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Tonic GABA B receptor-mediated postsynaptic currents are not present in WT or GAT1 KO CA1 pyramidal cells. A: no postsynaptic outward currents were present in WT as tested by the GABA B receptor antagonist CGP 62349 (8 M). This experiment was performed in the presence of NO-711 (10 M). In an untreated GAT1 KO slice (right), a tonic postsynaptic GABA B current was also absent. The pyramidal cells were recorded with a K-based intracellular solution-filled electrode and held at –50 mV. Slices were perfused with kynurenic acid (3 mM) and picrotoxin (50 M) to block ionotropic glutamate and GABA A receptors, respectively. B: baclofen (10 M) induced a robust GABA B receptor-mediated current, which was revealed during antagonism by CGP 62349 (8 M). The GABA B current reached 55 pA in the exemplar WT cell and 60 pA in the GAT1 KO cell. Scale bars apply to all traces in A and B. C: summary of postsynaptic GABA B currents in CA1 pyramidal cells. The WT data consist of the pooled results from 5 cells where NO-711 (n 2) or NO-711 0.8 M GABA (n 3) was present. No tonic GABA B currents were observed (1.3 1.4 pA, n 5). When NO-711 and GABA were replaced by 10 M baclofen, the GABA B currents reached 50.4 13.9 pA (n 9). For GAT1 KO slices, the GABA B current reached 1.3 1.4 pA (n 3) in the absence and 41.2 5.9 pA (n 7) in the presence of baclofen (10 M). The baclofen-induced currents were not different between WT vs. GAT1 KO (P 0.05). Error bars indicate SE.
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After its release from interneurons in the CNS, the major inhibitory neurotransmitter GABA is taken up by GABA transporters (GATs). The predominant neuronal GABA transporter GAT1 is localized in GABAergic axons and nerve terminals, where it is thought to influence GABAergic synaptic transmission, but the details of this regulation are unclear. To a...
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Context 1
... of such elevated [GABA]o, we also expected postsynaptic GABA B receptors to be tonically activated in CA1 pyramidal cells. We tested this hypothesis using K-methylsulfate-filled pipettes to record from neurons voltage-clamped at -50 mV while the GABA B receptor antagonist CGP 62349 was injected into the bath to yield a final concentration of 8 M (Fig. 5A). Surprisingly, nei- ther NO-711 (10 M, n 2) nor NO-711 plus 0.8 M GABA (n 3) induced any detectable postsynaptic GABA B 4. Miniature IPSCs (mIPSCs) in GAT1 KO mice have reduced fre- quencies compared with WT but similar kinetics. A: mIPSCs were recorded in TTX (1 M) in WT and GAT1 KO CA1 pyramidal cells. In GAT1 KOs, the mIPSCs had ...
Context 2
... WT and GAT1 KO cells. Longer inter-event intervals were observed for GAT1 KO mIPSCs. currents in WT slices (Fig. 5A, 1.3 1.4 pA, the 5 cells pooled). Similarly, a tonic postsynaptic GABA B receptor current could not be detected in GAT1 KO slices (1.3 0.9 pA, n 3). To ascertain the presence of functional postsyn- aptic GABA B receptors (Fig. 5B), we perfused the GABA B agonist baclofen (10 M). This induced a robust CGP 62349-sensitive current of 50.4 13.9 pA in WT (n 9) and 41.2 5.9 pA in GAT1 KO (n 7). Although the GAT1 KO baclofen response was somewhat smaller, this difference did not reach statistical significance (i. e., P 0.05, when compared with WT, unpaired t-test ...
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The inhibitory γ-aminobutyric acid–containing (GABAergic) neurons of the thalamic reticular and perigeniculate nuclei are
involved in the generation of normal and abnormal synchronized activity in thalamocortical networks. An important factor controlling
the generation of activity in this system is the amplitude and duration of inhibitory postsynap...
Citations
... In astrocytes, GAT-1 and GAT-3 function to regulate GABA uptake (Ghirardini et al., 2018). Activation of TRPA1 induces a Na + transient, which reduces GABA uptake by decreasing the driving force of the GABA transporter (GAT) (Jensen et al., 2003). In addition, transient Ca 2+ influx activates the Na + /Ca 2+ exchanger and produces a further elevation of intracellular Na + , thereby reducing GABA uptake (Hilge, 2012). ...
Ultrasound is highly biopermeable and can non-invasively penetrate deep into the brain. Stimulation with patterned low-intensity ultrasound can induce sustained inhibition of neural activity in humans and animals, with potential implications for research and therapeutics. Although mechanosensitive channels are involved, the cellular and molecular mechanisms underlying neuromodulation by ultrasound remain unknown. To investigate the mechanism of action of ultrasound stimulation, we studied the effects of two types of patterned ultrasound on synaptic transmission and neural network activity using whole-cell recordings in primary cultured hippocampal cells. Single-shot pulsed-wave (PW)
or continuous-wave (CW) ultrasound had no effect on neural activity. By contrast,
although repetitive CW stimulation also had no effect, repetitive PW stimulation
persistently reduced spontaneous recurrent burst firing. This inhibitory effect
was dependent on extrasynaptic—but not synaptic—GABAA receptors, and
the effect was abolished under astrocyte-free conditions. Pharmacological
activation of astrocytic TRPA1 channels mimicked the effects of ultrasound by increasing the tonic GABAA current induced by ambient GABA. Pharmacological blockade of TRPA1 channels abolished the inhibitory effect of ultrasound. These findings suggest that the repetitive PW low-intensity ultrasound used in our study does not have a direct effect on neural function but instead exerts its sustained neuromodulatory effect through modulation of ambient GABA levels via channels with characteristics of TRPA1, which is expressed in astrocytes.
... 14 To address this gap, we obtained chronic wireless ECoG and EMG recordings from mice lacking zero, one, or two copies of the Slc6a1 gene, corresponding to 100%, ~65%, and <2% rates of GAT-1-mediated [ 3 H]GABA uptake, respectively. 15 We observed a gradient of epileptic abnormalities with progressive GAT-1 deficiency. Wild-type GAT-1 +/+ mice had normal ECoG, whereas GAT-1 +/− mice had frequent SWDs associated with motor arrest (Figure 2A). ...
The solute carrier family 6 member 1 (SLC6A1) gene encodes GAT‐1, a γ‐aminobutyric acid transporter expressed on astrocytes and inhibitory neurons. Mutations in SLC6A1 are associated with epilepsy and developmental disorders, including motor and social impairments, but variant‐specific animal models are needed to elucidate mechanisms. Here, we report electrocorticographic (ECoG) recordings and clinical data from a patient with a variant in SLC6A1 that encodes GAT‐1 with a serine‐to‐leucine substitution at amino acid 295 (S295L), who was diagnosed with childhood absence epilepsy. Next, we show that mice bearing the S295L mutation (GAT‐1S295L/+) have spike‐and‐wave discharges with motor arrest consistent with absence‐type seizures, similar to GAT‐1+/− mice. GAT‐1S295L/+ and GAT‐1+/− mice follow the same pattern of pharmacosensitivity, being bidirectionally modulated by ethosuximide (200 mg/kg ip) and the GAT‐1 antagonist NO‐711 (10 mg/kg ip). By contrast, GAT‐1−/− mice were insensitive to both ethosuximide and NO‐711 at the doses tested. In conclusion, ECoG findings in GAT‐1S295L/+ mice phenocopy GAT‐1 haploinsufficiency and provide a useful preclinical model for drug screening and gene therapy investigations.
... In hippocampus, synaptically released GABA is the main source of extracellular GABA [GABA] e . GAT1 operating in uptake mode is the dominant transporter that keeps [GABA] e low and prevents activation of extrasynaptic GABA A and GABA B Rs (Jensen et al., 2003;Glykys and Mody, 2007). In the cerebral cortex however, GAT-3-mediated GABA efflux can be observed even under resting conditions (Kinney, 2005;Kirmse and Kirischuk, 2006). ...
The efficiency of neocortical information processing critically depends on the balance between the glutamatergic (excitatory, E) and GABAergic (inhibitory, I) synaptic transmission. A transient imbalance of the E/I-ratio during early development might lead to neuropsychiatric disorders later in life. The transgenic glutamic acid decarboxylase 67-green fluorescent protein (GAD67-GFP) mouse line (KI) was developed to selectively visualize GABAergic interneurons in the CNS. However, haplodeficiency of the GAD67 enzyme, the main GABA synthetizing enzyme in the brain, temporarily leads to a low GABA level in the developing brain of these animals. However, KI mice did not demonstrate any epileptic activity and only few and mild behavioral deficits. In the present study we investigated how the developing somatosensory cortex of KI-mice compensates the reduced GABA level to prevent brain hyperexcitability. Whole-cell patch clamp recordings from layer 2/3 pyramidal neurons at P14 and at P21 revealed a reduced frequency of miniature inhibitory postsynaptic currents (mIPSCs) in KI mice without any change in amplitude or kinetics. Interestingly, mEPSC frequencies were also decreased, while the E/I-ratio was nevertheless shifted toward excitation. Surprisingly, multi-electrode-recordings (MEA) from acute slices revealed a decreased spontaneous neuronal network activity in KI mice compared to wild-type (WT) littermates, pointing to a compensatory mechanism that prevents hyperexcitability. Blockade of GABA B receptors (GABA B Rs) with CGP55845 strongly increased the frequency of mEPSCs in KI, but failed to affect mIPSCs in any genotype or age. It also induced a membrane depolarization in P14 KI, but not in P21 KI or WT mice. MEA recordings in presence of CGP55845 revealed comparable levels of network activity in both genotypes, indicating that tonically activated GABA B Rs balance neuronal activity in P14 KI cortex despite the reduced GABA levels. Blockade of GABA transporter 3 (GAT-3) reproduced the CGP55845 effects suggesting that tonic activation of GABA B Rs is mediated by ambient GABA released via GAT-3 operating in reverse mode. We conclude that GAT-3-mediated GABA release leads to tonic activation of both pre- and postsynaptic GABA B Rs and restricts neuronal excitability in the developing cortex to compensate for reduced neuronal GABA synthesis. Since GAT-3 is predominantly located in astrocytes, GAD67 haplodeficiency may potentially stimulate astrocytic GABA synthesis through GAD67-independent pathways.
... Impairment and loss of any part of this complex system can alter cellular function and behavior. [29][30][31] Chronic stress, for example, can alter GABAergic inhibitory neuronal function in the basolateral amygdala (BLA), resulting in hyperexcitability of outflow of principal excitatory neurons culminating in fear and anxiety. 10,32 Reduced GABA A receptor-mediated inhibition has been suggested as a mechanism of hyperexcitability in patients with Fragile X syndrome. ...
Hyperexcitability is a major mechanism implicated in several neuropsychiatric disorders, such as organophosphate-induced status epilepticus (SE), primary epilepsy, stroke, spinal cord injury, traumatic brain injury, schizophrenia, and autism spectrum disorders. Underlying mechanisms are diverse, but a functional impairment and loss of GABAergic inhibitory neurons are common features in many of these disorders. While novel therapies abound to correct for the loss of GABAergic inhibitory neurons, it has been difficult at best to improve the activities of daily living for the majority of patients. Alpha-linolenic acid (ALA) is an essential omega-3 polyunsaturated fatty acid found in plants. ALA exerts pleiotropic effects in the brain that attenuate injury in chronic and acute brain disease models. However, the effect of ALA on GABAergic neurotransmission in hyperexcitable brain regions involved in neuropsychiatric disorders, such as the basolateral amygdala (BLA) and CA1 subfield of the hippocampus, is unknown. Administration of a single dose of ALA (1500 nmol/kg) subcutaneously increased the charge transfer of inhibitory postsynaptic potential currents mediated by GABAA receptors in pyramidal neurons by 52% in the BLA and by 92% in the CA1 compared to vehicle animals a day later. Similar results were obtained in pyramidal neurons from the BLA and CA1 when ALA was bath-applied in slices from naïve animals. Importantly, pretreatment with the high-affinity, selective TrkB inhibitor, k252, completely abolished the ALA-induced increase in GABAergic neurotransmission in the BLA and CA1, suggesting a brain-derived neurotrophic factor (BDNF)-mediated mechanism. Addition of mature BDNF (20 ng/mL) significantly increased GABAA receptor inhibitory activity in the BLA and CA1 pyramidal neurons similar to the results obtained with ALA. ALA may be an effective treatment for neuropsychiatric disorders where hyperexcitability is a major feature.
... In addition to the enzymes involved in glutamate and GABA metabolism, it has also been investigated to what extent manipulation of the cellular transporters for the two amino acid neurotransmitters may influence epileptiform activity. Thus, deletion of GAT1 in mice leads to increased sensitivity toward induced seizures (Chiu et al., 2005;Jensen et al., 2003), whereas human mutations in GAT1, causing dysfunctional GABA uptake, are associated with epilepsy, autism, and intellectual disability (Goodspeed et al., 2020;Mermer et al., 2021). Curiously, overexpression of GAT1 in mice, also increases susceptibility toward induced seizures Zhao et al., 2003), with subsequent impaired cognition . ...
Since it was first generally accepted that the two amino acids glutamate and GABA act as principal neurotransmitters, several landmark discoveries relating to this function have been uncovered. Synaptic homeostasis of these two transmitters involves several cell types working in close collaboration and is facilitated by specialized cellular processes. Notably, glutamate and GABA are extensively recycled between neurons and astrocytes in a process known as the glutamate/GABA‐glutamine cycle, which is essential to maintain synaptic transmission. The glutamate/GABA‐glutamine cycle is intimately coupled to cellular energy metabolism and relies on the metabolic function of both neurons and astrocytes. Importantly, astrocytes display unique metabolic features allowing extensive metabolite release, hereby providing metabolic support for neurons. Furthermore, astrocytes undergo complex metabolic adaptations in response to injury and pathology, which may greatly affect the glutamate/GABA‐glutamine cycle and synaptic transmission during disease. In this Milestone Review we outline major discoveries in relation to synaptic balancing of glutamate and GABA signaling, including cellular uptake, metabolism, and recycling. We provide a special focus on how astrocyte function and metabolism contribute to sustain neuronal transmission through metabolite transfer. Recent advances on cellular glutamate and GABA homeostasis are reviewed in the context of brain pathology, including glutamate toxicity and neurodegeneration. Finally, we consider how pathological astrocyte metabolism may serve as a potential target of metabolic intervention. Integrating the multitude of fine‐tuned cellular processes supporting neurotransmitter recycling, will aid the next generation of major discoveries on brain glutamate and GABA homeostasis. image
... Furthermore, oligodendrocytes and microglia also express GAT1 [30,31], but the quantitative importance of these cell types for GABA uptake remains unknown. Deletion of GAT1 in mice does not affect viability or longevity, but leads to altered behavior and elevated tonic inhibition [32][33][34], whereas GAT3 deletion supposedly is lethal [35]. These observations suggest that astrocyte GAT3 function is essential, and that astrocyte GABA uptake via GAT3, to a large extent, may be able to compensate for the absence of GAT1. ...
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.
... In a previous study, the expression of these GABA transporters was noted to be elevated in CA1 and DG regions of PTZ-kindled rats resulting in diminished GABA levels to interact with GABA A receptors subsequently leading to a deficit in GABA inhibition [57]. In the current study, the mRNA expression of GAT-1 and GAT-3 were downregulated by tiagabine which leading to decreased GABA uptake, augmented GABAergic inhibition and anti-kindling effects [58]. ...
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.
... The above-mentioned data demonstrates that the [GABA] i of astrocytes is region dependent and may be as high, at about 10 mM. The extracellular GABA concentration ([GABA] o ) is region dependent as well and amounts to 0.25 µM in the cortex [40], 0.8 µM in the hippocampus [41] and 3.5 µM in thalamus [34]. Such a strong transmembrane GABA gradient might enable GABA release, both via GABA-permeable channels, following the chemical gradient, or via secondary active GABA transporters, using the electrochemical driving force. ...
... In addition to the GABA gradient, the transmembrane gradients of Na + (quadratic impact) and Cl − determine the electromotive driving force and, in turn, the direction of GABA transport. [40] and 0.8 µM in the hippocampus [41] favor the GAT3-mediated release under resting conditions in the former (GAT rev~− 70 mV) but not in the latter (GAT rev~− 35 mV). (D) To change the direction of GAT-mediated transport in the hippocampus, either [Na + ] i has to be increased to about 25-30 mM (gray arrow; for instance, as a result of EAAT-mediated glutamate uptake [21]) or [GABA] i , it has to be elevated to about 4 mM (dashed arrow) [28]. ...
... These estimates demonstrate that GAT reversal under resting conditions is more probable in the cerebral cortex [20,76]. Moreover, the relative position of the reversal potential, and thus the direction and strength of the astrocytic tonic GABA release can be modulated by [Na + ] i transients, occurring for example via the activation of EAATs ( Figure 3A In the hippocampus, the main source of extracellular GABA is supposed to have a neuronal origin, and GAT1 operating in the uptake mode is the dominant GAT isoform [41,82]. However, further studies revealed that astrocytic GAT3 contributes to setting the GABA levels in the extracellular space [83]. ...
Astrocytes are the most abundant glial cells in the central nervous system (CNS) mediating a variety of homeostatic functions, such as spatial K+ buffering or neurotransmitter reuptake. In addition, astrocytes are capable of releasing several biologically active substances, including glutamate and GABA. Astrocyte-mediated GABA release has been a matter of debate because the expression level of the main GABA synthesizing enzyme glutamate decarboxylase is quite low in astrocytes, suggesting that low intracellular GABA concentration ([GABA]i) might be insufficient to support a non-vesicular GABA release. However, recent studies demonstrated that, at least in some regions of the CNS, [GABA]i in astrocytes might reach several millimoles both under physiological and especially pathophysiological conditions, thereby enabling GABA release from astrocytes via GABA-permeable anion channels and/or via GABA transporters operating in reverse mode. In this review, we summarize experimental data supporting both forms of GABA release from astrocytes in health and disease, paying special attention to possible feedback mechanisms that might govern the fine-tuning of astrocytic GABA release and, in turn, the tonic GABAA receptor-mediated inhibition in the CNS.
... The content of GAD can reflect GABA concentration to some extent, and GAD67, an important isozyme, is involved in the synthesis of GABA (Sloviter et al., 1996). GAT-1 is a GABA transporter that regulates GABA reuptake to reduce GABA levels (Jensen et al., 2003). Excitatory transmission in neurons may be regulated by the number and composition of Glu receptors. ...
Background: Central fatigue (CF) is a subjective sense of tiredness associated with cognitive and memory disorders, accompanied by reduced physical endurance and negative emotions, such as anxiety and depression. Disease progression and prognosis with regards to CF have been unfavorable and possibly contribute to dementia, schizophrenia, and other diseases. Additionally, effective treatments for CF are lacking. KangPiLao decoction (KPLD) has been widely applied in clinical treatment and is composed of six Chinese herbal medicines, some of which have confirmed anti-fatigue effects. While glutamic acid (Glu) is the main excitatory transmitter in the central nervous system (CNS), gamma-aminobutyric acid (GABA) is the major inhibitory transmitter. Both are involved in emotional, cognitive, and memory functions. This research was designed to explore how KPLD regulates cognitive and emotional disorders in rats with CF and to identify the relationship between the regulatory effect and the GABA/Glu pathway.
Methods: The compounds comprising KPLD were analyzed using high-performance liquid chromatography-mass spectrometry. Sixty Wistar rats were randomly divided into six groups. The modified multiple platform method was used to induce CF. Cognitive, emotional, and fatigue states were evaluated by performing behavioral tests (Morris water maze [MWM], open-field test [OFT], and grip strength test). Histomorphology, western blotting, immunohistochemistry, and RT-qPCR were performed to investigate protein and mRNA expression levels in the hippocampus and prefrontal cortexes involved in the GABA/Glu pathway.
Results: Rats with CF exhibited impaired spatial cognition and increased negative emotions in the MWM and OFT. KPLD enabled the improvement of these symptoms, especially in the high-concentration group. Western blotting and RT-qPCR demonstrated that the expression of GABAARα1, GABAARγ2, GABABR1, and GAD67 in rats with CF was higher, whereas GAT-1 and NMDAR2B were lower in the hippocampus and prefrontal cortex. KPLD decreased the expression of GABAARα1, GABABR1, GABAARγ2, and GAD67 in the hippocampus and prefrontal cortex and enhanced the expression of NR2B in the prefrontal cortex.
Conclusion: KPLD significantly improved cognitive and emotional disorders in rats with CF by regulating the GABA/Glu pathway. Overall, KPLD may be a promising candidate for developing a drug for treating CF.
... All patients were diagnosed with MAE presenting with sudden falls and demonstrated a strong EEG correlate with absence seizure based on previous studies (Goodspeed et al., 2020;Cai et al., 2019;Poliquin et al., 2021). Electrophysiological features are similar to those described in GAT-1 deficient mice (Chiu et al., 2005;Jensen et al., 2003). All patients had 2 to 4 Hz SWDs that were dominant in frontal or centro-occipital regions. ...
... At the pre-synaptic terminal, GAT-1 is responsible for the re-uptake of GABA from the synaptic cleft. Although the data of the GABA reuptake from GAT-1 variant knockin mice is not available, it has been reported that in GAT-1-deficient knockout mice (Jensen et al., 2003), GABA reuptake is compromised, resulting in both increased ambient GABA levels and spontaneous spike-wave discharges. Increased ambient GABA would cause enhanced tonic inhibition, which is likely mediated by GABA A receptor δ subunit containing receptors (Cope et al., 2009). ...
Objective
Mutations 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.
Methods
We 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.
Results
We 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.
Interpretation
SLC6A1 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.
