Electrical activity between skin cells regulates melanoma initiation

Oncogenes can only initiate tumors in certain cellular contexts, which is referred to as oncogenic competence. In melanoma, whether cells in the microenvironment can endow such competence remains unclear. Using a combination of zebrafish transgenesis coupled with human tissues, we demonstrate that GABAergic signaling between keratinocytes and melanocytes promotes melanoma initiation by BRAFV600E. GABA is synthesized in melanoma cells, which then acts on GABA-A receptors on keratinocytes. Electron microscopy demonstrates synapse-like structures between keratinocytes and melanoma cells, and multi-electrode array analysis shows that GABA acts to inhibit electrical activity in melanoma/keratinocyte co-cultures. Genetic and pharmacologic perturbation of GABA synthesis abrogates melanoma initiation in vivo. These data suggest that electrical activity across the skin microenvironment determines the ability of oncogenes to initiate melanoma.

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

Dopamine (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.

Astrocyte metabolism of the medium-chain fatty acids octanoic acid and decanoic acid promotes GABA synthesis in neurons via elevated glutamine supply

The medium-chain fatty acids octanoic acid (C8) and decanoic acid (C10) are gaining attention as beneficial brain fuels in several neurological disorders. The protective effects of C8 and C10 have been proposed to be driven by hepatic production of ketone bodies. However, plasma ketone levels correlates poorly with the cerebral effects of C8 and C10, suggesting that additional mechanism are in place. Here we investigated cellular C8 and C10 metabolism in the brain and explored how the protective effects of C8 and C10 may be linked to cellular metabolism. Using dynamic isotope labeling, with [U-13C]C8 and [U-13C]C10 as metabolic substrates, we show that both C8 and C10 are oxidatively metabolized in mouse brain slices. The 13C enrichment from metabolism of [U-13C]C8 and [U-13C]C10 was particularly prominent in glutamine, suggesting that C8 and C10 metabolism primarily occurs in astrocytes. This finding was corroborated in cultured astrocytes in which C8 increased the respiration linked to ATP production, whereas C10 elevated the mitochondrial proton leak. When C8 and C10 were provided together as metabolic substrates in brain slices, metabolism of C10 was predominant over that of C8. Furthermore, metabolism of both [U-13C]C8 and [U-13C]C10 was unaffected by etomoxir indicating that it is independent of carnitine palmitoyltransferase I (CPT-1). Finally, we show that inhibition of glutamine synthesis selectively reduced 13C accumulation in GABA from [U-13C]C8 and [U-13C]C10 metabolism in brain slices, demonstrating that the glutamine generated from astrocyte C8 and C10 metabolism is utilized for neuronal GABA synthesis. Collectively, the results show that cerebral C8 and C10 metabolism is linked to the metabolic coupling of neurons and astrocytes, which may serve as a protective metabolic mechanism of C8 and C10 supplementation in neurological disorders.

Effect of Anti-inflammatory Treatment with AMD3100 and CX3CR1 Deficiency on GABAA Receptor Subunit and Expression of Glutamate Decarboxylase Isoforms After Stroke

Following stroke, attenuation of detrimental inflammatory pathways might be a promising strategy to improve long-term outcome. In particular, cascades driven by pro-inflammatory chemokines interact with neurotransmitter systems such as the GABAergic system. This crosstalk might be of relevance for mechanisms of neuronal plasticity, however, detailed studies are lacking. The purpose of this study was to determine if treatment with 1,1′-[1,4-phenylenebis(methylene)]bis[1,4,8,11-tetraazacyclotetradecane] (AMD3100), an antagonist to the C-X-C chemokine receptor type 4 (CXCR4) and partial allosteric agonist to CXCR7 (AMD3100) alone or in combination with C-X3-C chemokine receptor type 1 (CX3CR1) deficiency, affect the expression of GABAA subunits and glutamate decarboxylase (GAD) isoforms. Heterozygous, CX3CR1-deficient mice and wild-type littermates were subjected to photothrombosis (PT). Treatment with AMD3100 (0.5 mg/kg twice daily i.p.) was administered starting from day 2 after induction of PT until day 14 after the insult. At this time point, GABAA receptor subunits (α3, β3, δ), GAD65 and GAD67, and CXCR4 were analyzed from the peri-infarct tissue and homotypic brain regions of the contralateral hemisphere by quantitative real-time PCR and Western Blot. Fourteen days after PT, CX3CR1 deficiency resulted in a significant decrease of the three GABAA receptor subunits in both the lesioned and the contralateral hemisphere compared to sham-operated mice. Treatment with AMD3100 promoted the down-regulation of GABAA subunits and GAD67 in the ipsilateral peri-infarct area, while the β3 subunit and the GAD isoforms were up-regulated in homotypic regions of the contralateral cortex. Changes in GABAA receptor subunits and GABA synthesis suggest that the CXCR4/7 and CX3CR1 signaling pathways are involved in the regulation of GABAergic neurotransmission in the post-ischemic brain.

Redefining differential roles of MAO-A in dopamine degradation and MAO-B in tonic GABA synthesis

Monoamine oxidase (MAO) is believed to mediate the degradation of monoamine neurotransmitters, including dopamine, in the brain. Between the two types of MAO, MAO-B has been believed to be involved in dopamine degradation, which supports the idea that the therapeutic efficacy of MAO-B inhibitors in Parkinson’s disease can be attributed to an increase in extracellular dopamine concentration. However, this belief has been controversial. Here, by utilizing in vivo phasic and basal electrochemical monitoring of extracellular dopamine with fast-scan cyclic voltammetry and multiple-cyclic square wave voltammetry and ex vivo fluorescence imaging of dopamine with GRABDA2m, we demonstrate that MAO-A, but not MAO-B, mainly contributes to striatal dopamine degradation. In contrast, our whole-cell patch-clamp results demonstrated that MAO-B, but not MAO-A, was responsible for astrocytic GABA-mediated tonic inhibitory currents in the rat striatum. We conclude that, in contrast to the traditional belief, MAO-A and MAO-B have profoundly different roles: MAO-A regulates dopamine levels, whereas MAO-B controls tonic GABA levels.

GABA Regulates Phenolics Accumulation in Soybean Sprouts under NaCl Stress

NaCl stress causes oxidative stress in plants; γ-aminobutyric acid (GABA) could alleviate such abiotic stress by enhancing the synthesis of phenolics, but the underlying mechanism is not clear. We investigated the effects of GABA on phenolics accumulation in soybean sprouts under NaCl stress by measuring changes in the content of physiological biochemicals and phenolic substances, in the activity and gene expression of key enzymes, and in antioxidant capacity. GABA reduced the oxidative damage in soybean sprouts caused by NaCl stress and enhanced the content of total phenolics, phenolic acids, and isoflavones by 16.58%, 22.47%, and 3.75%, respectively. It also increased the activities and expression of phenylalanine ammonia lyase, cinnamic acid 4-hydroxylase, and 4-coumarate coenzyme A ligase. Furthermore, GABA increased the activity of antioxidant enzymes and the antioxidant capacity. These events were inhibited by 3-mercaptopropionate (an inhibitor for GABA synthesis), indicating that GABA mediated phenolics accumulation and antioxidant system enhancement in soybean sprouts under NaCl stress.

Molecular Basis of GABA Hypofunction in Adolescent Schizophrenia-Like Animals

Schizophrenia is a neurodevelopmental disorder that NMDA receptor (NMDAR) hypofunction appears centrally involved. Schizophrenia typically emerges in adolescence or early adulthood. Electrophysiological and several neurochemical changes have linked the GABA deficits to abnormal behaviors induced by NMDAR hypofunction. However, few studies have systematically investigated the molecular basis of GABA deficits, especially during adolescence. To address this issue, we transiently administrated MK-801 to mice on PND 10, which exhibited schizophrenia-relevant deficits in adolescence. Slice recording showed reduced GABA transmission and PVI+ hypofunction, indicating GABAergic hypofunction. Cortical proteomic evaluation combined with analysis of single cell data from the Allen Brain showed that various metabolic processes were enriched in top ranks and differentially altered in excitatory neurons, GABAergic interneurons, and glial cells. Notably, the GABA-related amino acid metabolic process was disturbed in both astrocytes and interneurons, in which we found a downregulated set of GABA-related proteins (GAD65, SYNPR, DBI, GAT3, SN1, and CPT1A). They synergistically regulate GABA synthesis, release, reuptake, and replenishment. Their downregulation indicates impaired GABA cycle and homeostasis regulated by interneuron-astrocyte communication in adolescence. Our findings on molecular basis of GABA deficits could provide potential drug targets of GABAergic rescue for early prevention and intervention.

Glutamate Decarboxylase from Lactic Acid Bacteria—A Key Enzyme in GABA Synthesis

Glutamate decarboxylase (l-glutamate-1-carboxylase, GAD; EC 4.1.1.15) is a pyridoxal-5’-phosphate-dependent enzyme that catalyzes the irreversible α-decarboxylation of l-glutamic acid to γ-aminobutyric acid (GABA) and CO2. The enzyme is widely distributed in eukaryotes as well as prokaryotes, where it—together with its reaction product GABA—fulfils very different physiological functions. The occurrence of gad genes encoding GAD has been shown for many microorganisms, and GABA-producing lactic acid bacteria (LAB) have been a focus of research during recent years. A wide range of traditional foods produced by fermentation based on LAB offer the potential of providing new functional food products enriched with GABA that may offer certain health-benefits. Different GAD enzymes and genes from several strains of LAB have been isolated and characterized recently. GABA-producing LAB, the biochemical properties of their GAD enzymes, and possible applications are reviewed here.

Blocking Astrocytic GABA Restores Synaptic Plasticity in Prefrontal Cortex of Rat Model of Depression

A decrease in synaptic plasticity and/or a change in excitation/inhibition balance have been suggested as mechanisms underlying major depression disorder. However, given the crucial role of astrocytes in balancing synaptic function, particular attention should be given to the contribution of astrocytes in these mechanisms, especially since previous findings show that astrocytes are affected and exhibit reactive-like features in depression. Moreover, it has been shown that reactive astrocytes increase the synthesis and release of GABA, contributing significantly to tonic GABA inhibition. In this study we found decreased plasticity and increased tonic GABA inhibition in the prelimbic area in acute slices from the medial prefrontal cortex in the Flinders Sensitive Line (FSL) rat model of depression. The tonic inhibition can be reduced by either blocking astrocytic intracellular Ca2+ signaling or by reducing astrocytic GABA through inhibition of the synthesizing enzyme MAO-B with Selegiline. Blocking GABA synthesis also restores the impaired synaptic plasticity in the FSL prefrontal cortex, providing a new antidepressant mechanism of Selegiline.