GABA Transporter GAT-1 Research Articles

Glial alterations in the glutamatergic and GABAergic signaling pathways in a mouse model of Lafora disease, a severe form of progressive myoclonus epilepsy.

Lafora disease (LD; OMIM#254780) is a rare form of progressive myoclonus epilepsy characterized by the accumulation of insoluble deposits of aberrant glycogen (polyglucosans), named Lafora bodies (LBs), in the brain but also in peripheral tissues. It is assumed that the accumulation of LBs is related to the appearance of the characteristic pathological features of the disease. In mouse models of LD, we and others have reported an increase in the levels of reactive astrocytes and activated microglia, which triggers the expression of the different pro-inflammatory mediators. Recently, we have demonstrated that the TNF and IL-6 inflammatory signaling pathways are the main mediators of the neuroinflammatory phenotype associated with the disease. In this work, we present evidence that the activation of these pathways produces a dysregulation in the levels of different subunits of the excitatory ionotropic glutamatergic receptors (phopho-GluN2B, phospho-GluA2, GluK2) and also an increase in the levels of the GABA transporter GAT1 in the hippocampus of the Epm2b-/- mice. In addition, we present evidence of the presence of activated forms of the Src and Lyn protein kinases in this area. These effects may increase the excitatory glutamatergic signaling and decrease the inhibitory GABAergic tone, leading to hyper-excitability. More importantly, the enhanced production of these subunits occurs in non-neuronal cells such as activated microglia and reactive astrocytes, pointing out a key role of glia in the pathophysiology of LD.

Neuroinflammation alters GABAergic neurotransmission in hyperammonemia and hepatic encephalopathy, leading to motor incoordination. Mechanisms and therapeutic implications.

Enhanced GABAergic neurotransmission contributes to impairment of motor coordination and gait and of cognitive function in different pathologies, including hyperammonemia and hepatic encephalopathy. Neuroinflammation is a main contributor to enhancement of GABAergic neurotransmission through increased activation of different pathways. For example, enhanced activation of the TNFα-TNFR1-NF-κB-glutaminase-GAT3 pathway and the TNFα-TNFR1-S1PR2-CCL2-BDNF-TrkB pathway in cerebellum of hyperammonemic rats enhances GABAergic neurotransmission. This is mediated by mechanisms affecting GABA synthesizing enzymes GAD67 and GAD65, total and extracellular GABA levels, membrane expression of GABAA receptor subunits, of GABA transporters GAT1 and GAT three and of chloride co-transporters. Reducing neuroinflammation reverses these changes, normalizes GABAergic neurotransmission and restores motor coordination. There is an interplay between GABAergic neurotransmission and neuroinflammation, which modulate each other and altogether modulate motor coordination and cognitive function. In this way, neuroinflammation may be also reduced by reducing GABAergic neurotransmission, which may also improve cognitive and motor function in pathologies associated to neuroinflammation and enhanced GABAergic neurotransmission such as hyperammonemia, hepatic encephalopathy or Parkinson's disease. This provides therapeutic targets that may be modulated to improve cognitive and motor function and other alterations such as fatigue in a wide range of pathologies. As a proof of concept it has been shown that antagonists of GABAA receptors such as bicuculline reduces neuroinflammation and improves cognitive and motor function impairment in rat models of hyperammonemia and hepatic encephalopathy. Antagonists of GABAA receptors are not ideal therapeutic tools because they can induce secondary effects. As a more effective treatment to reduce GABAergic neurotransmission new compounds modulating it by other mechanisms are being developed. Golexanolone reduces GABAergic neurotransmission by reducing the potentiation of GABAA receptor activation by neurosteroids such as allopregnanolone. Golexanolone reduces neuroinflammation and GABAergic neurotransmission in animal models of hyperammonemia, hepatic encephalopathy and cholestasis and this is associated with improvement of fatigue, cognitive impairment and motor incoordination. This type of compounds may be useful therapeutic tools to improve cognitive and motor function in different pathologies associated with neuroinflammation and increased GABAergic neurotransmission.

Manganese- and zinc-coordinated interaction of Schistosoma japonicum glutathione S-transferase with neurotransmitter transporters GlyT1 and GAT3 in vitro

Glutathione S-transferases (GSTs) are a family of multifunctional isoenzymes involved in the neutralization of toxic compounds, drug resistance and several other cellular functions. The glutathione S-transferase enzyme of Schistosoma japonicum (SjGST-26) plays a role in human schistosomiasis and is also a frequently used fusion partner in mammalian and bacterial expression and pull-down systems. GSTs seem not to be naturally associated with metal ions. Exceptionally, in vitro, metal binding sites have been previously described in some schistosome GSTs; however, their possible physiological role is unclear. Molecules of several neurotransmitter transporters also contain a regulatory zinc binding site, which affects their transport cycle. Here we show that among several metals, manganese and zinc are able to induce a specific protein interaction of SjGST-26 with the glycine transporter GlyT1 and the GABA transporter GAT3 in vitro. The results suggest that metal-binding sites on SjGST-26 and neurotransmitter transporters might function in metal-coordinated interactions with other metalloproteins. Our results additionally indicate that the presence of metal ions in SjGST-26-based GST protein pull-down assays may lead to a false-positive interaction if the potential interacting target is the metalloprotein.

ANALYSIS OF THE FUNCTIONAL STATE OF ASTROGLIA IN THE HIPPOCAMPUS OF RATS GENETICALLY PRONE TO AUDIOGENIC SEIZURES

It was shown that abnormalities in astrocyte functions can cause alterations in neuronal excitability and promote the development of epilepsy, however, this question claims further investigation. The aim of the present work was to analyze the functional state of astrocytes in the hippocampus of Krushinsky-Molodkina (KM) rats genetically predisposed to the reflex audiogenic seizures. Adult naïve animals with fully-established audiogenic sensitivity were recruited in our experiments. Rats of parental Wistar strain were used as a control. Obtained results revealed unchanged protein expression of such astocytic markers as GFAP, ALDH1L1, and NFIA suggesting the absence of the reactive astrogliosis in the hippocampus of KM rats. SPARC and aquaporin 4 expression also did not differ from the control indicating no violations in astroglia regulation of synaptogenesis and water balance. Analysis of glial proteins responsible for reuptake and metabolism of neurotransmitters revealed normal expression of GABA transporter GAT-3 and glutamine synthetase, while glutamate transporters EAAT1 and 2 were significantly elevated pointing on enhanced activity of glutamate removal from synapses. In addition, decreased expression of the key glycolytic enzyme aldolase C, probably, indicated insufficient activity of glucose metabolism in astrocytes. Thus, obtained data pointed on genetically determined alterations of astroglia functions in the hippocampus of rats with inherited reflex epilepsy.

EXPLORING THE ROLE OF GAT1 AND GABA IN TEMOZOLOMIDE TREATED GLIOMA

Abstract Approximately 3000 Canadians a year are diagnosed with the most aggressive and fatal form of brain cancer called glioma. Even with surgical resection, chemotherapy, and radiotherapy these patients have an average survival of less than 20 months. Research in the past has primarily focused on identifying and targeting specific genetic mutations in the tumor. However, it is apparent that the environment surrounding the tumor can influence tumor growth, tumor spread into the brain, and drug resistance. The brain presents many challenges to glioma treatment, one of which is the unique environment in which gliomas grow, which contributes to poor patient outcomes. The interaction between tumor cells, surrounding normal brain tissue, and immune cells supports the aggressiveness of glioma. Using a model of chemosensitive and chemoresistant tumors, we have identified an increase in the GABA transporter GAT1 in the chemosensitive tumor treated with the standard chemotherapy temozolomide. GAT1 is primarily expressed on neurons, but has also been shown to be expressed on astrocytes and immune cells such as macrophages. Our lab has identified that macrophages tend to concentrate in the same region as GAT1 expressing cells within the tumor environment. In addition, multiple papers have shown that an increase in GABA promotes a glioma-promoting immune microenvironment. Thus, we believe that treatment with temozolomide causes an increase in environmental GAT1 expression, resulting in a reuptake of GABA, and promoting a glioma-inhibitory macrophage phenotype. We believe that understanding how the glioma-inhibitory macrophage phenotype occurs will help improve patient outcomes.

Molecular basis for substrate recognition and transport of human GABA transporter GAT1.

γ-Aminobutyric acid (GABA), an important inhibitory neurotransmitter in the central nervous system, is recycled through specific GABA transporters (GATs). GAT1, which is mainly expressed in the presynaptic terminals of axons, is a potential drug target of neurological disorders due to its essential role in GABA transport. Here we report four cryogenic electron microscopy structures of human GAT1, at resolutions of 2.2-3.2 Å. GAT1 in substrate-free form or in complex with the antiepileptic drug tiagabine exhibits an inward-open conformation. In the presence of GABA or nipecotic acid, inward-occluded structures are captured. The GABA-bound structure reveals an interaction network bridged by hydrogen bonds and ion coordination for GABA recognition. The substrate-free structure unwinds the last helical turn of transmembrane helix TM1a to release sodium ions and substrate. Complemented by structure-guided biochemical analyses, our studies reveal detailed mechanism of GABA recognition and transport, and elucidate mode of action of the inhibitors, nipecotic acid and tiagabine.

Cryo-EM structure of GABA transporter 1 reveals substrate recognition and transport mechanism

The inhibitory neurotransmitter γ-aminobutyric acid (GABA) is cleared from the synaptic cleft by the sodium- and chloride-coupled GABA transporter GAT1. Inhibition of GAT1 prolongs the GABAergic signaling at the synapse and is a strategy to treat certain forms of epilepsy. In this study, we present the cryo-electron microscopy structure of Rattus norvegicus GABA transporter 1 (rGAT1) at a resolution of 3.1 Å. The structure elucidation was facilitated by epitope transfer of a fragment-antigen binding (Fab) interaction site from the Drosophila dopamine transporter (dDAT) to rGAT1. The structure reveals rGAT1 in a cytosol-facing conformation, with a linear density in the primary binding site that accommodates a molecule of GABA, a displaced ion density proximal to Na site 1 and a bound chloride ion. A unique insertion in TM10 aids the formation of a compact, closed extracellular gate. Besides yielding mechanistic insights into ion and substrate recognition, our study will enable the rational design of specific antiepileptics.

A comparative review on the well-studied GAT1 and the understudied BGT-1 in the brain.

γ-aminobutyric acid (GABA) is the primary inhibitory neurotransmitter in the central nervous system (CNS). Its homeostasis is maintained by neuronal and glial GABA transporters (GATs). The four GATs identified in humans are GAT1 (SLC6A1), GAT2 (SLC6A13), GAT3 (SLC6A11), and betaine/GABA transporter-1 BGT-1 (SLC6A12) which are all members of the solute carrier 6 (SLC6) family of sodium-dependent transporters. While GAT1 has been investigated extensively, the other GABA transporters are less studied and their role in CNS is not clearly defined. Altered GABAergic neurotransmission is involved in different diseases, but the importance of the different transporters remained understudied and limits drug targeting. In this review, the well-studied GABA transporter GAT1 is compared with the less-studied BGT-1 with the aim to leverage the knowledge on GAT1 to shed new light on the open questions concerning BGT-1. The most recent knowledge on transporter structure, functions, expression, and localization is discussed along with their specific role as drug targets for neurological and neurodegenerative disorders. We review and discuss data on the binding sites for Na+, Cl-, substrates, and inhibitors by building on the recent cryo-EM structure of GAT1 to highlight specific molecular determinants of transporter functions. The role of the two proteins in GABA homeostasis is investigated by looking at the transport coupling mechanism, as well as structural and kinetic transport models. Furthermore, we review information on selective inhibitors together with the pharmacophore hypothesis of transporter substrates.

PDZ interaction of the GABA transporter GAT1 with the syntenin-1 in Neuro-2a cells

The GABA transporter GAT1 regulates brain inhibitory neurotransmission and it is considered a potential therapeutic target for the treatment of wide spectrum of neurological diseases including epilepsy, stroke and autism. Syntenin-1 binds to syntaxin 1A, which is known to regulate the plasma membrane insertion of several neurotransmitter transporters. Previously, a direct interaction of syntenin-1 with the glycine transporter GlyT2 was reported. Here, we show that the GABA transporter GAT1 also directly interacts with syntenin-1, involving both unidentified protein interaction interface and the GAT1 C-terminal PDZ binding motif interacting mainly with syntenin-1 PDZ domain 1. The PDZ interaction was eliminated by the mutation of GAT1 isoleucine 599 and tyrosine 598 located in PDZ positions 0 and -1, respectively. This indicates an unconventional PDZ interaction and possible regulation of the transporter PDZ motif via tyrosine phosphorylation. Whole syntenin-1 protein fused to GST protein and immobilised on glutathione resin coprecipitated intact GAT1 transporter from an extract of GAT1 transfected neuroblastoma N2a cells. This coprecipitation was inhibited by tyrosine phosphatases inhibitor pervanadate. The fluorescence tagged GAT1 and syntenin-1 colocalized upon coexpression in N2a cells. The above results show that syntenin-1 might be, in addition to GlyT2, directly involved in the trafficking of GAT1 transporter.

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.

Enhancing GAT-3 in thalamic astrocytes promotes resilience to brain injury in rodents.

Inflammatory processes induced by brain injury are important for recovery; however, when uncontrolled, inflammation can be deleterious, likely explaining why most anti-inflammatory treatments have failed to improve neurological outcomes after brain injury in clinical trials. In the thalamus, chronic activation of glial cells, a proxy of inflammation, has been suggested as an indicator of increased seizure risk and cognitive deficits that develop after cortical injury. Furthermore, lesions in the thalamus, more than other brain regions, have been reported in patients with viral infections associated with neurological deficits, such as SARS-CoV-2. However, the extent to which thalamic inflammation is a driver or by-product of neurological deficits remains unknown. Here, we found that thalamic inflammation in mice was sufficient to phenocopy the cellular and circuit hyperexcitability, enhanced seizure risk, and disruptions in cortical rhythms that develop after cortical injury. In our model, down-regulation of the GABA transporter GAT-3 in thalamic astrocytes mediated this neurological dysfunction. In addition, GAT-3 was decreased in regions of thalamic reactive astrocytes in mouse models of cortical injury. Enhancing GAT-3 in thalamic astrocytes prevented seizure risk, restored cortical states, and was protective against severe chemoconvulsant-induced seizures and mortality in a mouse model of traumatic brain injury, emphasizing the potential of therapeutically targeting this pathway. Together, our results identified a potential therapeutic target for reducing negative outcomes after brain injury.

Inhibitory co-transmission from midbrain dopamine neurons relies on presynaptic GABA uptake.

SUMMARYDopamine (DA)-releasing neurons in the substantia nigra pars compacta (SNcDA) inhibit target cells in the striatum through postsynaptic activation of γ-aminobutyric acid (GABA) receptors. However, the molecular mechanisms responsible for GABAergic signaling remain unclear, as SNcDA neurons lack enzymes typically required to produce GABA or package it into synaptic vesicles. Here, we show that aldehyde dehydrogenase 1a1 (Aldh1a1), an enzyme proposed to function as a GABA synthetic enzyme in SNcDA neurons, does not produce GABA for synaptic transmission. Instead, we demonstrate that SNcDA axons obtain GABA exclusively through presynaptic uptake using the membrane GABA transporter Gat1 (encoded by Slc6a1). GABA is then packaged for vesicular release using the vesicular monoamine transporter Vmat2. Our data therefore show that presynaptic transmitter recycling can substitute for de novo GABA synthesis and that Vmat2 contributes to vesicular GABA transport, expanding the range of molecular mechanisms available to neurons to support inhibitory synaptic communication.

Hyperammonemia Enhances GABAergic Neurotransmission in Hippocampus: Underlying Mechanisms and Modulation by Extracellular cGMP.

Rats with chronic hyperammonemia reproduce the cognitive and motor impairment present in patients with hepatic encephalopathy. It has been proposed that enhanced GABAergic neurotransmission in hippocampus may contribute to impaired learning and memory in hyperammonemic rats. However, there are no direct evidences of the effects of hyperammonemia on GABAergic neurotransmission in hippocampus or on the underlying mechanisms. The aims of this work were to assess if chronic hyperammonemia enhances the function of GABAA receptors in hippocampus and to identify the underlying mechanisms. Activation of GABAA receptors is enhanced in hippocampus of hyperammonemic rats, as analyzed in a multielectrode array system. Hyperammonemia reduces membrane expression of the GABA transporters GAT1 and GAT3, which is associated with increased extracellular GABA concentration. Hyperammonemia also increases gephyrin levels and phosphorylation of the β3 subunit of GABAA receptor, which are associated with increased membrane expression of the GABAA receptor subunits α1, α2, γ2, β3, and δ. Enhanced levels of extracellular GABA and increased membrane expression of GABAA receptors would be responsible for the enhanced GABAergic neurotransmission in hippocampus of hyperammonemic rats. Increasing extracellular cGMP reverses the increase in GABAA receptors activation by normalizing the membrane expression of GABA transporters and GABAA receptors. The increased GABAergic neurotransmission in hippocampus would contribute to cognitive impairment in hyperammonemic rats. The results reported suggest that reducing GABAergic tone in hippocampus by increasing extracellular cGMP or by other means may be useful to improve cognitive function in hyperammonemia and in cirrhotic patients with minimal or clinical hepatic encephalopathy.

Astrocytes in the ventral pallidum extinguish heroin seeking through GAT-3 upregulation and morphological plasticity at D1-MSN terminals

GABAergic projections from the nucleus accumbens core to the dorsolateral ventral pallidum are necessary for drug-conditioned cues to initiate relapse-like drug seeking. Astrocytes in the ventral pallidum are situated perisynaptically and regulate GABA transmission through expression of GABA uptake transporters, but whether they are involved in regulating drug seeking is unknown. To determine the contribution of ventral pallidal astrocytes to heroin seeking, we labeled astrocytes in male and female rats with a membrane-bound fluorescent tag and used confocal microscopy to quantify astroglial expression of the GABA transporter GAT-3 and astrocyte synaptic proximity after withdrawal from heroin self-administration and during 15 min of cued heroin seeking. We found that GAT-3 was upregulated in rats that had extinguished heroin seeking, but not in animals that were withdrawn from heroin without extinction training or in rats that extinguished sucrose seeking. When GAT-3 upregulation was reversed using a vivo-morpholino oligo, heroin seeking was restored in the extinguished context and extinction of cued heroin seeking was disrupted compared to control animals. Although astrocyte synaptic proximity was not altered overall after heroin withdrawal, examination of astrocyte proximity to accumbens D1- or D2-expressing afferents revealed a selective increase in astrocyte proximity with D1-expressing terminals during extinction of heroin self-administration. Experimentally-induced reduction of astrocyte synaptic proximity through knockdown of the astrocyte-selective actin-binding protein ezrin also markedly disrupted extinction of heroin seeking. Notably, GAT-3 or ezrin knockdown had no impact on context- or cue-induced seeking in sucrose-trained animals. These data show that astrocytes in the ventral pallidum undergo plasticity after extinction of heroin use that reduces seeking and highlight the importance of astrocyte-neuron interactions in shaping behaviors associated with opioid use disorder.

Late adolescence mortality in mice with brain-specific deletion of the volume-regulated anion channel subunit LRRC8A.

The leucine-rich repeat-containing family 8 member A (LRRC8A) is an essential subunit of the volume-regulated anion channel (VRAC). VRAC is critical for cell volume control, but its broader physiological functions remain under investigation. Recent studies in the field indicate that Lrrc8a disruption in brain astrocytes reduces neuronal excitability, impairs synaptic plasticity and memory, and protects against cerebral ischemia. In the present work, we generated the brain-wide conditional LRRC8A knock-out mice (LRRC8A bKO) using NestinCre-driven Lrrc8aflox/flox excision in neurons, astrocytes, and oligodendroglia. LRRC8A bKO animals were born close to the expected Mendelian ratio and developed without overt histological abnormalities, but, surprisingly, all died between 5 and 9 weeks of age with a seizure phenotype, which was confirmed by video and EEG recordings. Brain slice electrophysiology detected changes in the excitability of pyramidal cells and modified GABAergic inputs in the hippocampal CA1 region of LRRC8A bKO. LRRC8A-null hippocampi showed increased immunoreactivity of the astrocytic marker GFAP, indicating reactive astrogliosis. We also found decreased whole-brain protein levels of the GABA transporter GAT-1, the glutamate transporter GLT-1, and the astrocytic enzyme glutamine synthetase. Complementary HPLC assays identified reduction in the tissue levels of the glutamate and GABA precursor glutamine. Together, these findings suggest that VRAC provides vital control of brain excitability in mouse adolescence. VRAC deletion leads to a lethal phenotype involving progressive astrogliosis and dysregulation of astrocytic uptake and supply of amino acid neurotransmitters and their precursors.

A Computational Study of Astrocytic GABA Release at the Glutamatergic Synapse: EAAT-2 and GAT-3 Coupled Dynamics.

Neurotransmitter dynamics within neuronal synapses can be controlled by astrocytes and reflect key contributors to neuronal activity. In particular, Glutamate (Glu) released by activated neurons is predominantly removed from the synaptic space by perisynaptic astrocytic transporters EAAT-2 (GLT-1). In previous work, we showed that the time course of Glu transport is affected by ionic concentration gradients either side of the astrocytic membrane and has the propensity for influencing postsynaptic neuronal excitability. Experimental findings co-localize GABA transporters GAT-3 with EAAT-2 on the perisynaptic astrocytic membrane. While these transporters are unlikely to facilitate the uptake of synaptic GABA, this paper presents simulation results which demonstrate the coupling of EAAT-2 and GAT-3, giving rise to the ionic-dependent reversed transport of GAT-3. The resulting efflux of GABA from the astrocyte to the synaptic space reflects an important astrocytic mechanism for modulation of hyperexcitability. Key results also illustrate an astrocytic-mediated modulation of synaptic neuronal excitation by released GABA at the glutamatergic synapse.

Astroglia and Obsessive Compulsive Disorder.

Obsessive compulsive disorder (OCD) has a prevalence rate of 1-3% in the general population and has been ranked as one of the top ten leading causes of illness-related disability (American Psychiatric Association 2013; Kessler et al. 2005). OCD is characterized by persistent intrusive thoughts (obsessions) and repetitive behaviors (compulsions) (Leckman et al. 1997). There are various OCD-related disorders, including Tourette syndrome (TS), grooming disorders (e.g., skin-picking, trichotillomania), and autism spectrum disorders (ASD) that share considerable overlapping features with OCD (Browne et al. 2014). Although the neurobiological basis of OCD still remains obscure, neuroimaging studies in patients with OCD and OCD-related disorders have consistently identified hyperactivity in orbitofrontal cortex and striatum (Cerliani et al. 2015; Hou et al. 2014; Jung et al. 2017; Neuner et al. 2014). However, the cellular and synaptic abnormalities underlying this hyperactivity are unclear. The most prominent theory regarding the underlying mechanisms of OCD and OCD-related disorders is an increased excitation to inhibition (E/I) ratio due to increased glutamatergic excitation or reduced GABAergic inhibition (Albin and Mink 2006; Rubenstein and Merzenich 2003; Wu et al. 2012). A proper E/I ratio is achieved by factors expressed in neuron and glia. In astrocytes, both the glutamate transporter GLT1 and GABA transporter GAT-3 are critical for regulating the E/I balance (Aida et al. 2015; Aizawa et al. 2020; Boddum et al. 2016; Cui et al. 2014; Kersanté et al. 2013; Kiryk et al. 2008; Matos et al. 2018; Scimemi 2014; Sugimoto et al. 2018; Sugiyama et al. 2017; Tanaka et al. 1997; Zhao et al. 2018). Although astrocyte dysfunction has not been directly explored in OCD patients, several animal studies have found that astrocytes are involved in the pathophysiology of OCD. In this chapter, I highlight recent studies in which astrocyte dysfunction contributed to E/I imbalance, leading to pathological repetitive behaviors shared between patients with OCD, TS, and ASD.

Astrocytic GABA Accumulation in Experimental Temporal Lobe Epilepsy.

An imbalance of excitation and inhibition has been associated with the pathophysiology of epilepsy. Loss of GABAergic interneurons and/or synaptic inhibition has been shown in various epilepsy models and in human epilepsy. Despite this loss, several studies reported preserved or increased tonic GABAA receptor-mediated currents in epilepsy, raising the question of the source of the inhibitory transmitter. We used the unilateral intracortical kainate mouse model of temporal lobe epilepsy (TLE) with hippocampal sclerosis (HS) to answer this question. In our model we observed profound loss of interneurons in the sclerotic hippocampal CA1 region and dentate gyrus already 5 days after epilepsy induction. Consistent with the literature, the absence of interneurons caused no reduction of tonic inhibition of CA1 pyramidal neurons. In dentate granule cells the inhibitory currents were even increased in epileptic tissue. Intriguingly, immunostaining of brain sections from epileptic mice with antibodies against GABA revealed strong and progressive accumulation of the neurotransmitter in reactive astrocytes. Pharmacological inhibition of the astrocytic GABA transporter GAT3 did not affect tonic inhibition in the sclerotic hippocampus, suggesting that this transporter is not responsible for astrocytic GABA accumulation or release. Immunostaining further indicated that both decarboxylation of glutamate and putrescine degradation accounted for the increased GABA levels in reactive astrocytes. Together, our data provide evidence that the preserved tonic inhibitory currents in the epileptic brain are mediated by GABA overproduction and release from astrocytes. A deeper understanding of the underlying mechanisms may lead to new strategies for antiepileptic drug therapy.

Depression-like behaviors induced by defective PTPRT activity through dysregulated synaptic functions and neurogenesis.

PTPRT has been known to regulate synaptic formation and dendritic arborization of hippocampal neurons. PTPRT-/- null and PTPRT-D401A mutant mice displayed enhanced depression-like behaviors compared with wild-type mice. Transient knockdown of PTPRT in the dentate gyrus enhanced the depression-like behaviors of wild-type mice, whereas rescued expression of PTPRT ameliorated the behaviors of PTPRT-null mice. Chronic stress exposure reduced expression of PTPRT in the hippocampus of mice. In PTPRT-deficient mice the expression of GluR2 (also known as GRIA2) was attenuated as a consequence of dysregulated tyrosine phosphorylation, and the long-term potentiation at perforant-dentate gyrus synapses was augmented. The inhibitory synaptic transmission of the dentate gyrus and hippocampal GABA concentration were reduced in PTPRT-deficient mice. In addition, the hippocampal expression of GABA transporter GAT3 (also known as SLC6A11) was decreased, and its tyrosine phosphorylation was increased in PTPRT-deficient mice. PTPRT-deficient mice displayed reduced numbers and neurite length of newborn granule cells in the dentate gyrus and had attenuated neurogenic ability of embryonic hippocampal neural stem cells. In conclusion, our findings show that the physiological roles of PTPRT in hippocampal neurogenesis, as well as synaptic functions, are involved in the pathogenesis of depressive disorder.

Distribution of GAT1 GABA Transporter Levels in the Bötzinger Complex at the Early Stages of Postnatal Development in Rats with Prenatal Serotonin Deficiency

Objective. To study the distribution of GAT1 GABA transporter in the Botzinger complex at different time points in early postnatal development in rats in normal conditions and after prenatal serotonin deficiency. Materials and methods. Studies were performed on Wistar laboratory rats. Decreases in endogenous serotonin levels in the embryonic period were produced by inhibition of tryptophan hydroxylase with para-chlorophenylalanine (PCPA). GAT1 transporter protein was detected using an immunohistochemical reaction with primary rabbit anti-GABA transporter 1 polyclonal antibodies (AbCam, UK). Brains were studied on postnatal days 5, 10, and 20. Results. At the early periods of postnatal development, the Botzinger complex of control animals showed oscillations in the GAT1 GABA transporter level. At one week of life, the GAT1 level was high, both in networks of processes and terminals and in synapses. During the second week of life, the GAT1 level decreased, and by the end of the third week it again increased, reaching the initial level. Serotonin deficiency during the prenatal period induced a significant increase in the GAT1 level in the neuropil of the Botzinger complex in the experimental animals at all time points of postnatal development. Conclusions. Prenatal serotonin deficiency led to a significant increase in the level of GAT1 GABA transporter in the early periods of postnatal development, which may lead to changes in GABA transmission and, as a result, disruption of the balance between inhibitory and excitatory effects in the respiratory nucleus.