Loss of MeCP2 increases GABA uptake by astrocytes to suppress tonic inhibition of CA1 pyramidal neurons

Rett syndrome (RTT) is a neurodevelopmental disorder characterized a spectrum of phenotypes affecting neuronal and glial populations. Recent work by Dong et al. (Dong Q, Kim J, Nguyen L, Bu Q, Chang Q. J Neurosci 40: 6250–6261, 2020) suggests that augmented GABA uptake by astrocytes diminishes tonic inhibition in the hippocampus and contributes to increased seizure propensity in RTT. Here, I will review evidence supporting this possibility and critically evaluate how increased expression of a GABA transporter might contribute to this mechanism.

Molecular Determinants and Pharmacological Analysis for a Class of Competitive Non-transported Bicyclic Inhibitors of the Betaine/GABA Transporter BGT1

The betaine/GABA transporter 1 (BGT1) is a member of the GABA transporter (GAT) family with still elusive function, largely due to a lack of potent and selective tool compounds. Based on modeling, we here present the design, synthesis and pharmacological evaluation of five novel conformationally restricted cyclic GABA analogs related to the previously reported highly potent and selective BGT1 inhibitor (1S,2S,5R)-5-aminobicyclo[3.1.0]hexane-2-carboxylic acid (bicyclo-GABA). Using [3H]GABA radioligand uptake assays at the four human GATs recombinantly expressed in mammalian cell lines, we identified bicyclo-GABA and its N-methylated analog (2) as the most potent and selective BGT1 inhibitors. Additional pharmacological characterization in a fluorescence-based membrane potential assay showed that bicyclo-GABA and 2 are competitive inhibitors, not substrates, at BGT1, which was validated by a Schild analysis for bicyclo-GABA (pKB value of 6.4). To further elaborate on the selectivity profile both compounds were tested at recombinant α1β2γ2 GABAA receptors. Whereas bicyclo-GABA showed low micromolar agonistic activity, the N-methylated 2 was completely devoid of activity at GABAA receptors. To further reveal the binding mode of bicyclo-GABA and 2 binding hypotheses of the compounds were obtained from in silico-guided mutagenesis studies followed by pharmacological evaluation at selected BGT1 mutants. This identified the non-conserved BGT1 residues Q299 and E52 as the molecular determinants driving BGT1 activity and selectivity. The binding mode of bicyclo-GABA was further validated by the introduction of activity into the corresponding GAT3 mutant L314Q (38 times potency increase cf. wildtype). Altogether, our data reveal the molecular determinants for the activity of bicyclic GABA analogs, that despite their small size act as competitive inhibitors of BGT1. These compounds may serve as valuable tools to selectively and potently target BGT1 in order to decipher its elusive pharmacological role in the brain and periphery such as the liver and kidneys.

Reactive astrocytes augment hippocampal inhibitory tone via GABA transporter-3/4 to facilitate synaptic balance in Alzheimer’s disease

Background and Purpose: Cognitive decline is a major symptom in Alzheimer’s disease (AD), which is closely associated with synaptic excitatory-inhibitory imbalance. Here, we investigated whether astrocytic mechanisms involving the astrocyte-specific GABA transporter 3/4 (GAT3/4) play a role in altering the synaptic balance in AD and whether these mechanisms correlate with presynaptic cannabinoid type-1 receptors (CB1-Rs). Experimental approach: Using the APPNL-F/NL-F knock-in mouse model of AD, aged-matched to wild-type mice, we performed in vitro electrophysiological whole-cell recordings combined with immunohistochemistry in the CA1 and dentate gyrus (DG) regions of the hippocampus. Comparative neuroanatomy experiments were also performed in post-mortem brain tissue from human AD patients, age-matched to healthy controls. Results: We observed a higher expression of GABA content and GAT3/4 co-localised with reactive astrocytes, which enhanced tonic inhibition in the CA1, and DG of APPNL-F/NL-F mice compared to the age-matched wild-type animals. Blocking GAT3/4 - associated tonic inhibition in APPNL-F/NL-F mice resulted in an enhanced frequency of synaptic excitation, suggesting a presynaptic mechanism. These data also correlated with an up-regulation of CB1-Rs in astrocytes and cholecystokinin (CCK)-containing interneurons, which also enhanced tonic inhibition in the AD model, but did not affect GAT3/4 -associated tonic inhibition. The neuroanatomical results were mirrored in post-mortem tissue of AD patients. Conclusions: Our data suggest that reactive astrocytes lead to augmented tonic inhibition in the hippocampus, which probably plays an important presynaptic compensatory role in attempting to restore AD-associated neuronal hyperactivity. Therefore, reducing tonic inhibition through GAT3/4 may not be a good therapeutic strategy for AD.

Epileptic Mechanisms Shared by Alzheimer’s Disease: Viewed via the Unique Lens of Genetic Epilepsy

Our recent work on genetic epilepsy (GE) has identified common mechanisms between GE and neurodegenerative diseases including Alzheimer’s disease (AD). Although both disorders are seemingly unrelated and occur at opposite ends of the age spectrum, it is likely there are shared mechanisms and studies on GE could provide unique insights into AD pathogenesis. Neurodegenerative diseases are typically late-onset disorders, but the underlying pathology may have already occurred long before the clinical symptoms emerge. Pathophysiology in the early phase of these diseases is understudied but critical for developing mechanism-based treatment. In AD, increased seizure susceptibility and silent epileptiform activity due to disrupted excitatory/inhibitory (E/I) balance has been identified much earlier than cognition deficit. Increased epileptiform activity is likely a main pathology in the early phase that directly contributes to impaired cognition. It is an enormous challenge to model the early phase of pathology with conventional AD mouse models due to the chronic disease course, let alone the complex interplay between subclinical nonconvulsive epileptiform activity, AD pathology, and cognition deficit. We have extensively studied GE, especially with gene mutations that affect the GABA pathway such as mutations in GABAA receptors and GABA transporter 1. We believe that some mouse models developed for studying GE and insights gained from GE could provide unique opportunity to understand AD. These include the pathology in early phase of AD, endoplasmic reticulum (ER) stress, and E/I imbalance as well as the contribution to cognitive deficit. In this review, we will focus on the overlapping mechanisms between GE and AD, the insights from mutations affecting GABAA receptors, and GABA transporter 1. We will detail mechanisms of E/I imbalance and the toxic epileptiform generation in AD, and the complex interplay between ER stress, impaired membrane protein trafficking, and synaptic physiology in both GE and AD.

Astrocytic GABA Transporter controls sleep by modulating GABAergic signaling in Drosophila circadian neurons

A precise balance between sleep and wakefulness is essential to sustain a good quality of life and optimal brain function. GABA is known to play a key and conserved role in sleep control, and GABAergic tone should therefore be tightly controlled in sleep circuits. Here we examined the role of the astrocytic GABA transporter (GAT) in sleep regulation using Drosophila melanogaster. We found that a hypomorphic gat mutation (gat33-1) increased sleep amount, decreased sleep latency, and increased sleep consolidation. Interestingly, sleep defects were suppressed when gat33-1 was combined with a mutation disrupting wide-awake (wake), a gene that regulates the cell-surface levels of the GABAA receptor Resistance to Dieldrin (RDL) in the wake-promoting large ventral lateral neurons (l-LNvs). Moreover, RNAi knockdown of rdl and its modulator dnlg4 in these circadian neurons also suppressed gat33-1 sleep phenotypes. Brain immunohistochemistry showed that GAT-expressing astrocytes were located near RDL-positive l-LNvs cell bodies and dendritic processes. We conclude that astrocytic GAT decreases GABAergic tone and RDL activation in arousal promoting LNvs, thus determining proper sleep amount and quality in Drosophila

Dual Role for Astroglial Copper-Assisted Polyamine Metabolism during Intense Network Activity

Astrocytes serve essential roles in human brain function and diseases. Growing evidence indicates that astrocytes are central players of the feedback modulation of excitatory Glu signalling during epileptiform activity via Glu-GABA exchange. The underlying mechanism results in the increase of tonic inhibition by reverse operation of the astroglial GABA transporter, induced by Glu-Na+ symport. GABA, released from astrocytes, is synthesized from the polyamine (PA) putrescine and this process involves copper amino oxidase. Through this pathway, putrescine can be considered as an important source of inhibitory signaling that counterbalances epileptic discharges. Putrescine, however, is also a precursor for spermine that is known to enhance gap junction channel communication and, consequently, supports long-range Ca2+ signaling and contributes to spreading of excitatory activity through the astrocytic syncytium. Recently, we presented the possibility of neuron-glia redox coupling through copper (Cu+/Cu2+) signaling and oxidative putrescine catabolism. In the current work, we explore whether the Cu+/Cu2+ homeostasis is involved in astrocytic control on neuronal excitability by regulating PA catabolism. We provide supporting experimental data underlying this hypothesis. We show that the blockade of copper transporter (CTR1) by AgNO3 (3.6 µM) prevents GABA transporter-mediated tonic inhibitory currents, indicating causal relationship between copper (Cu+/Cu2+) uptake and the catabolism of putrescine to GABA in astrocytes. In addition, we show that MnCl2 (20 μM), an inhibitor of the divalent metal transporter DMT1, also prevents the astrocytic Glu-GABA exchange. Furthermore, we observed that facilitation of copper uptake by added CuCl2 (2 µM) boosts tonic inhibitory currents. These findings corroborate the hypothesis that modulation of neuron-glia coupling by copper uptake drives putrescine → GABA transformation, which leads to subsequent Glu-GABA exchange and tonic inhibition. Findings may in turn highlight the potential role of copper signaling in fine-tuning the activity of the tripartite synapse.

GABA transporter sustains IL-1β production in macrophages

Accumulating evidence shows that nervous system governs host immune responses; however, how γ-aminobutyric acid (GABA)ergic system shapes the function of innate immune cells is poorly defined. Here, we demonstrate that GABA transporter (GAT2) modulates the macrophage function. GAT2 deficiency lowers the production of interleukin-1β (IL-1β) in proinflammatory macrophages. Mechanistically, GAT2 deficiency boosts the betaine/S-adenosylmethionine (SAM)/hypoxanthine metabolic pathway to inhibit transcription factor KID3 expression through the increased DNA methylation in its promoter region. KID3 regulates oxidative phosphorylation (OXPHOS) via targeting the expression of OXPHOS-related genes and is also critical for NLRP3–ASC–caspase-1 complex formation. Likewise, GAT2 deficiency attenuates macrophage-mediated inflammatory responses in vivo, including lipopolysaccharide-induced sepsis, infection-induced pneumonia, and high-fat diet-induced obesity. Together, we propose that targeting GABAergic system (e.g., GABA transporter) could provide previously unidentified therapeutic opportunities for the macrophage-associated diseases.

“Activity of a subset of vesicular GABA-transporter neurons in the ventral zona incerta anticipate sleep onset”

Study Objectives Sleep and wake are opposing behavioral states controlled by the activity of specific neurons that need to be located and mapped. To better understand how a waking brain falls asleep it is necessary to identify activity of individual phenotype-specific neurons, especially neurons that anticipate sleep onset. In freely-behaving mice, we used microendoscopy to monitor calcium (Ca 2+) fluorescence in individual hypothalamic neurons expressing the vesicular GABA transporter (vGAT), a validated marker of GABA neurons. Methods vGAT-Cre mice (male=3; female=2) transfected with rAAV-FLEX-GCaMP6M in the lateral hypothalamus were imaged 30 days later during multiple episodes of waking (W), non-rapid-eye movement sleep (NREMS) or REMS (REMS). Results 372 vGAT neurons were recorded in the zona incerta. 23.9% of the vGAT neurons showed maximal fluorescence during wake (classified as wake-max), 4% were NREM-max, 56.2% REM-max and 5.9% wake/REM max, while 9.9% were state indifferent. In the NREM-max group, Ca 2+ fluorescence began to increase before onset of NREM sleep, remained high throughout NREM sleep, and declined in REM sleep. Conclusions We found that 60.2% of the vGAT GABA neurons in the zona incerta had activity that was biased towards sleep (NREM and REMS). A subset of vGAT neurons (NREM-max) became active in advance of sleep onset and may induce sleep by inhibiting the activity of the arousal neurons. Abnormal activation of the NREM-max neurons may drive sleep attacks and hypersomnia.