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 MdALMT1–7). 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.

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.

The Dual Role of the GABAA Receptor in Peripheral Inflammation and Neuroinflammation: A Study in Hyperammonemic Rats

Cognitive and motor impairment in minimal hepatic encephalopathy (MHE) are mediated by neuroinflammation, which is induced by hyperammonemia and peripheral inflammation. GABAergic neurotransmission in the cerebellum is altered in rats with chronic hyperammonemia. The mechanisms by which hyperammonemia induces neuroinflammation remain unknown. We hypothesized that GABAA receptors can modulate cerebellar neuroinflammation. The GABAA antagonist bicuculline was administrated daily (i.p.) for four weeks in control and hyperammonemic rats. Its effects on peripheral inflammation and on neuroinflammation as well as glutamate and GABA neurotransmission in the cerebellum were assessed. In hyperammonemic rats, bicuculline decreases IL-6 and TNFα and increases IL-10 in the plasma, reduces astrocyte activation, induces the microglia M2 phenotype, and reduces IL-1β and TNFα in the cerebellum. However, in control rats, bicuculline increases IL-6 and decreases IL-10 plasma levels and induces microglial activation. Bicuculline restores the membrane expression of some glutamate and GABA transporters restoring the extracellular levels of GABA in hyperammonemic rats. Blocking GABAA receptors improves peripheral inflammation and cerebellar neuroinflammation, restoring neurotransmission in hyperammonemic rats, whereas it induces inflammation and neuroinflammation in controls. This suggests a complex interaction between GABAergic and immune systems. The modulation of GABAA receptors could be a suitable target for improving neuroinflammation in MHE.

Synthesis and biological evaluation of novel N-substituted nipecotic acid derivatives with tricyclic cage structures in the lipophilic domain as GABA uptake inhibitors

A new class of GABA reuptake inhibitors with sterically demanding, highly rigid tricyclic cage structures as the lipophilic domain was synthesized and investigated in regard to their biological activity at the murine GABA transporters (mGAT1–mGAT4). The construction of these compounds, consisting of nipecotic acid, a symmetric tricyclic amine, and a plain hydrocarbon linker connecting the two subunits via their amino nitrogens, was accomplished via reductive amination of a nipecotic acid derivative with an N-alkyl substituent displaying a terminal aldehyde function with tricyclic secondary amines. The target compounds varied with regard to spacer length, the bridge size of one of the bridges, and the substituents of the tricyclic skeleton to study the impact of these changes on their potency. Among the tested compounds nipecotic acid ethyl ester derivates with phenyl residues attached to the cage subunit showed reasonable inhibitory potency and subtype selectivity in favor of mGAT3 and mGAT4, respectively.

A Targeted Bioinformatics Assessment of Adrenocortical Carcinoma Reveals Prognostic Implications of GABA System Gene Expression

Adrenocortical carcinoma (ACC) is a rare but deadly cancer for which few treatments exist. Here, we have undertaken a targeted bioinformatics study of The Cancer Genome Atlas (TCGA) ACC dataset focusing on the 30 genes encoding the γ-aminobutyric acid (GABA) system—an under-studied, evolutionarily-conserved system that is an emerging potential player in cancer progression. Our analysis identified a subset of ACC patients whose tumors expressed a distinct GABA system transcriptome. Transcript levels of ABAT (encoding a key GABA shunt enzyme), were upregulated in over 40% of tumors, and this correlated with several favorable clinical outcomes including patient survival; while enrichment and ontology analysis implicated two cancer-related biological pathways involved in metastasis and immune response. The phenotype associated with ABAT upregulation revealed a potential metabolic heterogeneity among ACC tumors associated with enhanced mitochondrial metabolism. Furthermore, many GABAA receptor subunit-encoding transcripts were expressed, including two (GABRB2 and GABRD) prognostic for patient survival. Transcripts encoding GABAB receptor subunits and GABA transporters were also ubiquitously expressed. The GABA system transcriptome of ACC tumors is largely mirrored in the ACC NCI-H295R cell line, suggesting that this cell line may be appropriate for future functional studies investigating the role of the GABA system in ACC cell growth phenotypes and metabolism.

Nonlinearities between inhibition and T-type calcium channel activity bidirectionally regulate thalamic oscillations

Absence seizures result from 3 to 5 Hz generalized thalamocortical oscillations that depend on highly regulated inhibitory neurotransmission in the thalamus. Efficient reuptake of the inhibitory neurotransmitter GABA is essential, and reuptake failure worsens human seizures. Here, we show that blocking GABA transporters (GATs) in acute rat brain slices containing key parts of the thalamocortical seizure network modulates epileptiform activity. As expected, we found that blocking either GAT1 or GAT3 prolonged oscillations. However, blocking both GATs unexpectedly suppressed oscillations. Integrating experimental observations into single-neuron and network-level computational models shows how a non-linear dependence of T-type calcium channel gating on GABAB receptor activity regulates network oscillations. Receptor activity that is either too brief or too protracted fails to sufficiently open T-type channels necessary for sustaining oscillations. Only within a narrow range does prolonging GABAB receptor activity promote channel opening and intensify oscillations. These results have implications for therapeutics that modulate inhibition kinetics.