GABA transporter mGat4 is involved in multiple neural functions in mice.

γ-Aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system. The termination of GABA transmission is through the action of GABA transporters (GATs). mGAT4 (encoded by Slc6a11) is another GAT besides GAT1 (encoded by Slc6a1) that functions in GABA reuptake in CNS. Research on the function of mGAT4 is still in its infancy. We developed an mGat4 knockout mouse model (mGat4-/- mice) and performed a series of behavioral analyses for the first time to study the effect of mGat4 on biological processes in CNS. Our results indicated that homozygous mGat4-/- mice had less depression, anxiety-like behavior and more social activities than their wild-type littermate controls. However, they had weight loss and showed motor incoordination and imbalance. Meanwhile, mGat4-/- mice showed increased pain threshold and hypoalgesia behavior in nociceptive stimulus and learning and memory impairments. The expression of multiple components of the GABAergic system including GAD67, GABAA and KCC2 was altered. There is little or no compensatory change in mGat1. In a word, mGat4 may play a key role in normal motor coordination, sensation, emotion, learning and memory and could be the potential target of neurological disorders.

γ-Aminobutyric acid transporter and GABAA receptor mechanisms in Slc6a1+/A288V and Slc6a1+/S295L mice associated with developmental and epileptic encephalopathies.

We have previously characterized the molecular mechanisms for variants in γ-aminobutyric acid transporter 1-encoding solute carrier family 6-member 1 (SLC6A1) in vitro and concluded that a partial or complete loss of γ-aminobutyric acid uptake due to impaired protein trafficking is the primary aetiology. Impairment of γ-aminobutyric acid transporter 1 function could cause compensatory changes in the expression of γ-aminobutyric acid receptors, which, in turn, modify disease pathophysiology and phenotype. Here we used different approaches including radioactive 3H γ-aminobutyric acid uptake in cells and synaptosomes, immunohistochemistry and confocal microscopy as well as brain slice surface protein biotinylation to characterize Slc6a1+/A288V and Slc6a1+/S295L mice, representative of a partial or a complete loss of function of SLC6A1 mutations, respectively. We employed the γ-aminobutyric acid transporter 1-specific inhibitor [3H]tiagabine binding and GABAA receptor subunit-specific radioligand binding to profile the γ-aminobutyric acid transporter 1 and GABAA receptor expression in major brain regions such as cortex, cerebellum, hippocampus and thalamus. We also determined the total and surface expression of γ-aminobutyric acid transporter 1, γ-aminobutyric acid transporter 3 and expression of GABAA receptor in the major brain regions in the knockin mice. We found that γ-aminobutyric acid transporter 1 protein was markedly reduced in cortex, hippocampus, thalamus and cerebellum in both mutant mouse lines. Consistent with the findings of reduced γ-aminobutyric acid uptake for both γ-aminobutyric acid transporter 1(A288V) and γ-aminobutyric acid transporter 1(S295L), both the total and the γ-aminobutyric acid transporter 1-mediated 3H γ-aminobutyric acid reuptake was reduced. We found that γ-aminobutyric acid transporter 3 is only abundantly expressed in the thalamus and there was no compensatory increase of γ-aminobutyric acid transporter 3 in either of the mutant mouse lines. γ-Aminobutyric acid transporter 1 was reduced in both somatic regions and nonsomatic regions in both mouse models, in which a ring-like structure was identified only in the Slc6a1+/A288V mouse, suggesting more γ-aminobutyric acid transporter 1 retention inside endoplasmic reticulum in the Slc6a1+/A288V mouse. The [3H]tiagabine binding was similar in both mouse models despite the difference in γ-aminobutyric acid uptake function and γ-aminobutyric acid transporter 1 protein expression for both mutations. There were no differences in GABAA receptor subtype expression, except for a small increase in the expression of α5 subunits of GABAA receptor in the hippocampus of Slc6a1S295L homozygous mice, suggesting a potential interaction between the expression of this GABAA receptor subtype and the mutant γ-aminobutyric acid transporter 1. The study provides the first comprehensive characterization of the SLC6A1 mutations in vivo in two representative mouse models. Because both γ-aminobutyric acid transporter 1 and GABAA receptors are targets for anti-seizure medications, the findings from this study can help guide tailored treatment options based on the expression and function of γ-aminobutyric acid transporter 1 and GABAA receptor in SLC6A1 mutation-mediated neurodevelopmental and epileptic encephalopathies.

A new method for isolation and purification of fusion-competent inhibitory synaptic vesicles.

Synaptic vesicles specific to inhibitory GABA-releasing neurons are critical for regulating neuronal excitability. To study the specific molecular composition, architecture, and function of inhibitory synaptic vesicles, we have developed a new method to isolate and purify GABA synaptic vesicles from mouse brains. GABA synaptic vesicles were immunoisolated from mouse brain tissue using an engineered fragment antigen-binding region (Fab) against the vesicular GABA transporter (vGAT) and purified. Western blot analysis confirmed that the GABA synaptic vesicles were specifically enriched for vGAT and largely depleted of contaminants from other synaptic vesicle types, such as vesicular glutamate transporter (vGLUT1), and other cellular organelles. This degree of purity was achieved despite the relatively low abundance of vGAT vesicles compared to the total synaptic vesicle pool in mammalian brains. Cryo-electron microscopy images of these isolated GABA synaptic vesicles revealed intact morphology with circular shape and protruding proteinaceous densities. The GABA synaptic vesicles are functional, as assessed by a hybrid (ex vivo/in vitro) vesicle fusion assay, and they undergo synchronized fusion with synthetic plasma membrane mimic vesicles in response to Ca2+-triggering, but, as a negative control, not to Mg2+-triggering. Our immunoisolation method could also be applied to other types of vesicles.

Astrocytic GABAergic Regulation in Alcohol Use and Major Depressive Disorders.

Gamma-aminobutyric acid (GABA) is the major inhibitory neurotransmitter in the central nervous system (CNS). Most GABAergic neurons synthesize GABA from glutamate and release it in the synaptic cleft in the CNS. However, astrocytes can also synthesize and release GABA, activating GABA receptors in the neighboring neurons in physiological and pathological conditions. As the primary homeostatic glial cells in the brain, astrocytes play a crucial role in regulating GABA homeostasis and synaptic neurotransmission. Accumulating evidence demonstrates that astrocytic GABA dysregulation is implicated in psychiatric disorders, including alcohol use disorder (AUD) and major depressive disorder (MDD), the most prevalent co-occurring psychiatric disorders. Several current medications and emerging pharmacological agents targeting GABA levels are in clinical trials for treating AUD and MDD. This review offers a concise summary of the role of astrocytic GABA regulation in AUD and MDD. We also provide an overview of the current understanding and areas of debate regarding the mechanisms by which astrocytes regulate GABA in the CNS and their potential significance in the molecular basis of AUD and MDD, paving the way toward future research directions and potential therapeutic target areas within this field.

Corrigendum: Cerebellar and basal ganglia inputs define three main nuclei in the mouse ventral motor thalamus.

[This corrects the article DOI: 10.3389/fnana.2023.1242839.].

TMEM230 modulates seizure in epilepsy: evidence from temporal lobe epilepsy patients and mouse models.

Transmembrane protein (TMEM230) is located in secretory/recycling vesicles, including synaptic vesicles in neurons. However, the functional relationship between TMEM230 and epilepsy is still a mystery. The aims of this study were to investigate the expression of TMEM230 in patients with temporal lobe epilepsy (TLE) and two different mice models of chronic epilepsy, and to determine the probable roles of TMEM230 in epilepsy. Our results showed that TMEM230 expression was increased in the temporal neocortex of epileptic patients and the hippocampus and cortex of epileptic mice compared with that in the control tissues. Moreover, TMEM230 was mainly expressed in the neurons in both humans and mice epileptic brain. TMEM230 co-localized with glutamate vesicular transporter 1 (VGLUT-1), but not with vesicular GABA transporter (VGAT). Mechanistically, coimmunoprecipitation confirmed that TMEM230 interacted with VGLUT-1, but not with VGAT in the hippocampus of epileptic mice. Lentivirus mediated overexpression of TMEM230 increased mice susceptibility to epilepsy and behavioural phenotypes of epileptic seizures during the kainite (KA)-induced chronic phase of epileptic seizures and the pentylenetetrazole (PTZ) kindling process, whereas lentivirus-mediated TMEM230 downregulation had the opposite effect. These results shed light on the functions of TMEM230 in neurons, suggesting that TMEM230 may play a critical role in the regulation of epileptic activity via influencing excitatory neurotransmission.

Action Sequence Learning Is Impaired in Genetically Modified Mice with the Suppressed GABAergic Transmission from the Thalamic Reticular Nucleus to the Thalamus.

The thalamic reticular nucleus (TRN) is a thin sheet of GABAergic neurons surrounding the thalamus, and it regulates the activity of thalamic relay neurons. The TRN has been reported to be involved in sensory gating, attentional regulation, and some other functions. However, little is known about the contribution of the TRN to sequence learning. In the present study, we examined whether the TRN is involved in reward-based learning of action sequence with no eliciting stimuli (operant conditioning), by analyzing the performance of male and female Avp-Vgat-/- mice (Vgatflox/flox mice crossed to an Avp-Cre driver line) on tasks conducted in an operant box having three levers. Our histological and electrophysiological data demonstrated that in adult Avp-Vgat-/- mice, vesicular GABA transporter (VGAT) was absent in most TRN neurons and the GABAergic transmission from the TRN to the thalamus was largely suppressed. The performance on a task in which mice needed to press an active lever for food reward showed that simple operant learning of lever pressing and learning of win-stay and lose-shift strategies are not affected in Avp-Vgat-/- mice. In contrast, the performance on a task in which mice needed to press three levers in a correct order for food reward showed that learning of the order of lever pressing (action sequence learning) was impaired in Avp-Vgat-/- mice. These results suggest that the TRN plays an important role in action sequence learning.

E2730, an uncompetitive γ-aminobutyric acid transporter-1 inhibitor, suppresses epileptic seizures in a rat model of chronic mesial temporal lobe epilepsy.

OBJECTIVE: More than one third of mesial temporal lobe epilepsy (MTLE) patients are resistant to current antiseizure medications (ASMs), and half experience mild-to-moderate adverse effects of ASMs. There is therefore a strong need to develop and test novel ASMs. The objective of this work is to evaluate the pharmacokinetics and neurological toxicity of E2730, a novel uncompetitive inhibitor of γ-aminobutyric acid transporter-1, and to test its seizure suppression effects in a rat model of chronic MTLE. METHODS: We first examined plasma levels and adverse neurological effects of E2730 in healthy Wistar rats. Adult male rats were implanted with osmotic pumps delivering either 10, 20, or 100 mg/kg/day of E2730 subcutaneously for 1 week. Blood sampling and behavioral assessments were performed at several timepoints. We next examined whether E2730 suppressed seizures in rats with chronic MTLE. These rats were exposed to kainic acid-induced status epilepticus, and 9 weeks later, when chronic epilepsy was established, were assigned to receive one of the three doses of E2730 or vehicle for 1 week in a randomized crossover design. Continuous video-electroencephalographic monitoring was acquired during the treatment period to evaluate epileptic seizures. RESULTS: Plasma levels following continuous infusion of E2730 showed a clear dose-related increase in concentration. The drug was well tolerated at all doses, and any sedation or neuromotor impairment was mild and transient, resolving within 48 h of treatment initiation. Remarkably, E2730 treatment in chronically epileptic rats led to seizure suppression in a dose-dependent manner, with 65% of rats becoming seizure-free at the highest dose tested. Mean seizure class did not differ between the treatment groups. SIGNIFICANCE: This study shows that continuous subcutaneous infusion of E2730 over 7 days results in a marked, dose-dependent suppression of spontaneous recurrent seizures, with minimal adverse neurological effects, in a rat model of chronic MTLE. E2730 shows strong promise as an effective new ASM to be translated into clinical trials.

Cerebellar and basal ganglia inputs define three main nuclei in the mouse ventral motor thalamus.

The thalamus is a central link between cortical and subcortical brain motor systems. Axons from the deep nuclei of the cerebellum (DCN), or the output nuclei of the basal ganglia system (substantia nigra reticulata, SNr; and internal pallidum GPi/ENT) monosynaptically innervate the thalamus, prominently some nuclei of the ventral nuclear group. In turn, axons from these ventral nuclei innervate the motor and premotor areas of the cortex, where their input is critical for planning, execution and learning of rapid and precise movements. Mice have in recent years become a widely used model in motor system research. However, information on the distribution of cerebellar and basal ganglia inputs in the rodent thalamus remains poorly defined. Here, we mapped the distribution of inputs from DCN, SNr, and GPi/ENT to the ventral nuclei of the mouse thalamus. Immunolabeling for glutamatergic and GABAergic neurotransmission markers delineated two distinct main territories, characterized each by the presence of large vesicular glutamate transporter type 2 (vGLUT2) puncta or vesicular GABA transporter (vGAT) puncta. Anterograde labeling of axons from DCN revealed that they reach virtually all parts of the ventral nuclei, albeit its axonal varicosities (putative boutons) in the vGAT-rich sector are consistently smaller than those in the vGLUT2-rich sector. In contrast, the SNr axons innervate the whole vGAT-rich sector, but not the vGLUT2-rich sector. The GPi/ENT axons were found to innervate only a small zone of the vGAT-rich sector which is also targeted by the other two input systems. Because inputs fundamentally define thalamic cell functioning, we propose a new delineation of the mouse ventral motor nuclei that is consistent with the distribution of DCN, SNr and GPi/ENT inputs and resembles the general layout of the ventral motor nuclei in primates.

Lipopolysaccharide augments microglial GABA uptake by increasing GABA transporter-1 trafficking and bestrophin-1 expression.

Gamma-aminobutyric acid (GABA), the principal inhibitory neurotransmitter in the brain, affects numerous immune cell functions. Microglia, the brain's resident innate immune cells, regulate GABA signaling through GABA receptors and express the complete GABAergic machinery for GABA synthesis, uptake, and release. Here, the use of primary microglial cell cultures and ex vivo brain tissue sections allowed for demonstrating that treatment with lipopolysaccharide (LPS) increased microglial GABA uptake as well as GABA transporter (GAT)-1 trafficking. This effect was not entirely abolished by treatment with GAT inhibitors (GAT-Is). Notably, LPS also induced microglial upregulation of bestrophin-1 (BEST-1), a Ca2+ -activated Cl- channel permeable to GABA. Combined administration of GAT-Is and a BEST-1 inhibitor completely abolished LPS-induced microglial GABA uptake. Interestingly, increased microglial GAT-1 membrane turnover via syntaxin 1A was detected in LPS-treated cultures after BEST-1 blockade. Altogether, these findings provided evidence for a novel mechanism through which LPS may trigger the inflammatory response by directly altering microglial GABA clearance and identified the GAT-1/BEST-1 interplay as a potential novel mechanism involved in brain inflammation.

Bile acid interactions with neurotransmitter transporters.

Synthesized in the liver from cholesterol, the bile acids (BAs) primary role is emulsifying fats to facilitate their absorption. BAs can cross the blood-brain barrier (BBB) and be synthesized in the brain. Recent evidence suggests a role for BAs in the gut-brain signaling by modulating the activity of various neuronal receptors and transporters, including the dopamine transporter (DAT). In this study, we investigated the effects of BAs and their relationship with substrates in three transporters of the solute carrier 6 family. The exposure to obeticholic acid (OCA), a semi-synthetic BA, elicits an inward current (IBA) in the DAT, the GABA transporter 1 (GAT1), and the glycine transporter 1 (GlyT1b); this current is proportional to the current generated by the substrate, respective to the transporter. Interestingly, a second consecutive OCA application to the transporter fails to elicit a response. The full displacement of BAs from the transporter occurs only after exposure to a saturating concentration of a substrate. In DAT, perfusion of secondary substrates norepinephrine (NE) and serotonin (5-HT) results in a second OCA current, decreased in amplitude and proportional to their affinity. Moreover, co-application of 5-HT or NE with OCA in DAT, and GABA with OCA in GAT1, did not alter the apparent affinity or the Imax, similar to what was previously reported in DAT in the presence of DA and OCA. The findings support the previous molecular model that suggested the ability of BAs to lock the transporter in an occluded conformation. The physiological significance is that it could possibly avoid the accumulation of small depolarizations in the cells expressing the neurotransmitter transporter. This achieves better transport efficiency in the presence of a saturating concentration of the neurotransmitter and enhances the action of the neurotransmitter on their receptors when they are present at reduced concentrations due to decreased availability of transporters.

Visualizing GABA transporters in vivo: an overview of reported radioligands and future directions.

By clearing GABA from the synaptic cleft, GABA transporters (GATs) play an essential role in inhibitory neurotransmission. Consequently, in vivo visualization of GATs can be a valuable diagnostic tool and biomarker for various psychiatric and neurological disorders. Not surprisingly, in recent years several research attempts to develop a radioligand have been conducted, but so far none have led to suitable radioligands that allow imaging of GATs. Here, we provide an overview of the radioligands that were developed with a focus on GAT1, since this is the most abundant transporter and most of the research concerns this GAT subtype. Initially, we focus on the field of GAT1 inhibitors, after which we discuss the development of GAT1 radioligands based on these inhibitors. We hypothesize that the radioligands developed so far have been unsuccessful due to the zwitterionic nature of their nipecotic acid moiety. To overcome this problem, the use of non-classical GAT inhibitors as basis for GAT1 radioligands or the use of carboxylic acid bioisosteres may be considered. As the latter structural modification has already been used in the field of GAT1 inhibitors, this option seems particularly viable and could lead to the development of more successful GAT1 radioligands in the future.

Laminar-Specific Alterations in Calbindin-Positive Boutons in the Prefrontal Cortex of Subjects With Schizophrenia.

BACKGROUND: Cognitive deficits in schizophrenia are associated with altered GABA (gamma-aminobutyric acid) neurotransmission in the prefrontal cortex (PFC). GABA neurotransmission requires GABA synthesis by 2 isoforms of glutamic acid decarboxylase (GAD65 and GAD67) and packaging by the vesicular GABA transporter (vGAT). Current postmortem findings suggest that GAD67 messenger RNA is lower in a subset of the calbindin-expressing (CB+) class of GABA neurons in schizophrenia. Hence, we assessed if CB+ GABA neuron boutons are affected in schizophrenia. METHODS: For 20 matched pairs of subjects with schizophrenia and unaffected comparison subjects, PFC tissue sections were immunolabeled for vGAT, CB, GAD67, and GAD65. The density of CB+ GABA boutons and levels of the 4 proteins per bouton were quantified. RESULTS: Some CB+ GABA boutons contained both GAD65 and GAD67 (GAD65+/GAD67+), whereas others contained only GAD65 (GAD65+) or GAD67 (GAD67+). In schizophrenia, vGAT+/CB+/GAD65+/GAD67+ bouton density was not altered, vGAT+/CB+/GAD65+ bouton density was 86% higher in layers 2/superficial 3 (L2/3s), and vGAT+/CB+/GAD67+ bouton density was 36% lower in L5-6. Bouton GAD levels were differentially altered across bouton types and layers. In schizophrenia, the sum of GAD65 and GAD67 levels in vGAT+/CB+/GAD65+/GAD67+ boutons was 36% lower in L6, GAD65 levels were 51% higher in vGAT+/CB+/GAD65+ boutons in L2, and GAD67 levels in vGAT+/CB+/GAD67+ boutons were 30% to 46% lower in L2/3s-6. CONCLUSIONS: These findings indicate that schizophrenia-associated alterations in the strength of inhibition from CB+ GABA neurons in the PFC differ across cortical layers and bouton classes, suggesting complex contributions to PFC dysfunction and cognitive impairments in schizophrenia.

Understanding the function of the GABAergic system and its potential role in rheumatoid arthritis.

Rheumatoid arthritis (RA) is a highly disabling chronic autoimmune disease. Multiple factors contribute to the complex pathological process of RA, in which an abnormal autoimmune response, high survival of inflammatory cells, and excessive release of inflammatory factors lead to a severe chronic inflammatory response. Clinical management of RA remains limited; therefore, exploring and discovering new mechanisms of action could enhance clinical benefits for patients with RA. Important bidirectional communication occurs between the brain and immune system in inflammatory diseases such as RA, and circulating immune complexes can cause neuroinflammatory responses in the brain. The gamma-aminobutyric acid (GABA)ergic system is a part of the nervous system that primarily comprises GABA, GABA-related receptors, and GABA transporter (GAT) systems. GABA is an inhibitory neurotransmitter that binds to GABA receptors in the presence of GATs to exert a variety of pathophysiological regulatory effects, with its predominant role being neural signaling. Nonetheless, the GABAergic system may also have immunomodulatory effects. GABA/GABA-A receptors may inhibit the progression of inflammation in RA and GATs may promote inflammation. GABA-B receptors may also act as susceptibility genes for RA, regulating the inflammatory response of RA via immune cells. Furthermore, the GABAergic system may modulate the abnormal pain response in RA patients. We also summarized the latest clinical applications of the GABAergic system and provided an outlook on its clinical application in RA. However, direct studies on the GABAergic system and RA are still lacking; therefore, we hope to provide potential therapeutic options and a theoretical basis for RA treatment by summarizing any potential associations.

Experimental and Bioinformatic Insights into the Effects of Epileptogenic Variants on the Function and Trafficking of the GABA Transporter GAT-1.

In this article, we identified a novel epileptogenic variant (G307R) of the gene SLC6A1, which encodes the GABA transporter GAT-1. Our main goal was to investigate the pathogenic mechanisms of this variant, located near the neurotransmitter permeation pathway, and compare it with other variants located either in the permeation pathway or close to the lipid bilayer. The mutants G307R and A334P, close to the gates of the transporter, could be glycosylated with variable efficiency and reached the membrane, albeit inactive. Mutants located in the center of the permeation pathway (G297R) or close to the lipid bilayer (A128V, G550R) were retained in the endoplasmic reticulum. Applying an Elastic Network Model, to these and to other previously characterized variants, we found that G307R and A334P significantly perturb the structure and dynamics of the intracellular gate, which can explain their reduced activity, while for A228V and G362R, the reduced translocation to the membrane quantitatively accounts for the reduced activity. The addition of a chemical chaperone (4-phenylbutyric acid, PBA), which improves protein folding, increased the activity of GAT-1WT, as well as most of the assayed variants, including G307R, suggesting that PBA might also assist the conformational changes occurring during the alternative access transport cycle.

A novel approach for vital visualization and studying of neurons containing Ca2+ -permeable AMPA receptors.

Calcium-permeable AMPA receptors (CP-AMPARs) play a pivotal role in brain functioning in health and disease. They are involved in synaptic plasticity, synaptogenesis, and neuronal circuits development. However, the functions of neurons expressing CP-AMPARs and their role in the modulation of network activity remain elusive since reliable and accurate visualization methods are absent. Here we developed an approach allowing the vital identification of neurons containing CP-AMPARs. The proposed method relies on evaluating Ca2+ influx in neurons during activation of AMPARs in the presence of NMDAR and KAR antagonists, and blockers of voltage-gated Ca2+ channels. Using this method, we studied the properties of CP-AMPARs-containing neurons. We showed that the overwhelming majority of neurons containing CP-AMPARs are GABAergic, and they are distinguished by higher amplitudes of the calcium responses to applications of the agonists. Furthermore, about 30% of CP-AMPARs-containing neurons demonstrate the presence of GluK1-containing KARs. Although CP-AMPARs-containing neurons are characterized by more significant Ca2+ influx during the activation of AMPARs than other neurons, AMPAR-mediated Na+ influx is similar in these two groups. We revealed that neurons containing CP-AMPARs demonstrate weak GABA(A)R-mediated inhibition because of the low percentage of GABAergic synapses on the soma of these cells. However, our data show that weak GABA(A)R-mediated inhibition is inherent to all GABAergic neurons in the culture and cannot be considered a unique feature of CP-AMPARs-containing neurons. We believe that the suggested approach will help to understand the role of CP-AMPARs in the mammalian nervous system in more detail.

Quantitative analysis of the GABAergic innervation of the soma and axon initial segment of pyramidal cells in the human and mouse neocortex.

Perisomatic GABAergic innervation in the cerebral cortex is carried out mostly by basket and chandelier cells, which differentially participate in the control of pyramidal cell action potential output and synchronization. These cells establish multiple synapses with the cell body (and proximal dendrites) and the axon initial segment (AIS) of pyramidal neurons, respectively. Using multiple immunofluorescence, confocal microscopy and 3D quantification techniques, we have estimated the number and density of GABAergic boutons on the cell body and AIS of pyramidal neurons located through cortical layers of the human and mouse neocortex. The results revealed, in both species, that there is clear variability across layers regarding the density and number of perisomatic GABAergic boutons. We found a positive linear correlation between the surface area of the soma, or the AIS, and the number of GABAergic terminals in apposition to these 2 neuronal domains. Furthermore, the density of perisomatic GABAergic boutons was higher in the human cortex than in the mouse. These results suggest a selectivity for the GABAergic innervation of the cell body and AIS that might be related to the different functional attributes of the microcircuits in which neurons from different layers are involved in both human and mouse.

Age, Education Years, and Biochemical Factors Are Associated with Selective Neuronal Changes in the Elderly Hippocampus.

Brain aging involves regional alterations of specific cellular subpopulations in the human hippocampus: a network hub for memory consolidation. The present study investigates whether age, sex, education years, and the concentration of neuropathological and inflammatory proteins influence neuronal-type marker expression in the elderly hippocampus. We analyzed the digital images (1 µm/pixel) of postmortem hippocampal sections from 19 non-demented individuals (from 78 to 99 years). This material was obtained from the "Aging Dementia and TBI Study" open database. Brain samples were processed through in situ hybridization (ISH) for the immunodetection of VGLUT1 (glutamatergic transporter) and GAT1 (GABAergic transporter) and mRNAs and Luminex protein quantifications. After image acquisition, we delineated the dentate gyrus, CA 3/2, and CA1 hippocampal subdivisions. Then, we estimated the area fraction in which the ISH markers were expressed. Increased VGLUT1 was observed in multiple hippocampal subfields at late ages. This glutamatergic marker is positively correlated with beta-amyloid and tau proteins and negatively correlated with interleukin-7 levels. Additionally, education years are positively correlated with GAT1 in the hippocampus of elderly women. This GABAergic marker expression is associated with interferon-gamma and brain-derived neurotrophic factor levels. These associations can help to explain how hippocampal sub-regions and neurotransmitter systems undergo distinct physiological changes during normal aging.

4-Phenylbutyrate restored γ-aminobutyric acid uptake and reduced seizures in SLC6A1 patient variant-bearing cell and mouse models.

We have studied the molecular mechanisms of variants in solute carrier Family 6 Member 1 associated with neurodevelopmental disorders, including various epilepsy syndromes, autism and intellectual disability. Based on functional assays of solute carrier Family 6 Member 1 variants, we conclude that partial or complete loss of γ-amino butyric acid uptake due to reduced membrane γ-amino butyric acid transporter 1 trafficking is the primary aetiology. Importantly, we identified common patterns of the mutant γ-amino butyric acid transporter 1 protein trafficking from biogenesis, oligomerization, glycosylation and translocation to the cell membrane across variants in different cell types such as astrocytes and neurons. We hypothesize that therapeutic approaches to facilitate membrane trafficking would increase γ-amino butyric acid transporter 1 protein membrane expression and function. 4-Phenylbutyrate is a Food and Drug Administration-approved drug for paediatric use and is orally bioavailable. 4-Phenylbutyrate shows promise in the treatment of cystic fibrosis. The common cellular mechanisms shared by the mutant γ-amino butyric acid transporter 1 and cystic fibrosis transmembrane conductance regulator led us to hypothesize that 4-phenylbutyrate could be a potential treatment option for solute carrier Family 6 Member 1 mutations. We examined the impact of 4-phenylbutyrate across a library of variants in cell and knockin mouse models. Because γ-amino butyric acid transporter 1 is expressed in both neurons and astrocytes, and γ-amino butyric acid transporter 1 deficiency in astrocytes has been hypothesized to underlie seizure generation, we tested the effect of 4-phenylbutyrate in both neurons and astrocytes with a focus on astrocytes. We demonstrated existence of the mutant γ-amino butyric acid transporter 1 retaining wildtype γ-amino butyric acid transporter 1, suggesting the mutant protein causes aberrant protein oligomerization and trafficking. 4-Phenylbutyrate increased γ-amino butyric acid uptake in both mouse and human astrocytes and neurons bearing the variants. Importantly, 4-phenylbutyrate alone increased γ-amino butyric acid transporter 1 expression and suppressed spike wave discharges in heterozygous knockin mice. Although the mechanisms of action for 4-phenylbutyrate are still unclear, with multiple possibly being involved, it is likely that 4-phenylbutyrate can facilitate the forward trafficking of the wildtype γ-amino butyric acid transporter 1 regardless of rescuing the mutant γ-amino butyric acid transporter 1, thus increasing γ-amino butyric acid uptake. All patients with solute carrier Family 6 Member 1 variants are heterozygous and carry one wildtype allele, suggesting a great opportunity for treatment development leveraging wildtype protein trafficking. The study opens a novel avenue of treatment development for genetic epilepsy via drug repurposing.

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

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.