GABA Uptake Research Articles

Spontaneous Calcium Transients Recorded from Striatal Astrocytes in a Preclinical Model of Autism

Autism spectrum disorder (ASD) is known as a group of neurodevelopmental conditions including stereotyped and repetitive behaviors, besides social and sensorimotor deficits. Anatomical and functional evidence indicates atypical maturation of the striatum. Astrocytes regulate the maturation and plasticity of synaptic circuits, and impaired calcium signaling is associated with repetitive behaviors and atypical social interaction. Spontaneous calcium transients (SCT) recorded in the striatal astrocytes of the rat were investigated in the preclinical model of ASD by prenatal exposure to valproic acid (VPA). Our results showed sensorimotor delay, augmented glial fibrillary acidic protein -a typical intermediate filament protein expressed by astrocytes- and diminished expression of GABAA-ρ3 through development, and increased frequency of SCT with a reduced latency that resulted in a diminished amplitude in the VPA model. The convulsant picrotoxin, a GABAA (γ-aminobutyric acid type A) receptor antagonist, reduced the frequency of SCT in both experimental groups but rescued this parameter to control levels in the preclinical ASD model. The amplitude and latency of SCT were decreased by picrotoxin in both experimental groups. Nipecotic acid, a GABA uptake inhibitor, reduced the mean amplitude only for the control group. Nevertheless, nipecotic acid increased the frequency but diminished the latency in both experimental groups. Thus, we conclude that striatal astrocytes exhibit SCT modulated by GABAA-mediated signaling, and prenatal exposure to VPA disturbs this tuning.

Somatostatin Interneurons Recruit Pre- and Postsynaptic GABAB Receptors in the Adult Mouse Dentate Gyrus.

The integration of spatial information in the mammalian dentate gyrus (DG) is critical to navigation. Indeed, DG granule cells (DGCs) rely upon finely balanced inhibitory neurotransmission in order to respond appropriately to specific spatial inputs. This inhibition arises from a heterogeneous population of local GABAergic interneurons (INs) that activate both fast, ionotropic GABAA receptors (GABAAR) and slow, metabotropic GABAB receptors (GABABR), respectively. GABABRs in turn inhibit pre- and postsynaptic neuronal compartments via temporally long-lasting G-protein-dependent mechanisms. The relative contribution of each IN subtype to network level GABABR signal setting remains unknown. However, within the DG, the somatostatin (SSt) expressing IN subtype is considered crucial in coordinating appropriate feedback inhibition on to DGCs. Therefore, we virally delivered channelrhodopsin 2 to the DG in order to obtain control of this specific SSt IN subpopulation in male and female adult mice. Using a combination of optogenetic activation and pharmacology, we show that SSt INs strongly recruit postsynaptic GABABRs to drive greater inhibition in DGCs than GABAARs at physiological membrane potentials. Furthermore, we show that in the adult mouse DG, postsynaptic GABABR signaling is predominantly regulated by neuronal GABA uptake and less so by astrocytic mechanisms. Finally, we confirm that activation of SSt INs can also recruit presynaptic GABABRs, as has been shown in neocortical circuits. Together, these data reveal that GABABR signaling allows SSt INs to control DG activity and may constitute a key mechanism for gating spatial information flow within hippocampal circuits.

The mitochondrial aspartate/glutamate carrier does not transport GABA

ɣ-aminobutyric acid (GABA) is a four‑carbon amino acid acting as the main inhibitory transmitter in the invertebrate and vertebrate nervous systems. The metabolism of GABA is well compartmentalized in the cell and the uptake of cytosolic GABA into the mitochondrial matrix is required for its degradation. A previous study carried out in the fruit fly Drosophila melanogaster indicated that the mitochondrial aspartate/glutamate carrier (AGC) is responsible for mitochondrial GABA accumulation. Here, we investigated the transport of GABA catalysed by the human and D. melanogaster AGC proteins through a well-established method for the study of the substrate specificity and the kinetic parameters of the mitochondrial carriers. In this experimental system, the D. melanogaster spliced AGC isoforms (Aralar1-PA and Aralar1-PE) and the human AGC isoforms (AGC1/aralar1 and AGC2/citrin) are unable to transport GABA both in homo- and in hetero-exchange with either glutamate or aspartate, i.e. the canonical substrates of AGC. Moreover, GABA has no inhibitory effect on the exchange activities catalysed by the investigated AGCs. Our data demonstrate that AGC does not transport GABA and the molecular identity of the GABA transporter in human and D. melanogaster mitochondria remains unknown.

Unveiling the crucial role of betaine: modulation of GABA homeostasis via SLC6A1 transporter (GAT1)

Betaine is an endogenous osmolyte that exhibits therapeutic potential by mitigating various neurological disorders. However, the underlying cellular and molecular mechanisms responsible for its neuroprotective effects remain puzzling.In this study, we describe a possible mechanism behind the positive impact of betaine in preserving neurons from excitotoxicity. Here we demonstrate that betaine at low concentration modulates the GABA uptake by GAT1 (slc6a1), the predominant GABA transporter in the central nervous system. This modulation occurs through the temporal inhibition of the transporter, wherein prolonged occupancy by betaine impedes the swift transition of the transporter to the inward conformation. Importantly, the modulatory effect of betaine on GAT1 is reversible, as the blocking of GAT1 disappears with increased extracellular GABA. Using electrophysiology, mass spectroscopy, radiolabelled cellular assay, and molecular dynamics simulation we demonstrate that betaine has a dual role in GAT1: at mM concentration acts as a slow substrate, and at µM as a temporal blocker of GABA, when it is below its K0.5. Given this unique modulatory characteristic and lack of any harmful side effects, betaine emerges as a promising neuromodulator of the inhibitory pathways improving GABA homeostasis via GAT1, thereby conferring neuroprotection against excitotoxicity.

A transporter's doom or destiny: SLC6A1 in health and disease, novel molecular targets and emerging therapeutic prospects.

As the first member of the solute carrier 6 (SLC6) protein family, the γ-aminobutyric acid (GABA) transporter 1 (GAT1, SLC6A1), plays a pivotal role in the uptake of GABA from the synaptic cleft into neurons and astrocytes. This process facilitates the subsequent storage of GABA in presynaptic vesicles. The human SLC6A1 gene is highly susceptible to missense mutations, leading to severe clinical outcomes, such as epilepsy, in the afflicted patients. The molecular mechanisms of SLC6A1-associated disorders are discerned to some degree; many SLC6A1 mutations are now known to impair protein folding, and consequently fail to reach the plasma membrane. Inherently, once inside the endoplasmic reticulum (ER), GAT1 abides by a complex cascade of events that enable efficient intracellular trafficking. This involves association with specialized molecular chaperones responsible for steering the protein folding process, oligomerization, sorting through the Golgi apparatus, and ultimately delivery to the cell surface. The entire process is subject to stringent quality control mechanisms at multiple checkpoints. While the majority of the existing loss-of-function SLC6A1 variants interfere with folding and membrane targeting, certain mutants retain abundant surface expression. In either scenario, suppressed GAT1 activity disrupts GABAergic neurotransmission, preceding the disease manifestation in individuals harboring these mutations. The nervous system is enthralling and calls for systematic, groundbreaking research efforts to dissect the precise molecular factors associated with the onset of complex neurological disorders, and uncover additional non-canonical therapeutic targets. Recent research has given hope for some of the misfolded SLC6A1 variants, which can be salvaged by small molecules, i.e., chemical and pharmacological chaperones, acting on multiple upstream targets in the secretory pathway. We here highlight the significance of pharmacochaperoning as a therapeutic strategy for the treatment of SLC6A1-related disorders.

Spinal GABA transporter 1 contributes to evoked-pain related behavior but not resting pain after incision injury.

The inhibitory function of GABA at the spinal level and its central modulation in the brain are essential for pain perception. However, in post-surgical pain, the exact mechanism and modes of action of GABAergic transmission have been poorly studied. This work aimed to investigate GABA synthesis and uptake in the incisional pain model in a time-dependent manner. Here, we combined assays for mechanical and heat stimuli-induced withdrawal reflexes with video-based assessments and assays for non-evoked (NEP, guarding of affected hind paw) and movement-evoked (MEP, gait pattern) pain-related behaviors in a plantar incision model in male rats to phenotype the effects of the inhibition of the GABA transporter (GAT-1), using a specific antagonist (NO711). Further, we determined the expression profile of spinal dorsal horn GAT-1 and glutamate decarboxylase 65/67 (GAD65/67) by protein expression analyses at four time points post-incision. Four hours after incision, we detected an evoked pain phenotype (mechanical, heat and movement), which transiently ameliorated dose-dependently following spinal inhibition of GAT-1. However, the NEP-phenotype was not affected. Four hours after incision, GAT-1 expression was significantly increased, whereas GAD67 expression was significantly reduced. Our data suggest that GAT-1 plays a role in balancing spinal GABAergic signaling in the spinal dorsal horn shortly after incision, resulting in the evoked pain phenotype. Increased GAT-1 expression leads to increased GABA uptake from the synaptic cleft and reduces tonic GABAergic inhibition at the post-synapse. Inhibition of GAT-1 transiently reversed this imbalance and ameliorated the evoked pain phenotype.

SLC6A1 variant pathogenicity, molecular function and phenotype: a genetic and clinical analysis.

Genetic variants in the SLC6A1 gene can cause a broad phenotypic disease spectrum by altering the protein function. Thus, systematically curated clinically relevant genotype-phenotype associations are needed to understand the disease mechanism and improve therapeutic decision-making. We aggregated genetic and clinical data from 172 individuals with likely pathogenic/pathogenic (lp/p) SLC6A1 variants and functional data for 184 variants (14.1% lp/p). Clinical and functional data were available for a subset of 126 individuals. We explored the potential associations of variant positions on the GAT1 3D structure with variant pathogenicity, altered molecular function and phenotype severity using bioinformatic approaches. The GAT1 transmembrane domains 1, 6 and extracellular loop 4 (EL4) were enriched for patient over population variants. Across functionally tested missense variants (n = 156), the spatial proximity from the ligand was associated with loss-of-function in the GAT1 transporter activity. For variants with complete loss of in vitro GABA uptake, we found a 4.6-fold enrichment in patients having severe disease versus non-severe disease (P = 2.9 × 10-3, 95% confidence interval: 1.5-15.3). In summary, we delineated associations between the 3D structure and variant pathogenicity, variant function and phenotype in SLC6A1-related disorders. This knowledge supports biology-informed variant interpretation and research on GAT1 function. All our data can be interactively explored in the SLC6A1 portal (https://slc6a1-portal.broadinstitute.org/).

Structural and stereochemical determinants for hGAT3 inhibition: development of novel conformationally constrained and substituted analogs of (S)-isoserine

AbstractThe GABA transporter 3 (GAT3) is a member of the GABA transporter (GAT) family proposed to have a role in regulating tonic inhibition. The GAT3-preferring substrate (S)-isoserine has shown beneficial effects in a mouse model of stroke accompanied by an increased GAT3 expression, indicating a molecular mechanism mediated by GAT3. However, (S)-isoserine is not ideally suited for in vivo studies due to a lack of selectivity and brain permeability. To elucidate the structural determinants of (S)-isoserine for GAT3 inhibition, and to optimize and inform further ligand development, we here present the design, synthesis and pharmacological evaluation of a series of conformationally constrained isoserine analogs with defined stereochemistry. Using [3H]GABA uptake assays at recombinant human GAT3, we identified the azetidine and pyrrolidine analogs (2S,2´S)-6 and (2S,2´S)-7 as the most potent inhibitors. To further elaborate on the selectivity profile both compounds were tested at all GATs, the taurine transporter (TauT) and GABAA receptors. Although (2S,2´S)-6 and (2S,2´S)-7 are comparable to (S)-isoserine with respect to potency, the selectivity vs. the taurine transporter was significantly improved (at least 6 and 53 times more activity at hGAT3, respectively). A subsequent comprehensive structure-activity study showed that different connectivity approaches, stereochemical variations, simple or larger α- and N-substituents, and even minor size enlargement of the heterocyclic ring all abrogated GAT3 inhibition, indicating very strict stereochemical and size requirements. The observed structure activity relationships may guide future ligand optimization and the novel ligands ((2S,2´S)-6 and (2S,2´S)-7) can serve as valuable tools to validate the proposed GAT3-mediated effect of (S)-isoserine such as in functional recovery after stroke and thus help corroborate the relevance of targeting GAT3 and tonic inhibition in relevant brain pathologies.

Discovery of E2730, a novel selective uncompetitive GAT1 inhibitor, as a candidate for anti-seizure medication.

As of 2022, 36 anti-seizure medications (ASMs) have been licensed for the treatment of epilepsy, however, adverse effects (AEs) are commonly reported. Therefore, ASMs with a wide margin between therapeutic effects and AEs are preferred over ASMs that are associated with a narrow margin between efficacy and risk of AEs. E2730 was discovered using in vivo phenotypic screening and characterized as an uncompetitive, yet selective, inhibitor of γ-aminobutyric acid (GABA) transporter 1 (GAT1). Here, we describe the preclinical characteristics of E2730. Anti-seizure effects of E2730 were evaluated in several animal models of epilepsy: corneal kindling, 6 Hz-44 mA psychomotor seizure, amygdala kindling, Fragile X syndrome, and Dravet syndrome models. Effects of E2730 on motor coordination were assessed in accelerating rotarod tests. The mechanism of action of E2730 was explored by [3 H]E2730 binding assay. The GAT1-selectivity over other GABA transporters was examined by GABA uptake assay of GAT1, GAT2, GAT3, or betaine/GABA transporter 1 (BGT-1) stably expressing HEK293 cells. To further investigate the mechanism for E2730-mediated inhibition of GAT1, in vivo microdialysis and in vitro GABA uptake assays were conducted under conditions of different GABA concentrations. E2730 showed anti-seizure effects in the assessed animal models with an approximately >20-‍fold margin between efficacy and motor incoordination. [3 H]E2730 binding on brain synaptosomal membrane was abolished in GAT1-deficient mice, and E2730 selectively inhibited GAT1-mediated GABA uptake over other GABA transporters. In addition, results of GABA uptake assays showed that E2730-mediated inhibition of GAT1 positively correlated to the level of ambient GABA in vitro. E2730 also increased extracellular GABA concentration in hyperactivated conditions but not under basal levels in vivo. E2730 is a novel, selective, uncompetitive GAT1 inhibitor, which acts selectively under the condition of increasing synaptic activity, contributing to a wide margin between therapeutic effect and motor incoordination.

424 Clinical Translational Approach to Targeted Therapy in SLC6A1-related Neurodevelopmental Disorder

OBJECTIVES/GOALS: SLC6A1-Related Neurodevelopmental Disorder (SLC6A1-NDD) is a leading genetic cause of epilepsy and autism. Haploinsufficiency of SLC6A1 leads to reduced uptake of GABA from the synaptic cleft, and increased extracellular GABA in mice. It is a candidate for gene transfer therapy, but translational read outs are needed. EEG is a promising biomarker. METHODS/STUDY POPULATION: The SLC6A1-NDD program includes a specialty clinic and prospective cohort study run in parallel with pre-clinical gene therapy development. Characterization and pre-clinical testing of a homozygous knock-out, heterozygous knock-out, and two humanized knock-in models are on-going, including EEG analyses before and after treatment. Patients with a confirmed diagnosis of SLC6A1-NDD are seen annually in a specialty clinic and a subset participate in the cohort study which collects standardized questionnaires, EEGs, and MR Spectroscopy to measure glutamate and GABA. Gene Therapy Program investigators meet weekly to discuss progress on pre-clinical and clinical trial readiness on SLC6A1-NDD and to align efforts on translational read outs, including EEG, in both humans and the pre-clinical models. RESULTS/ANTICIPATED RESULTS: We have enrolled the full cohort of 20 participants in the prospective SLC6A1-NDD cohort study. Preliminary results have shown that all but 1 individual has a history of developmental delay, and 8 of the 24 individuals in our clinical cohort had at least one episode of developmental regression. Over 90% have epilepsy, and 17/20 in the cohort study have intermittent rhythmic delta activity on EEG. The full knock out mice have behavioral and learning deficits and abnormal electrical brain activity on telemetry, including bursts of spike trains, analogous to epileptiform activity seen in humans. Next steps include quantitative analysis of both mouse and human EEG to develop a translational brain-based biomarker. We plan to assess delta power and investigate genotype-phenotype correlations in mice and humans. DISCUSSION/SIGNIFICANCE: With targeted therapies in development for SLC6A1-NDD, translational biomarkers that demonstrate engagement with the brain are critical. With clinical heterogeneity in SLC6A1-NDD, biomarkers can objectively capture change. Collaborative translational projects may improve efficiency in rare disease research to facilitate early phase trials.

Volume-transmitted GABA waves pace epileptiform rhythms in the hippocampal network

Mechanisms that entrain and pace rhythmic epileptiform discharges remain debated. Traditionally, the quest to understand them has focused on interneuronal networks driven by synaptic GABAergic connections. However, synchronized interneuronal discharges could also trigger the transient elevations of extracellular GABA across the tissue volume, thus raising tonic conductance (Gtonic) of synaptic and extrasynaptic GABA receptors in multiple cells. Here, we monitor extracellular GABA in hippocampal slices using patch-clamp GABA "sniffer" and a novel optical GABA sensor, showing that periodic epileptiform discharges are preceded by transient, region-wide waves of extracellular GABA. Neural network simulations that incorporate volume-transmitted GABA signals point to a cycle of GABA-driven network inhibition and disinhibition underpinning this relationship. We test and validate this hypothesis using simultaneous patch-clamp recordings from multiple neurons and selective optogenetic stimulation of fast-spiking interneurons. Critically, reducing GABA uptake in order to decelerate extracellular GABA fluctuations-without affecting synaptic GABAergic transmission or resting GABA levels-slows down rhythmic activity. Our findings thus unveil a key role of extrasynaptic, volume-transmitted GABA in pacing regenerative rhythmic activity in brain networks.

Astrocytes regulate inhibitory neurotransmission through GABA uptake, metabolism, and recycling.

Synaptic regulation of the primary inhibitory neurotransmitter γ-aminobutyric acid (GABA) is essential for brain function. Cerebral GABA homeostasis is tightly regulated through multiple mechanisms and is directly coupled to the metabolic collaboration between neurons and astrocytes. In this essay, we outline and discuss the fundamental roles of astrocytes in regulating synaptic GABA signaling. A major fraction of synaptic GABA is removed from the synapse by astrocytic uptake. Astrocytes utilize GABA as a metabolic substrate to support glutamine synthesis. The astrocyte-derived glutamine is subsequently transferred to neurons where it serves as the primary precursor of neuronal GABA synthesis. The flow of GABA and glutamine between neurons and astrocytes is collectively termed the GABA-glutamine cycle and is essential to sustain GABA synthesis and inhibitory signaling. In certain brain areas, astrocytes are even capable of synthesizing and releasing GABA to modulate inhibitory transmission. The majority of oxidative GABA metabolism in the brain takes place in astrocytes, which also leads to synthesis of the GABA-related metabolite γ-hydroxybutyric acid (GHB). The physiological roles of endogenous GHB remain unclear, but may be related to regulation of tonic inhibition and synaptic plasticity. Disrupted inhibitory signaling and dysfunctional astrocyte GABA handling are implicated in several diseases including epilepsy and Alzheimer's disease. Synaptic GABA homeostasis is under astrocytic control and astrocyte GABA uptake, metabolism, and recycling may therefore serve as relevant targets to ameliorate pathological inhibitory signaling.

NMDA Receptor Activation and Ca2+/PKC Signaling in Nicotine-Induced GABA Transport Shift in Embryonic Chick Retina.

Nicotinic receptors are present in the retina of different vertebrates, and in the chick retina, it is present during early development throughout to post-hatching. These receptors are activated by nicotine, an alkaloid with addictive and neurotransmitter release modulation properties, such as GABA signaling. Here we evaluated the mechanisms of nicotine signaling in the avian retina during the development of neuron-glia cells at a stage where synapses are peaking. Nicotine almost halved [3H]-GABA uptake, reducing it by 45% whilst increasing more than two-fold [3H]-GABA release in E12 embryonic chick retinas. Additionally, nicotine mediated a 33% increase in [3H]-D-aspartate release. MK-801 50μM blocked 66% of nicotine-induced [3H]-GABA release and Gö 6983 100nM prevented the nicotine-induced reduction in [3H]-GABA uptake by rescuing 40% of this neurotransmitter uptake, implicating NMDAR and PKC (respectively) in the nicotinic responses. In addition, NO-711 prevented [3H]-GABA uptake and release induced by nicotine. Furthermore, the relevance of calcium influx for PKC activation was evidenced through fura-2 imaging. We conclude that the shift of GABA transport mediated by nicotine promotes GABA release by inducing transporter reversal via nicotine-induced EAA release through EAATs, or by a direct effect of nicotine in activating nicotinic receptors permeable to calcium and promoting PKC pathway activation and shifting GAT-1 activity, both prompting calcium influx, and activation of the PKC pathway and shifting GAT-1 activity.

Drosophila melanogaster as a model for unraveling unique molecular features of epilepsy elicited by human GABA transporter 1 variants.

Mutations in the human γ-aminobutyric acid (GABA) transporter 1 (hGAT-1) can instigate myoclonic-atonic and other generalized epilepsies in the afflicted individuals. We systematically examined fifteen hGAT-1 disease variants, all of which dramatically reduced or completely abolished GABA uptake activity. Many of these loss-of-function variants were absent from their regular site of action at the cell surface, due to protein misfolding and/or impaired trafficking machinery (as verified by confocal microscopy and de-glycosylation experiments). A modest fraction of the mutants displayed correct targeting to the plasma membrane, but nonetheless rendered the mutated proteins devoid of GABA transport, possibly due to structural alterations in the GABA binding site/translocation pathway. We here focused on a folding-deficient A288V variant. In flies, A288V reiterated its impeded expression pattern, closely mimicking the ER-retention demonstrated in transfected HEK293 cells. Functionally, A288V presented a temperature-sensitive seizure phenotype in fruit flies. We employed diverse small molecules to restore the expression and activity of folding-deficient hGAT-1 epilepsy variants, in vitro (in HEK293 cells) and in vivo (in flies). We identified three compounds (chemical and pharmacological chaperones) conferring moderate rescue capacity for several variants. Our data grant crucial new insights into: (i) the molecular basis of epilepsy in patients harboring hGAT-1 mutations, and (ii) a proof-of-principle that protein folding deficits in disease-associated hGAT-1 variants can be corrected using the pharmacochaperoning approach. Such innovative pharmaco-therapeutic prospects inspire the rational design of novel drugs for alleviating the clinical symptoms triggered by the numerous emerging pathogenic mutations in hGAT-1.

Psychoactive Isoxazoles, Muscimol, and Isoxazole Derivatives from the Amanita (Agaricomycetes) Species: Review of New Trends in Synthesis, Dosage, and Biological Properties.

Herbal products found in nature can serve as great systems of study for drug design. The Amanita muscaria mushroom is native to many parts of the Northern Hemisphere and has a very distinctive appearance with its red cap and white spotted warts. The mushroom comprises several pharmacologically active alkaloids, including muscazone, muscarine, ibotenic acid, and muscimol, the latter two compounds being potent GABA agonists. Muscimol has served as a backbone in the design of GABA agonists devoid of effects on the GABA-metabolizing enzyme, GABA transaminase, and GABA uptake systems. In this sense, several analogs of muscimol have been synthesized and studied including THIP, THPO, iso-THIP, iso-THAZ and 4-PIOL which all interact with the GABA receptors much differently. The growing pharmacological and toxicological interest based on many conflicting opinions on the use of the neuroprotective role of muscimol analogs against some neurodegenerative diseases, its potent role in the treatment of cerebral ischemia and other socially significant health conditions provided the basis for this review.

Perinatal hypoxia and thalamus brain region: increased efficiency of antiepileptic drug levetiracetam to inhibit GABA release from nerve terminals

Levetiracetam (LV), 2S-(2-oxo-1-pyrrolidiny1) butanamide, is an antiepileptic drug. The exact mechanisms of anticonvulsant effects of LV remain unclear. In this study, rats (Wistar strain) underwent hypoxia and seizures at the age of 10–12 postnatal days (pd). [3H]GABA release was analysed in isolated from thalamus nerve terminals (synaptosomes) during development at the age of pd 17–19 and pd 24–26 (infantile stage), pd 38–40 (puberty) and pd 66–73 (young adults) in control and after perinatal hypoxia. The extracellular level of [3H]GABA in the preparation of thalamic synaptosomes increased during development at the age of pd 38–40 and pd 66–73 as compared to earlier ones. LV did not influence the extracellular level of [3H]GABA in control and after perinatal hypoxia at all studied ages. Exocytotic [3H]GABA release in control increased at the age of pd 24–26 as compared to pd 17–19. After hypoxia, exocytotic [3H]GABA release from synaptosomes also increased during development. LV elevated [3H]GABA release from thalamic synaptosomes at the age of pd 66–73 after hypoxia and during blockage of GABA uptake by NO-711 only. LV realizes its antiepileptic effects at the presynaptic site through an increase in exocytotic release of [3H]GABA in thalamic synaptosomes after perinatal hypoxia at pd 66–73. LV exhibited a more significant effect in thalamic synaptosomes after perinatal hypoxia than in control ones. The action of LV is age-dependent, and the drug was inert at the infantile stage that can be useful for an LV application strategy in child epilepsy therapy. Keywords: brain development, exocytosis, GABA, levetiracetam, perinatal hypoxia, thalamic synaptosomes

Astrocytic GABA transporter 1 deficit in novel SLC6A1 variants mediated epilepsy: Connected from protein destabilization to seizures in mice and humans

ObjectiveMutations in γ-aminobutyric acid (GABA) transporter 1 (GAT-1)-encoding SLC6A1 have been associated with myoclonic atonic epilepsy and other phenotypes. We determined the patho-mechanisms of the mutant GAT-1, in order to identify treatment targets. MethodsWe conducted whole-exome sequencing of patients with myoclonic atonic epilepsy (MAE) and characterized the seizure phenotypes and EEG patterns. We studied the protein stability and structural changes with homology modeling and machine learning tools. We characterized the function and trafficking of the mutant GAT-1 with 3H radioactive GABA uptake assay and confocal microscopy. We utilized different models including a knockin mouse and human astrocytes derived from induced pluripotent stem cells (iPSCs). We focused on astrocytes because of their direct impact of astrocytic GAT-1 in seizures. ResultsWe identified four novel SLC6A1 variants associated with MAE and 2 to 4 Hz spike-wave discharges as a common EEG feature. Machine learning tools predicted that the variant proteins are destabilized. The variant protein had reduced expression and reduced GABA uptake due to endoplasmic reticular retention. The consistent observation was made in cortical and thalamic astrocytes from variant-knockin mice and human iPSC-derived astrocytes. The Slc6a+/A288V mouse, representative of MAE, had increased 5–7 Hz spike-wave discharges and absence seizures. InterpretationSLC6A1 variants in various locations of the protein peptides can cause MAE with similar seizure phenotypes and EEG features. Reduced GABA uptake is due to decreased functional GAT-1, which, in thalamic astrocytes, could result in increased extracellular GABA accumulation and enhanced tonic inhibition, leading to seizures and abnormal EEGs.

Neuromodulation by selective angiotensin-converting enzyme 2 inhibitors

Here, neuromodulatory effects of selective angiotensin-converting enzyme 2 (ACE2) inhibitors were investigated. Two different types of small molecule ligands for ACE2 inhibition were selected using chemical genetic approach, they were synthesized using developed chemical method and tested using presynaptic rat brain nerve terminals (synaptosomes). EBC-36032 (1 µM) increased in a dose-dependent manner spontaneous and stimulated ROS generation in nerve terminals that was of non-mitochondrial origin. Another inhibitor EBC-36033 (MLN-4760) was inert regarding modulation of ROS generation. EBC-36032 and EBC-36033 (100 µM) did not modulate the exocytotic release of L-[14C]glutamate, whereas both inhibitors decreased the initial rate of uptake, but not accumulation (10 min) of L-[14C]glutamate by nerve terminals. EBC-36032 (100 µM) decreased the exocytotic release as well as the initial rate and accumulation of [3H]GABA by nerve terminals. EBC-36032 and EBC-36033 did not change the extracellular levels and transporter-mediated release of [3H]GABA and L-[14C]glutamate, and tonic leakage of [3H]GABA from nerve terminals. Therefore, synthesized selective ACE2 inhibitors decreased uptake of glutamate and GABA as well as exocytosis of GABA at the presynaptic level. The initial rate of glutamate uptake was the only parameter that was mitigated by both ACE2 inhibitors despite stereochemistry issues. In terms of ACE2-targeted antiviral/anti-SARS-CoV-2 and other therapies, novel ACE2 inhibitors should be checked on the subject of possible renin–angiotensin system (RAS)-independent neurological side effects.

High performance liquid chromatography-based method to analyze activity of GABA transporters in central nervous system

The GATs are the membrane proteins responsible for the uptake of GABA in the central nervous system. Alterations in GAT activity are implicated in several neurological diseases, including retinopathies. The present study describes an alternative method to determine GAT activity in tissue preparations of the central nervous system, using high performance liquid chromatography (HPLC) with fluorescence detection. The GABA concentration in the medium was determined using the o-phthaldehyde (OPA)-derivation protocol validated by the Brazilian Health Regulatory Agency (ANVISA) and the United States Food and Drug Administration (US-FDA). The GAT activity in the retinal preparations was determined through the evaluation of the GABA uptake, which was measured by assessing the difference between the initial and final concentrations of GABA in the incubation medium. The evaluation of the GAT kinetics returned values of Km = 382.5 ± 32.2 μM and Vmax = 34 nmol/mg of protein. The data also demonstrated that the GABA uptake was predominantly Na+- and temperature-dependent, and was also inhibited by incubation with nipecotic acid, a substrate of GABA transporters. Taken together, these findings confirm that our approach provided a specific measure of GAT activity in retinal tissue. The data presented here thus validate, for the first time, an alternative, simple and sensitive method for the evaluation of GAT activity using high performance chromatography on preparations of the central nervous system.

Amphiphilic anti-SARS-CoV-2 drug remdesivir incorporates into the lipid bilayer and nerve terminal membranes influencing excitatory and inhibitory neurotransmission

Remdesivir is a novel antiviral drug, which is active against the SARS-CoV-2 virus. Remdesivir is known to accumulate in the brain but it is not clear whether it influences the neurotransmission. Here we report diverse and pronounced effects of remdesivir on transportation and release of excitatory and inhibitory neurotransmitters in rat cortex nerve terminals (synaptosomes) in vitro. Direct incorporation of remdesivir molecules into the cellular membranes was shown by FTIR spectroscopy, planar phospholipid bilayer membranes and computational techniques. Remdesivir decreases depolarization-induced exocytotic release of L-[14C] glutamate and [3H] GABA, and also [3H] GABA uptake and extracellular level in synaptosomes in a dose-dependent manner. Fluorimetric studies confirmed remdesivir-induced impairment of exocytosis in nerve terminals and revealed a decrease in synaptic vesicle acidification. Our data suggest that remdesivir dosing during antiviral therapy should be precisely controlled to prevent possible neuromodulatory action at the presynaptic level. Further studies of neurotropic and membranotropic effects of remdesivir are necessary.