zonisamide (ZNS) in an astrocyte-microglia co-culture model of inflammation. Different concentrations of ZNS (10, 20, 40, 100 µg/ml) or TGB (1, 10, 20, 50 µg/ml) were added to primary rat astrocytes co-cultures with 5-10% (M5, physiological conditions) or 30-40% (M30, pathological inflammatory conditions) microglia for 24 h, aiming to study glial viability, microglial activation, connexin 43 (Cx43) expression and gap-junctional coupling. ZNS led to the reduction of glial viability by only 100 µg/ml under physiological conditions. By contrast, TGB revealed toxic effects with a significant, concentration-dependent reduction of glial viability under physiological and pathological conditions. After the incubation of M30 co-cultures with 20 µg/ml TGB, the microglial activation was significantly decreased and resting microglia slightly increased, suggesting possible anti-inflammatory features of TGB under inflammatory conditions. Otherwise, ZNS caused no significant changes of microglial phenotypes. The gap-junctional coupling was significantly decreased after the incubation of M5 co-cultures with 20 and 50 µg/ml TGB, which can be related to its anti-epileptic activity under noninflammatory conditions. A significant decrease of Cx43 expression and cell-cell coupling was found after the incubation of M30 co-cultures with 10 µg/ml ZNS, suggesting additional anti-seizure effects of ZNS with the disruption of glial gap-junctional communication under inflammatory conditions. TGB and ZNS differentially regulated the glial properties. Developing novel ASMs targeting glial cells may have future potential as an "add-on" therapy to classical ASMs targeting neurons.

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.

Development of Positron Emission Tomography Radiotracers for the GABA Transporter 1.

In vivo positron emission tomography (PET) imaging of the γ-aminobutyric acid (GABA) receptor complex has been accomplished using radiolabeled benzodiazepine derivatives, but development of specific presynaptic radioligands targeting the neuronal membrane GABA transporter type 1 (GAT-1) has been less successful. The availability of new structure-activity studies of GAT-1 inhibitors and the introduction of a GAT-1 inhibitor (tiagabine, Gabatril) into clinical use prompted us to reinvestigate the syntheses of PET ligands for this transporter. Initial synthesis and rodent PET studies of N-[11C]methylnipecotic acid confirmed the low brain uptake of that small and polar molecule. The common design approach to improve blood-brain barrier permeability of GAT-1 inhibitors is the attachment of a large lipophilic substituent. We selected an unsymmetrical bis-aromatic residue attached to the ring nitrogen by a vinyl ether spacer from a series recently reported by Wanner and coworkers. Nucleophilic aromatic substitution of an aryl chloride precursor with [18F]fluoride was used to prepare the desired candidate radiotracer ( R, E/ Z)-1-(2-((4-fluoro-2-(4-[18F]fluorobenzoyl)styryl)oxy)ethyl)piperidine-3-carboxylic acid (( R, E/ Z)-[18F]10). PET studies in rats showed no brain uptake, which was not altered by pretreatment of animals with the P-glycoprotein inhibitor cyclosporine A, indicating efflux by Pgp was not responsible. Subsequent PET imaging studies of ( R, E/ Z)-[18F]10 in rhesus monkey brain showed very low brain uptake. Finally, to test if the free carboxylic acid group was the likely cause of poor brain uptake, PET studies were done using the ethyl ester derivative of ( R, E/ Z)-[18F]10. Rapid and significant monkey brain uptake of the ester was observed, followed by a slow washout over 90 min. The blood-brain barrier permeability of the ester supports a hypothesis that the free acid function limits brain uptake of nipecotic acid-based GAT-1 radioligands, and future radiotracer efforts should investigate the use of carboxylic acid bioisosteres.