GABA Pathway Research Articles

Novel ultrashort-acting benzodiazepine remimazolam lowers shivering threshold in rabbits.

Shivering after surgery or during therapeutic hypothermia can lead to serious complications, such as myocardial infarction and respiratory failure. Although several anesthetics and opioids are shown to have anti-shivering effects, their sedative and respiratory side effects dampen the usefulness of these drugs for the prevention of shivering. In the present study, we explored the potential of a novel ultrashort-acting benzodiazepine, remimazolam, in the prevention of shivering using a rabbit model of hypothermia. Adult male Japanese white rabbits were anesthetized with isoflurane. The rabbits received saline (control), remimazolam (either 0.1 or 1 mg/kg/h), or remimazolam + flumazenil, a selective γ-aminobutyric acid (GABA) type A receptor antagonist (n = 6 each). Thirty minutes after discontinuation of the drugs, cooling was initiated by perfusing 10°C water via a plastic tube positioned in the colon until the animal shivered. Core body temperature and hemodynamic and physiological parameters were recorded. Remimazolam at 1 mg/kg/h significantly lowered the core temperature change during shivering (−2.50 ± 0.20°C vs. control: −1.00 ± 0.12°C, p = 0.0009). The effect of 1 mg/kg/h remimazolam on the core temperature change was abolished by flumazenil administration (−0.94 ± 0.16°C vs. control: −1.00 ± 0.12°C, p = 0.996). Most of the hemodynamic and physiological parameters did not differ significantly among groups during cooling. Remimazolam at a clinically relevant dose successfully suppressed shivering in rabbits via the GABA pathway even after its anesthetic effects likely disappeared. Remimazolam may have the potential to prevent shivering in patients undergoing surgery or therapeutic hypothermia.

In silico analysis of metabolic effects of bipolar disorder on prefrontal cortex identified altered GABA, glutamate-glutamine cycle, energy metabolism and amino acid synthesis pathways.

Bipolar disorder (BP) is a lifelong psychiatric condition, which often disrupts the daily life of the patients. It is characterized by unstable and periodic mood changes, which cause patients to display unusual shifts in mood, energy, activity levels, concentration and the ability to carry out day-to-day tasks. BP is a major psychiatric condition, and it is still undertreated. The causes and neural mechanisms of bipolar disorder are unclear, and diagnosis is still mostly based on psychiatric examination, furthermore the unstable character of the disorder makes diagnosis challenging. Identification of the molecular mechanisms underlying the disease may improve the diagnosis and treatment rates. Single nucleotide polymorphisms (SNP) and transcriptome profiles of patients were studied along with signalling pathways that are thought to be associated with bipolar disorder. Here, we present a computational approach that uses publicly available transcriptome data from bipolar disorder patients and healthy controls. Along with statistical analyses, data are integrated with a genome-scale metabolic model and protein-protein interaction network. Healthy individuals and bipolar disorder patients are compared based on their metabolic profiles. We hypothesize that energy metabolism alterations in bipolar disorder relate to perturbations in amino-acid metabolism and neuron-astrocyte exchange reactions. Due to changes in amino acid metabolism, neurotransmitters and their secretion from neurons and metabolic exchange pathways between neurons and astrocytes such as the glutamine-glutamate cycle are also altered. Changes in negatively charged (-1) KIV and KMV molecules are also observed, and it indicates that charge balance in the brain is highly altered in bipolar disorder. Due to this fact, we also hypothesize that positively charged lithium ions may stabilize the disturbed charge balance in neurons in addition to its effects on neurotransmission. To the best of our knowledge, our approach is unique as it is the first study using genome-scale metabolic models in neuropsychiatric research.

Transcriptome analysis of the pulp of citrus fruitlets suggests that domestication enhanced growth processes and reduced chemical defenses increasing palatability

To identify key traits brought about by citrus domestication, we have analyzed the transcriptomes of the pulp of developing fruitlets of inedible wild Ichang papeda (Citrus ichangensis), acidic Sun Chu Sha Kat mandarin (C. reticulata) and three palatable segregants of a cross between commercial Clementine (C. x clementina) and W. Murcott (C. x reticulata) mandarins, two pummelo/mandarin admixtures of worldwide distribution. RNA-seq comparison between the wild citrus and the ancestral sour mandarin identified 7267 differentially expressed genes, out of which 2342 were mapped to 117 KEGG pathways. From the remaining genes, a set of 2832 genes was functionally annotated and grouped into 45 user-defined categories. The data suggest that domestication promoted fundamental growth processes to the detriment of the production of chemical defenses, namely, alkaloids, terpenoids, phenylpropanoids, flavonoids, glucosinolates and cyanogenic glucosides. In the papeda, the generation of energy to support a more active secondary metabolism appears to be dependent upon upregulation of glycolysis, fatty acid degradation, Calvin cycle, oxidative phosphorylation, and ATP-citrate lyase and GABA pathways. In the acidic mandarin, downregulation of cytosolic citrate degradation was concomitant with vacuolar citrate accumulation. These changes affected nitrogen and carbon allocation in both species leading to major differences in organoleptic properties since the reduction of unpleasant secondary metabolites increases palatability while acidity reduces acceptability. The comparison between the segregants and the acidic mandarin identified 357 transcripts characterized by the occurrence in the three segregants of additional downregulation of secondary metabolites and basic structural cell wall components. The segregants also showed upregulation of genes involved in the synthesis of methyl anthranilate and furaneol, key substances of pleasant fruity aroma and flavor, and of sugar transporters relevant for sugar accumulation. Transcriptome and qPCR analysis in developing and ripe fruit of a set of genes previously associated with citric acid accumulation, demonstrated that lower acidity is linked to downregulation of these regulatory genes in the segregants. The results suggest that the transition of inedible papeda to sour mandarin implicated drastic gene expression reprograming of pivotal pathways of the primary and secondary metabolism, while palatable mandarins evolved through progressive refining of palatability properties, especially acidity.

γ-Aminobutyrate Improves the Postharvest Marketability of Horticultural Commodities: Advances and Prospects.

Postharvest deterioration can result in qualitative and quantitative changes in the marketability of horticultural commodities, as well as considerable economic loss to the industry. Low temperature and controlled atmosphere conditions (low O2 and elevated CO2) are extensively employed to prolong the postharvest life of these commodities. Nevertheless, they may suffer from chilling injury and other physiological disorders, as well as excessive water loss and bacterial/fungal decay. Research on the postharvest physiological, biochemical, and molecular responses of horticultural commodities indicates that low temperature/controlled atmosphere storage is associated with the promotion of γ-aminobutyrate (GABA) pathway activity, with or without the accumulation of GABA, delaying senescence, preserving quality and ameliorating chilling injury. Regardless of whether apple fruits are stored under low temperature/controlled atmosphere conditions or room temperature, elevated endogenous GABA or exogenous GABA maintains their quality by stimulating the activity of the GABA shunt (glutamate GABA succinic semialdehyde succinate) and the synthesis of malate, and delaying fruit ripening. This outcome is associated with changes in the genetic and biochemical regulation of key GABA pathway reactions. Flux estimates suggest that the GABA pool is derived primarily from glutamate, rather than polyamines, and that succinic semialdehyde is converted mainly to succinate, rather than γ-hydroxybutyrate. Exogenous GABA is a promising strategy for promoting the level of endogenous GABA and the activity of the GABA shunt in both intact and fresh-cut commodities, which increases carbon flux through respiratory pathways, restores or partially restores redox and energy levels, and improves postharvest marketability. The precise mechanisms whereby GABA interacts with other signaling molecules such as Ca2+, H2O2, polyamines, salicylic acid, nitric oxide and melatonin, or with phytohormones such as ethylene, abscisic acid and auxin remain unknown. The occurrence of the aluminum-activated malate transporter and the glutamate/aspartate/GABA exchanger in the tonoplast, respectively, offers prospects for reducing transpirational water in cut flowers and immature green fruit, and for altering the development, flavor and biotic resistance of apple fruits.

CsCuAOs and CsAMADH1 Are Required for Putrescine-Derived γ-Aminobutyric Acid Accumulation in Tea.

Polyamines are a potential source of γ-aminobutyric acid (GABA) in plants under abiotic stress. However, studies on GABA enrichment in tea mostly focus on the GABA shunt, while the correlation between polyamine degradation and GABA formation in tea is largely unknown. In this study, tea plants responded to exogenous putrescine, resulting in a significant increase in GABA content, while the glutamate level did not change. At the same time, five copper-containing amine oxidase (CuAO) and eight aminoaldehyde dehydrogenase (AMADH) genes involved in the putrescine-derived GABA pathway were identified from the Tea Plant Information Archive. Expression analysis indicated that CsCuAO1, CsCuAO3 as well as CsAMADH1 were induced to play an important function in response to exogenous putrescine. Thus, the three genes were cloned and the catalytic efficiency of soluble recombinant proteins was determined. CsCuAOs and CsAMADH1 exhibited indispensable functions in the GABA production from putrescine in vitro. Subcellular localization assays indicated that CsAMADH1 was localized in plastid, while both CsCuAO1 and CsCuAO3 were localized in peroxisome. In addition, the synergistic effects of CsCuAOs and CsAMADH1 were investigated by a transient co-expression system in Nicotiana benthamiana. Our data suggest that these three genes regulate the accumulation of GABA in tea by participating in the polyamine degradation pathway and improve the content of GABA in tea to a certain extent. The results will greatly contribute to the production of GABA tea.

Raloxifene prevents intracellular invasion of pathogenic bacteria through modulation of cell metabolic pathways.

Pathogenic bacteria can invade and survive inside phagocytic and non-phagocytic cells and use them as a sanctuary against antibiotics. Induction of various host protective mechanisms, including autophagy, can be a novel and effective method to combat intracellular bacteria. Recent studies report that raloxifene, a selective oestrogen receptor modulator, can induce cellular autophagy. To demonstrate the effect of raloxifene on intracellular invasion and proliferation of pathogenic bacteria and investigate raloxifene-induced metabolic changes in host cells. Autophagic induction was determined by the extent of nuclear fragmentation and expression levels of the LC3B protein. Intracellular invasion of MRSA strains into A549 lung epithelial cells and invasion of Mycobacterium abscessus into RAW264.7 macrophages were assessed by invasion and proliferation assays. Changes in host cell metabolism were examined by transcriptomic profiling using RNA sequencing. Our data demonstrate increased autophagy in cells upon raloxifene treatment, which contributed to prevention of bacterial invasion and proliferation. Transcriptomic profiling of host cells revealed changes in cholesterol-related pathways and consequent increases in oxidative stress-related genes and in autophagic induction through the TRIM and GABA pathways. Our results demonstrate raloxifene's potential as a broad-spectrum antibacterial agent through autophagic induction in host cells and prevention of intracellular invasion and proliferation of pathogenic bacteria.

Proteomics Study Reveals the Anti-Depressive Mechanisms and the Compatibility Advantage of Chaihu-Shugan-San in a Rat Model of Chronic Unpredictable Mild Stress

Background: Chaihu-Shugan-San is a classical prescription to treat depression. According to the traditional Chinese medicine (TCM) principle, the 2 decomposed recipes in Chaihu-Shugan-San exert synergistic effects, including Shu Gan (stagnated Gan-Qi dispersion) and Rou Gan (Gan nourishment to alleviate pain). However, the specific mechanism of Chaihu-Shugan-San on depression and its compatibility rule remain to be explored. Objective: We aimed to explore the anti-depression mechanisms and analyze the advantage of TCM compatibility of Chaihu-Shugan-San. Methods: The chronic unpredictable mild stress (CUMS) rat model was established. Antidepressant effects were evaluated by sucrose preference test (SPT), and forced swimming test (FST). Tandem Mass Tag (TMT)-based quantitative proteomics of the hippocampus was used to obtain differentially expressed proteins (DEPs). Bioinformatics analysis including Gene Ontology (GO), pathway enrichment, and protein-protein interaction (PPI) networks was utilized to study the DEPs connections. At last, the achieved key targets were verified by western blotting. Results: Chaihu-Shugan-San increased weight gain and food intake, as well as exhibited better therapeutic effects including enhanced sucrose preference and extended immobility time when compared with its decomposed recipes. Proteomics showed Chaihu-Shugan-San, Shu Gan, and Rou Gan regulated 110, 12, and 407 DEPs, respectively. Compared with Shu Gan or Rou Gan alone, the expression of 22 proteins was additionally changed by Chaihu-Shugan-San treatment, whereas the expression of 323 proteins whose expression was changed by Shu Gan or Rou Gan alone were not changed by Chaihu-Shugan-San treatment. Bioinformatics analysis demonstrated that Chaihu-Shugan-San affected neurotransmitter’s release and transmission cycle (e.g., γ-aminobutyric acid (GABA), glutamate, serotonin, norepinephrine, dopamine, and acetylcholine). GABA release pathway is also targeted by the 22 DEPs. Unexpectedly, only 2 pathways were enriched by the 323 DEPs: Metabolism and Cellular responses to external stimuli. Lastly, the expression of Gad2, Vamp2, and Pde2a was verified by western blotting. Conclusions: Chaihu-Shugan-San treats depression via multiple targets and pathways, which may include regulations of 110 DEPs and some neurotransmitter’s transmission cycle. Compared with Shu Gan and Rou Gan, the 22 Chaihu-Shugan-San advanced proteins and the affected GABA pathway may be the advantages of Chaihu-Shugan-San compatibility. This research offers data and theory support for the clinical application of Chaihu-Shugan-San.

Metabolite profiling reveals a connection between aldehyde dehydrogenase 1A3 and GABA metabolism in breast cancer metastasis

IntroductionAldehyde dehydrogenase 1A3 (ALDH1A3) is a cancer stem cell (CSC) marker and in breast cancer it is associated with triple-negative/basal-like subtypes and aggressive disease. Studies on the mechanisms of ALDH1A3 in cancer have primarily focused on gene expression changes induced by the enzyme; however, its effects on metabolism have thus far been unstudied and may reveal novel mechanisms of pathogenesis.ObjectiveDetermine how ALDH1A3 alters the metabolite profile in breast cancer cells and assess potential impacts.MethodTriple-negative MDA-MB-231 tumors and cells with manipulated ALDH1A3 levels were assessed by HPLC–MS metabolomics and metabolite data was integrated with transcriptome data. Mice harboring MDA-MB-231 tumors with or without altered ALDH1A3 expression were treated with γ-aminobutyric acid (GABA) or placebo. Effects on tumor growth, and lungs and brain metastasis were quantified by staining of fixed thin sections and quantitative PCR. Breast cancer patient datasets from TCGA, METABRIC and GEO were used to assess the co-expression of GABA pathway genes with ALDH1A3.ResultsIntegrated metabolomic and transcriptome data identified GABA metabolism as a primary dysregulated pathway in ALDH1A3 expressing breast tumors. Both ALDH1A3 and GABA treatment enhanced metastasis. Patient dataset analyses revealed expression association between ALDH1A3 and GABA pathway genes and corresponding increased risk of metastasis.ConclusionThis study revealed a novel pathway affected by ALDH1A3, GABA metabolism. Like ALDH1A3 expression, GABA treatment promotes metastasis. Given the clinical use of GABA mimics to relieve chemotherapy-induced peripheral nerve pain, further study of the effects of GABA in breast cancer progression is warranted.

Enhancing GABA inhibition is the next generation of medications for essential tremor.

γ-Aminobutyric acid (GABA) is the most prevalent inhibitory CNS neurotransmitter. Activating GABA-A receptors hyperpolarizes cells via Cl- influx, which inhibits action potentials. Although the exact pathophysiologies of tremor are incompletely understood, proposed neuroanatomy extensively implicates GABA pathways. Pathological studies and imaging studies also show GABA abnormalities in patients with ET. Most importantly, medications that activate GABA-A receptors, such as primidone, often improve tremor. Ongoing clinical trials and physiology research should further refine potential future GABAergic targets and treatments, which are currently the most promising targets for pharmacological intervention.

Comprehensive Genome-Wide Identification and Transcript Profiling of GABA Pathway Gene Family in Apple (Malus domestica).

γ-Aminobutyric Acid (GABA), a four-carbon non-protein amino acid, is a significant component of the free amino acid pool in most prokaryotic and eukaryotic organisms. GABA is involved in pH regulation, maintaining C/N balance, plant development and defence, as well as a compatible osmolyte and an alternative pathway for glutamate utilization via anion flux. Glutamate decarboxylase (GAD, EC 4.1.1.15) and GABA transaminase (GABA-T, EC 2.6.1.19) are two key enzymes involved in the synthesis and metabolism of GABA. Recently, GABA transporters (GATs), protein and aluminium-activated malate transporter (ALMT) proteins which function as GABA receptors, have been shown to be involved in GABA regulation. However, there is no report on the characterization of apple GABA pathway genes. In this study, we performed a genome-wide analysis and expression profiling of the GABA pathway gene family in the apple genome. A total of 24 genes were identified including five GAD genes (namely MdGAD 1–5), two GABA-T genes (namely MdGABA-T 1,2), 10 GAT genes (namely GAT 1–10) and seven ALMT genes (namely MdALMT 1–7). These genes were randomly distributed on 12 chromosomes. Phylogenetic analyses grouped GABA shunt genes into three clusters—cluster I, cluster II, and cluster III—which had three, four, and five genes, respectively. The expression profile analysis revealed significant MdGAD4 expression levels in both fruit and flower organs, except pollen. However, there were no significant differences in the expression of other GABA shunt genes in different tissues. This work provides the first characterization of the GABA shunt gene family in apple and suggests their importance in apple response to abiotic stress. These results can serve as a guide for future studies on the understanding and functional characterization of these gene families.

Fragile X Syndrome: Lessons Learned and What New Treatment Avenues Are on the Horizon

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and the leading single-gene form of autism spectrum disorder, encompassing cognitive, behavioral, and physical forms of clinical involvement. FXS is caused by large expansions of a noncoding CGG repeat (>200 repeats) in the FMR1 gene, at which point the gene is generally silenced. Absence of FMR1 protein (FMRP), important for synaptic development and maintenance, gives rise to the neurodevelopmental disorder. There is, at present, no therapeutic approach that directly reverses the loss of FMRP; however, there is an increasing number of potential treatments that target the pathways dysregulated in FXS, including those that address the enhanced activity of the mGluR5 pathway and deficits in GABA pathways. Based on studies of targeted therapeutics to date, the prospects are good for one or more effective therapies for FXS in the near future.

An aqueous extract of Canarium schweinfurthii attenuates seizures and potentiates sleep in mice: Evidence for involvement of GABA Pathway

About 30% of epileptic patients continue to have seizures. The present study investigates the anticonvulsant and sedative effects of an aqueous extract of C. schweinfurthii in mice. Anticonvulsant effects of C. schweinfurthii aqueous extract (0.01–300 mg/kg, p.o.) were tested against 4-aminopyridine (4-AP, 15 mg/kg, i.p.) -, pilocarpine (PILO, 380 mg/kg, i.p.) - and pentylenetetrazole (PTZ, 75 mg/kg, i.p.) -induced seizures, while sedative effects were tested on diazepam (35 mg/kg, i.p.)-induced sleep. Afterward, the most effective dose of the extract (11.9 mg/kg) was antagonized with N-methyl-β-carboline-3-carboxamide or flumazenil. In another set of experiments, mice were sacrificed for the estimation of GABA content and GABA-T activity in the cerebral cortex. The dose of the extract that protected 50% of mice (ED50) against 4-AP, PILO, and PTZ was respectively 4.43 mg/kg (versus 12.01 for phenobarbital), 9.59 mg/kg (vs 8.67 for diazepam), and 2.12 mg/kg (vs 0.20 for clonazepam). Further, the ED50 of the extract that increased the duration of sleep was 0.24 mg/kg (vs 0.84 for phenobarbital). N-methyl-β-carboline-3-carboxamide or flumazenil antagonized (p < 0.001) the anticonvulsant effect of C. schweinfurthii in PTZ-induced seizures and diazepam-induced sleep when compared to the negative control group. The extract at all doses increased (p < 0.001) the GABA content and decreased (p < 0.001) GABA-T activity. These findings suggest that C. schweinfurthii possesses anticonvulsant and sedative effects. These effects seem to be mediated via the modulation of the GABA neurotransmission. These data explain the use of this plant to treat epilepsy in Cameroon traditional medicine.

Anti-seizure effects of walnut peptides in mouse models of induced seizure: The involvement of GABA and nitric oxide pathways

Epilepsy is one of the foremost medical disorders. Oxidative stress is a well-known mechanism in epileptogenesis, and many studies suggest that oxidative stress affects the onset and evolution of epilepsy. Here we evaluated the walnut peptide extracts’ anti-seizure property in three different mouse seizure models including pentylenetetrazole-induced clonic seizure, chemical kindling, and maximal electroshock. Walnut peptides (20 mg/Kg) were administered by intraperitoneal (IP) injection of mice 60 min before seizure induction in the three models. To delineate the mechanisms of walnut peptides anti-seizure activity, we evaluated the impact of diazepam, flumazenil, and a NOS inhibitor on this activity. Intraperitoneal administration of walnut peptides significantly increased the seizure threshold. Our results also demonstrated that walnut peptides exert their anti-seizure properties through the modulation of benzodiazepine receptors. Thus, walnut peptides may be considered as a new anti-convulsion agent, which can reduce seizure occurrence and slow down seizure progression.

GABA shunt contribution to flavonoid biosynthesis and metabolism in tea plants (Camellia sinensis)

γ-Aminobutyric acid (GABA), a signal molecule, is regarded as the intersection node of carbon and nitrogen metabolism, and its contributions to flavonoid metabolism in tea plant growth and development remain unclear. The correlation between the GABA shunt and flavonoid metabolism in tea plants is worth to explore. Secondary metabolites and their correlations with the taste of tea soup made from tea plants (Camellia sinensis) during different seasons were investigated. Related secondary metabolites and transcript profiles of genes encoding enzymes in the GABA shunt, flavonoid pathway and polyamine biosynthesis were measured throughout the tea plant growth seasons and after exogenous GABA applications. In addition, the abundance of differentially expressed proteins was quantified after treatments with or without exogenous GABA. The tea leaves showed the highest metabolite concentrations in spring season. CsGAD, CsGABAT, CsSPMS, CsODC, CsF3H and CsCHS were found to be important genes in the GABA and anthocyanin biosynthesis pathways. GABA and anthocyanin concentrations showed a positive correlation, to some extent, CsF3H and CsCHS played important roles in the GABA and anthocyanin biosynthesis.

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.

Common molecular mechanisms of SLC6A1 variant-mediated neurodevelopmental disorders in astrocytes and neurons.

Solute carrier family 6 member 1 (SLC6A1) is abundantly expressed in the developing brain even before the CNS is formed. Its encoded GABA transporter 1 (GAT-1) is responsible for the reuptake of GABA into presynaptic neurons and glia, thereby modulating neurotransmission. GAT-1 is expressed globally in the brain, in both astrocytes and neurons. The GABA uptake function of GAT-1 in neurons cannot be compensated for by other GABA transporters, while the function in glia can be partially replaced by GABA transporter 3. Recently, many variants in SLC6A1 have been associated with a spectrum of epilepsy syndromes and neurodevelopmental disorders, including myoclonic atonic epilepsy, childhood absence epilepsy, autism, and intellectual disability, but the pathomechanisms associated with these phenotypes remain unclear. The presence of GAT-1 in both neurons and astrocytes further obscures the role of abnormal GAT-1 in the heterogeneous disease phenotype manifestations. Here we examine the impact on transporter trafficking and function of 22 SLC6A1 variants identified in patients with a broad spectrum of phenotypes. We also evaluate changes in protein expression and subcellular localization of the variant GAT-1 in various cell types, including neurons and astrocytes derived from human patient induced pluripotent stem cells. We found that a partial or complete loss-of-function represents a common disease mechanism, although the extent of GABA uptake reduction is variable. The reduced GABA uptake appears to be due to reduced cell surface expression of the variant transporter caused by variant protein misfolding, endoplasmic reticulum retention, and subsequent degradation. Although the extent of reduction of the total protein, surface protein, and the GABA uptake level of the variant transporters is variable, the loss of GABA uptake function and endoplasmic reticulum retention is consistent across induced pluripotent stem cell-derived cell types, including astrocytes and neurons, for the surveyed variants. Interestingly, we did not find a clear correlation of GABA uptake function and the disease phenotypes, such as myoclonic atonic epilepsy versus developmental delay, in this study. Together, our study suggests that impaired transporter protein trafficking and surface expression are the major disease-associated mechanisms associated with pathogenic SLC6A1 variants. Our results resemble findings from pathogenic variants in other genes affecting the GABA pathway, such as GABAA receptors. This study provides critical insight into therapeutic developments for SLC6A1 variant-mediated disorders and implicates that boosting transporter function by either genetic or pharmacological approaches would be beneficial.

Comprehensive characterization of neurotransmitters and neuronal signaling (NT) pathway alterations in colorectal cancer (CRC).

3537 Background: Aberrant NT signaling has been shown to activate uncontrolled proliferation and dissemination in several gastrointestinal cancer types. Neurotransmitters have been shown to affect endothelial cells and immune cells in the tumor microenvironment to promote tumor progression. We previously showed that single nucleotide polymorphisms in the dopamine and GABA pathways are associated with outcome in patients with metastatic CRC receiving first-line treatment. Here we further evaluated the distribution and molecular context of NT pathway alterations in CRC. Methods: A total of 7,595 CRC tumors tested at Caris Life Sciences (Phoenix, AZ) with NextGen Sequencing on DNA (Next Seq, 592 genes or NovaSeq, WES) and RNA (NovaSeq, WTS) were analyzed. ssGSEA (single-sample gene set enrichment analysis) was used to calculate pathway enrichment scores (ES) of 7 NT gene sets (GABA, nicotinic, muscarinic, dopamine (DA), reelin, glial cell line-derived neurotrophic factor and neurotrophins). X2/Fisher-Exact was used for comparison and significance was determined as p-value adjusted for multiple comparison of ( q) &lt; 0.05. Results: ES based on sample sites showed a substantial heterogeneity in NT enrichment. Notably, when compared to primary tumors, all 7 gene sets were significantly enriched in brain metastases (mets; ES ratio 1.14-1.55), while abdomen, liver, and peritoneal mets displayed significant decreases in most NT gene sets. DA was enriched in ovarian and lung mets (ES ratio: 1.18 and 1.09, respectively), the latter also showing increased neurotrophins ES (1.06) (all q &lt; 0.05). When investigating primary tumors grouped according to overall ES by unsupervised clustering, right-sided and CMS4 CRCs were more prevalent in the high ES cluster compared to the low ES cluster (32 vs 29%, P = 0.02 and 46 vs 30%, P &lt; 0.001, respectively). In addition, tumors in the high ES cluster showed lower prevalence of TMB-H (≥ 10mt/MB) (7 vs 10%), MSI-H (6 vs 10%) and PD-L1 (2 vs 6%), while higher CNA rates were noted in 9 genes (all q &lt; 0.05). High ES tumors showed significant positive associations with microenvironment infiltration of B cells, T cells (NK, CD4+ and CD8+ T cells, but not Treg), M2 Macrophages, Myeloid Dendritic Cell, Neutrophils, and an inverse association with M1 Macrophages, regardless of MSI status ( q &lt; 0.05). Conclusions: This is the first and most extensive molecular profiling study to investigate NT signaling pathway alterations in CRC. Our data show a distinct distribution of pathway enrichment according to metastatic site, distinct molecular features in high vs low ES clusters in primary tumors (including CMS subtypes, TMB, MSI and PD-L1 rates), and differential immune cell infiltration. These findings support the role of NT signaling in the metastatic spread of CRC and modulation of tumor immune microenvironment.

Exercise Improved the Anti-Epileptic Effect of Carbamazepine through GABA Enhancement in Epileptic Rats.

Carbamazepine (CBZ) is an anticonvulsant drug that usually is used for the treatment of seizures. The anti-epileptic and the anti-epileptogenic effect of exercise has been reported, as well. This study was aimed to evaluate the synergic effect of combined therapy of exercise and CBZ in epileptic rats, as well as the alternation of the GABA pathway as a possible involved mechanism. The seizure was induced by pentylenetetrazol (PTZ) injection. Animals were divided into sham, seizure, exercise (EX), CBZ (25, 50 and 75), EX + CBZ (25, 50 and 75). Treadmill forced running for 30min has been considered as the exercise 5days per week for four weeks. CBZ was injected in doses of 25, 50 and 75mg/kg, half an hour before seizure induction and 5h after doing exercise in the animals forced to exercise. Seizure severity reduced and latency increased in the EX + CBZ (25) and EX + CBZ (50) groups compared to the seizure group. The distribution of GAD65 in both hippocampal CA1 and CA3 areas increased in the EX + CBZ (75) group. GABAA receptor α1 was up-regulated in the CA3 area of the EX + CBZ (75) group. The distribution of GAD65 in the cortical area increased in EX, EX + CBZ (50), CBZ (75) and EX + CBZ (75) groups. GABAA receptor α1 was up-regulated in the neocortex of EX + CBZ (50), CBZ (75) and EX + CBZ (75) groups. Our findings suggested that exercise has improved the efficacy of CBZ and reduced the anti-epileptic dose. The enhancement of GABA signaling might be involved in the synergistic effect of exercise and CBZ.

Cingulate networks associated with gray matter loss in Parkinson's disease show high expression of cholinergic genes in the healthy brain.

Structural covariance networks are able to identify functionally organized brain regions by gray matter volume covariance across a population. We examined the transcriptomic signature of such anatomical networks in the healthy brain using postmortem microarray data from the Allen Human Brain Atlas. A previous study revealed that a posterior cingulate network and anterior cingulate network showed decreased gray matter in brains of Parkinson's disease patients. Therefore, we examined these two anatomical networks to understand the underlying molecular processes that may be involved in Parkinson's disease. Whole brain transcriptomics from the healthy brain revealed upregulation of genes associated with serotonin, GPCR, GABA, glutamate, and RAS‐signaling pathways. Our results also suggest involvement of the cholinergic circuit, in which genes NPPA, SOSTDC1, and TYRP1 may play a functional role. Finally, both networks were enriched for genes associated with neuropsychiatric disorders that overlap with Parkinson's disease symptoms. The identified genes and pathways contribute to healthy functions of the posterior and anterior cingulate networks and disruptions to these functions may in turn contribute to the pathological and clinical events observed in Parkinson's disease.

Adenosine and Kynurenic Acid Interactions: Possible Relevance for Schizophrenia Treatment?

Adenosine and Kynurenic Acid Interactions: Possible Relevance for Schizophrenia Treatment?