Novel pharmacological targets for GABAergic dysfunction in ADHD.

Attention deficit/hyperactivity disorder (ADHD) is a neurodevelopment disorder that affects approximately 5% of the population. The disorder is characterized by impulsivity, hyperactivity, and deficits in attention and cognition, although symptoms vary across patients due to the heterogenous and polygenic nature of the disorder. Stimulant medications are the standard of care treatment for ADHD patients, and their effectiveness has led to the dopaminergic hypothesis of ADHD in which deficits in dopaminergic signaling, especially in cortical brain regions, mechanistically underly ADHD pathophysiology. Despite their effectiveness in many individuals, almost one-third of patients do not respond to stimulant treatments and the long-term negative side effects of these medications remain unclear. Emerging clinical evidence is beginning to highlight an important role of dysregulated excitatory/inhibitory (E/I) balance in ADHD. These deficits in E/I balance are related to functional abnormalities in glutamate and Gamma-Aminobutyric Acid (GABA) signaling in the brain, with increasing emphasis placed on GABAergic interneurons driving specific aspects of ADHD pathophysiology. Recent genome-wide association studies (GWAS) have also highlighted how genes associated with GABA function are mutated in human populations with ADHD, resulting in the generation of several new genetic mouse models of ADHD. This review will discuss how GABAergic dysfunction underlies ADHD pathophysiology, and how specific receptors/proteins related to GABAergic interneuron dysfunction may be pharmacologically targeted to treat ADHD in subpopulations with specific comorbidities and symptom domains. This article is part of the Special Issue on "PFC circuit function in psychiatric disease and relevant models".

Alternative Splicing and Nonsense-Mediated Decay of a Zebrafish GABA Receptor Subunit Transcript.

The superfamily of Cys-loop ionotropic neurotransmitter receptors includes those that detect GABA, glutamate, glycine, and acetylcholine. There is ample evidence that many Cys-loop receptor subunit genes include alternatively spliced exons. In this study, we report a novel example of alternative splicing (AS): we show that the 68-bp exon 3 in the zebrafish gabrr2b gene-which codes for the ρ2b GABAAR subunit-is an alternative cassette exon. Skipping of gabrr2b exon 3 results in a downstream frame shift and a premature termination codon (PTC). We provide evidence in larval zebrafish that transcripts containing the PTC are subject to degradation through nonsense-mediated decay. We also compile reports of AS of homologous exons in other Cys-loop receptor genes in multiple species. Our data add to a large body of research demonstrating that exon 3 in Cys-loop receptor genes is a conserved site for AS, the effects of which can vary from novel splice-isoform generation to downregulation of gene expression through transcript degradation.

Abnormal axonal development and severe epileptic phenotype in Dynamin-1 (DNM1) encephalopathy.

Dynamin-1 (DNM1) is involved in synaptic vesicle recycling, and DNM1 mutations can lead to developmental and epileptic encephalopathy. The neuroimaging of DNM1 encephalopathy has not been reported in detail. We describe a severe phenotype of DNM1 encephalopathy showing characteristic neuroradiological features. In addition, we reviewed previously reported cases who have DNM1 pathogenic variants with white matter abnormalities. Our case presented drug-resistant seizures from 1 month of age and epileptic spasms at 2 years of age. Brain MRI showed no progression of myelination, progression of diffuse cerebral atrophy, and a thin corpus callosum. Proton magnetic resonance spectroscopy showed a decreased N-acetylaspartate peak and diffusion tensor imaging presented with less pyramidal decussation. Whole-exome sequencing revealed a recurrent de novo heterozygous variant of DNM1. So far, more than 50 cases of DNM1 encephalopathy have been reported. Among these patients, delayed myelination occurred in two cases of GTPase-domain DNM1 encephalopathy and in six cases of middle-domain DNM1 encephalopathy. The neuroimaging findings in this case suggest inadequate axonal development. DNM1 is involved in the release of synaptic vesicles with the inhibitory transmitter GABA, suggesting that GABAergic neuron dysfunction is the mechanism of refractory epilepsy in DNM1 encephalopathy. GABA-mediated signaling mechanisms play important roles in axonal development and GABAergic neuron dysfunction may be cause of white matter abnormalities in DNM1 encephalopathy.

Substituted Cysteine Modification and Protection with n-Alkyl-MTS Reagents Quantifies Steric Changes Induced by a Mutation in Anesthetic Binding Sites on GABA Type A Receptors.

Multiple approaches, including cryogenic electron microscopy (cryo-EM), indicate that the anesthetics etomidate and propofol modulate α1ß2/3γ2 GABAA receptors by binding in overlapping transmembrane inter-subunit sites near ßM286 and αL232 sidechains. High-precision approaches in functional receptors are needed for comparisons with cryo-EM. We previously used substituted cysteine modification and protection (SCAMP) with n-alkyl-methanethiosulfonate (MTS) reagents and electrophysiology in α1ß3M286Cγ2L receptors to estimate the distance from etomidate to ß3M286 with precision near 1.3 Å. Here, we address three more aims using this approach: (i) SCAMP with etomidate was tested in α1L232Cß3γ2L receptors; (ii) studies in α1L232Wß3M286Cγ2L receptors assessed whether α1L232W displaces etomidate relative to ß3M286C; and (iii) results with propofol were compared with those with etomidate. Voltage-clamp electrophysiology in Xenopus oocytes was used to assess persistent functional changes after exposing cysteine-substituted receptors to methyl-MTS through n-decyl-MTS. Overlap of modified cysteine sidechains with bound anesthetic was inferred when anesthetic co-application with alkyl-MTS reagent blocked the development of persistent effects. In α1L232Cß3γ2L receptors, only pentyl-MTS and hexyl-MTS induced persistent effects that were unaltered by etomidate co-application, precluding a direct estimate of intermolecular distance. In α1L232Wß3M286Cγ2L receptors, sidechain overlap with bound etomidate was inferred for modifications with ethyl-MTS through n-pentyl-MTS, with unambiguous cut-on and cut-off. Comparison with results in α1ß3M286Cγ2L reveals that α1L232W, which increases maximal sidechain length by 2.1 Å, displaces etomidate closer to ß3M286C by about 1.3 Å. Propofol results largely mirrored those with etomidate. These findings indicate that both etomidate and propofol bind within 1 Å of α1L232, consistent with cryo-EM structures. SIGNIFICANCE STATEMENT: We combined electrophysiology, cysteine substitutions, and n-alkyl-methanethiosulfonate modifiers in functional GABAA receptors to enable precise estimates of the distance between ß3M286C sidechains and anesthetics (etomidate and propofol) bound in transmembrane ß+/α- inter-subunit pockets. Comparing results in α1ß3M286Cγ2L and α1L232Wß3M286Cγ2L receptors reveals that α1L232W mutations displace both anesthetics toward ß3M286C, indicating that these anesthetics bind within 1 Å of the α1L232 sidechain in functional receptors, consistent with cryogenic electron microscopy structures derived under nonphysiologic conditions.

Computational Exploration of the Effects of Mutations on GABA Aminotransferase in GABA Aminotransferase Deficiency.

Gamma-aminobutyric acid (GABA) transaminase-also called GABA aminotransferase (GABA-AT)-deficiency is a rare autosomal recessive disorder characterized by a severe neonatal-infantile epileptic encephalopathy with symptoms such as seizures, hypotonia, hyperreflexia, developmental delay, and growth acceleration. GABA transaminase deficiency is caused by mutations in GABA-AT, the enzyme responsible for the catabolism of GABA. Mutations in multiple locations on GABA-AT have been reported and their locations have been shown to influence the onset of the disease and the severity of symptoms. We examined how GABA-AT mutations influence the structural stability of the enzyme and GABA-binding affinity using computational methodologies such as molecular dynamics simulation and binding free energy calculation to understand the underlying mechanism through which GABA-AT mutations cause GABA-AT deficiency. GABA-AT 3D model depiction was carried out together with seven individual mutated models of GABA-AT. The structural stability of all the predicted models was analyzed using several tools and web servers. All models were evaluated based on their phytochemical values. Additionally, 100 ns MD simulation was carried out and the mutated models were evaluated using RMSD, RMSF, Rg, and SASA. gmxMMPBSA free energy calculation was carried out. Moreover, RMSD and free energy calculations were also compared with those obtained using online web servers. Our study demonstrates that P152S, Q296H, and R92Q play a more critical role in the structural instability of GABA-AT compared with the other mutated models: G465R, L211F, L478P, and R220K.

SLC4A10 mutation causes a neurological disorder associated with impaired GABAergic transmission.

SLC4A10 is a plasma-membrane bound transporter that utilizes the Na+ gradient to drive cellular HCO3- uptake, thus mediating acid extrusion. In the mammalian brain, SLC4A10 is expressed in principal neurons and interneurons, as well as in epithelial cells of the choroid plexus, the organ regulating the production of CSF. Using next generation sequencing on samples from five unrelated families encompassing nine affected individuals, we show that biallelic SLC4A10 loss-of-function variants cause a clinically recognizable neurodevelopmental disorder in humans. The cardinal clinical features of the condition include hypotonia in infancy, delayed psychomotor development across all domains and intellectual impairment. Affected individuals commonly display traits associated with autistic spectrum disorder including anxiety, hyperactivity and stereotyped movements. In two cases isolated episodes of seizures were reported in the first few years of life, and a further affected child displayed bitemporal epileptogenic discharges on EEG without overt clinical seizures. While occipitofrontal circumference was reported to be normal at birth, progressive postnatal microcephaly evolved in 7 out of 10 affected individuals. Neuroradiological features included a relative preservation of brain volume compared to occipitofrontal circumference, characteristic narrow sometimes 'slit-like' lateral ventricles and corpus callosum abnormalities. Slc4a10 -/- mice, deficient for SLC4A10, also display small lateral brain ventricles and mild behavioural abnormalities including delayed habituation and alterations in the two-object novel object recognition task. Collapsed brain ventricles in both Slc4a10-/- mice and affected individuals suggest an important role of SLC4A10 in the production of the CSF. However, it is notable that despite diverse roles of the CSF in the developing and adult brain, the cortex of Slc4a10-/- mice appears grossly intact. Co-staining with synaptic markers revealed that in neurons, SLC4A10 localizes to inhibitory, but not excitatory, presynapses. These findings are supported by our functional studies, which show the release of the inhibitory neurotransmitter GABA is compromised in Slc4a10-/- mice, while the release of the excitatory neurotransmitter glutamate is preserved. Manipulation of intracellular pH partially rescues GABA release. Together our studies define a novel neurodevelopmental disorder associated with biallelic pathogenic variants in SLC4A10 and highlight the importance of further analyses of the consequences of SLC4A10 loss-of-function for brain development, synaptic transmission and network properties.

Histone lysine demethylase 3B regulates autophagy via transcriptional regulation of GABARAPL1 in acute myeloid leukemia cells.

Macroautophagy (hereafter referred to as autophagy) is a highly conserved self­digestion process that is critical for maintaining homeostasis in response to various stresses. The autophagy­related protein family, including the GABA type A receptor­associated protein (GABARAP) and microtubule­associated protein 1 light chain 3 subfamilies, is crucial for autophagosome biogenesis. Although the regulatory machinery of autophagy in the cytoplasm has been widely studied, its transcriptional and epigenetic regulatory mechanisms still require more targeted investigations. The present study identified histone lysine demethylase 3B (KDM3B) as a crucial component of autophagy on a panel of leukemia cell lines, including K562, THP1 and U937, resulting in transcriptional activation of the autophagy­related gene GABA type A receptor­associated protein like 1 (GABARAPL1). KDM3B expression promoted autophagosome formation and affected the autophagic flux in leukemia cells under the induction of external stimuli. Notably, RNA­sequencing and reverse transcription­quantitative PCR analysis showed that KDM3B knockout inhibited the expression of GABARAPL1. Chromatin immunoprecipitation­quantitative PCR and luciferase assay showed that KDM3B was associated with the GABARAPL1 gene promoter under stimulation and enhanced its transcription. The present findings demonstrated that KDM3B was critical for regulating the GABARAPL1 gene and influencing the process of autophagy in leukemia cells. These results provide a new insight for exploring the association between autophagy and KDM3B epigenetic regulation in leukemia.

GABAA Receptor ß3 Subunit Mutation N328D Heterozygous Knock-in Mice Have Lennox-Gastaut Syndrome.

Lennox-Gastaut Syndrome (LGS) is a developmental and epileptic encephalopathy (DEE) characterized by multiple seizure types, electroencephalogram (EEG) patterns, and cognitive decline. Its etiology has a prominent genetic component, including variants in GABRB3 that encodes the GABAA receptor (GABAAR) ß3 subunit. LGS has an unknown pathophysiology, and few animal models are available for studying LGS. The objective of this study was to evaluate Gabrb3+/N328D knock-in mice as a model for LGS. We generated a heterozygous knock-in mouse expressing Gabrb3 (c.A982G, p.N238D), a de novo mutation identified in a patient with LGS. We investigated Gabrb3+/N328D mice for features of LGS. In 2-4-month-old male and female C57BL/J6 wild-type and Gabrb3+/N328D mice, we investigated seizure severity using video-monitored EEG, cognitive impairment using a suite of behavioral tests, and profiled GABAAR subunit expression by Western blot. Gabrb3+/N328D mice showed spontaneous seizures and signs of cognitive impairment, including deficits in spatial learning, memory, and locomotion. Moreover, Gabrb3+/N328D mice showed reduced ß3 subunit expression in the cerebellum, hippocampus, and thalamus. This phenotype of epilepsy and neurological impairment resembles the LGS patient phenotype. We conclude that Gabrb3+/N328D mice provide a good model for investigating the pathophysiology and therapeutic intervention of LGS and DEEs.

Changes in Memory, Sedation, and Receptor Kinetics Imparted by the ß2-N265M and ß3-N265M GABAA Receptor Point Mutations.

Point mutations in the ß2 (N265S) and ß3 (N265M) subunits of γ-amino butyric acid type A receptors (GABAARs) that render them insensitive to the general anesthetics etomidate and propofol have been used to link modulation of ß2-GABAARs to sedation and ß3-GABAARs to surgical immobility. These mutations also alter GABA sensitivity, and mice carrying the ß3-N265M mutation have been reported to have impaired baseline memory. Here, we tested the effects of the ß2-N265M and ß3-N265M mutations on memory, movement, hotplate sensitivity, anxiety, etomidate-induced sedation, and intrinsic kinetics. We found that both ß2-N265M and ß3-N265M mice exhibited baseline deficits in the Context Preexposure Facilitation Effect learning paradigm. Exploratory activity was slightly greater in ß2-N265M mice, but there were no changes in either genotype in anxiety or hotplate sensitivity. ß2-N265M mice were highly resistant to etomidate-induced sedation, and heterozygous mice were partially resistant. In rapid solution exchange experiments, both mutations accelerated deactivation two- to three-fold compared to wild type receptors and prevented modulation by etomidate. This degree of change in the receptor deactivation rate is comparable to that produced by an amnestic dose of etomidate but in the opposite direction, indicating that intrinsic characteristics of GABAARs are optimally tuned under baseline conditions to support mnemonic function.

TorC1 and nitrogen catabolite repression control of integrated GABA shunt and retrograde pathway gene expression.

Despite our detailed understanding of how the lower GABA shunt and retrograde genes are regulated, there is a paucity of validated information concerning control of GAD1, the glutamate decarboxylase gene which catalyzes the first reaction of the GABA shunt. Further, integration of glutamate degradation via the GABA shunt has not been investigated. Here, we show that while GAD1 shares a response to rapamycin-inhibition of the TorC1 kinase, it does so independently of the Gln3 and Gat1 NCR-sensitive transcriptional activators that mediate transcription of the lower GABA shunt genes. We also show that GABA shunt gene expression increases dramatically in response to nickel ions. The α-ketoglutarate needed for the GABA shunt to cycle, thereby producing reduced pyridine nucleotides, derives from the retrograde pathway as shown by a similar high increase in the retrograde reporter, CIT2 when nickel is present in the medium. These observations demonstrate high integration of the GABA shunt, retrograde, peroxisomal glyoxylate cycle, and ß-oxidation pathways.

Biosynthetic Characterization, Heterologous Production, and Genomics-Guided Discovery of GABA-Containing Fungal Heptapeptides.

The biosynthetic gene cluster of γ-aminobutyric acid (GABA)-containing fungal cyclic heptapeptides unguisins A (1) and B (2) was identified in the fungus Aspergillus violaceofuscus CBS 115571. In vitro enzymatic reactions and gene deletion experiments revealed that the unguisin pathway involves the alanine racemase UngC to provide d-alanine, which is then accepted by the first adenylation domain of the nonribosomal peptide synthetase (NRPS) UngA. Intriguingly, the hydrolase UngD was found to transform unguisins into previously undescribed linear peptides. Subsequently, heterologous production of these peptides in Aspergillus oryzae was achieved, in which we established a methodology to readily introduce a large NRPS gene into the fungal host. Finally, genome mining revealed new unguisin congeners, each containing a (2R,3R)-ß-methylphenylalanine residue.

TET3 epigenetically controls feeding and stress response behaviors via AGRP neurons.

The TET family of dioxygenases promote DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC). Hypothalamic agouti-related peptide-expressing (AGRP-expressing) neurons play an essential role in driving feeding, while also modulating nonfeeding behaviors. Besides AGRP, these neurons produce neuropeptide Y (NPY) and the neurotransmitter GABA, which act in concert to stimulate food intake and decrease energy expenditure. Notably, AGRP, NPY, and GABA can also elicit anxiolytic effects. Here, we report that in adult mouse AGRP neurons, CRISPR-mediated genetic ablation of Tet3, not previously known to be involved in central control of appetite and metabolism, induced hyperphagia, obesity, and diabetes, in addition to a reduction of stress-like behaviors. TET3 deficiency activated AGRP neurons, simultaneously upregulated the expression of Agrp, Npy, and the vesicular GABA transporter Slc32a1, and impeded leptin signaling. In particular, we uncovered a dynamic association of TET3 with the Agrp promoter in response to leptin signaling, which induced 5hmC modification that was associated with a chromatin-modifying complex leading to transcription inhibition, and this regulation occurred in both the mouse models and human cells. Our results unmasked TET3 as a critical central regulator of appetite and energy metabolism and revealed its unexpected dual role in the control of feeding and other complex behaviors through AGRP neurons.

Exogenous GABA improves the resistance of apple seedlings to long-term drought stress by enhancing GABA shunt and secondary cell wall biosynthesis.

Drought stress is an important factor limiting apple production. γ-Aminobutyric acid (GABA) exists widely in plants and participates in the response to abiotic stress as a metabolite or signaling molecule. The role of exogenous GABA in apple plants, response to long-term drought stress remains unclear. Our study confirmed that exogenous GABA affects the drought resistance of apple plants under long-term drought stress. We found that 1 mM exogenous GABA improved the resistance of apple seedlings to long-term drought stress. The plants showed better growth, less reactive oxygen radical accumulation, less damage to cell membranes and greater active photosynthetic capacity. Under long-term drought stress, exogenous GABA facilitated GABA shunt, resulting in more accumulation of organic acids, namely citric acid, succinic acid and malic acid, in roots and stems of apple seedlings. In addition, exogenous GABA upregulated the expression of cellulose-related genes and lignin-related genes, and activated secondary cell wall-related transcription factors to synthesize more cellulose and lignin. A multiple factorial analysis confirmed that the GABA shunt and the biosynthesis of cellulose and lignin substantially contributed to the growth of apple seedlings with the application of exogenous GABA under long-term drought stress. Our results suggested that exogenous GABA improved the resistance of apple seedlings to long-term drought stress by enhancing GABA shunt and secondary cell wall biosynthesis.

Cell Surface Calcium-Sensing Receptor Heterodimers: Mutant Gene Dosage Affects Ca2+ Sensing but Not G Protein Interaction.

The calcium-sensing receptor is a homodimeric class C G protein-coupled receptor (GPCR) that senses extracellular Ca2+ (Ca2+ o ) via a dimeric extracellular Venus flytrap (VFT) unit that activates G protein-dependent signaling via twin Cysteine-rich domains linked to transmembrane heptahelical (HH) bundles. It plays a key role in the regulation of human calcium and thus mineral metabolism. However, the nature of interactions between VFT units and HH bundles, and the impacts of heterozygous or homozygous inactivating mutations, which have implications for disorders of calcium metabolism are not yet clearly defined. Herein we generated CaSR-GABAB1 and CaSR-GABAB2 chimeras subject to GABAB -dependent endoplasmic reticulum sorting to traffic mutant heterodimers to the cell surface. Transfected HEK-293 cells were assessed for Ca2+ o -stimulated Ca2+ i mobilization using mutations in either the VFT domains and/or HH bundle intraloop-2 or intraloop-3. When the same mutation was present in both VFT domains of receptor dimers, analogous to homozygous neonatal severe hyperparathyroidism (NSHPT), receptor function was markedly impaired. Mutant heterodimers containing one wild-type (WT) and one mutant VFT domain, however, corresponding to heterozygous familial hypocalciuric hypercalcemia type-1 (FHH-1), supported maximal signaling with reduced Ca2+ o potency. Thus two WT VFT domains were required for normal Ca2+ o potency and there was a pronounced gene-dosage effect. In contrast, a single WT HH bundle was insufficient for maximal signaling and there was no functional difference between heterodimers in which the mutation was present in one or both intraloops; ie, no gene-dosage effect. Finally, we observed that the Ca2+ o -stimulated CaSR operated exclusively via signaling in-trans and not via combined in-trans and in-cis signaling. We consider how receptor asymmetry may support the underlying mechanisms. © 2022 The Authors. Journal of Bone and Mineral Research published by Wiley Periodicals LLC on behalf of American Society for Bone and Mineral Research (ASBMR).

Whole Transcriptome Sequencing Reveals Drought Resistance-Related Genes in Upland Cotton.

China, particularly the cotton-growing province of Xinjiang, is experiencing acute agricultural water shortages, stifling the expansion of the cotton sector. Discovering drought resistance genes in cotton and generating high-quality, drought-resistant cotton varieties through molecular breeding procedures are therefore critical to the cotton industry's success. The drought-resistant cotton variety Xinluzhong No. 82 and the drought-sensitive cotton variety Kexin No. 1 were utilised in this study to uncover a batch of drought-resistant candidate genes using whole transcriptome sequencing. The following are the key research findings: A competing endogenous RNA network (ceRNA) was built using complete transcriptional sequencing to screen the core genes in the core pathway, and two drought-related candidate genes were discovered. It was found that γ-aminobutyric acid aminotransferase (GhGABA-T, Gohir.A11G156000) was upregulated at 0 h vs. 12 h and downregulated at 12 h vs. 24 h. L-Aspartate oxidase (GhAO, Gohir.A07G220600) was downregulated at 0 h vs. 12 h and upregulated at 12 h vs. 24 h. GABA-T is analogous to a pyridoxal phosphate-dependent transferase superfamily protein (POP2) in Arabidopsis thaliana and influences plant drought resistance by controlling γ-aminobutyric acid (GABA) concentration. The analogue of GhAO in A. thaliana is involved in the early steps of nicotinamide adenine dinucleotide (NAD) production as well as in plant antioxidant responses. This study revealed that gene expression regulatory networks can be used for rapid screening of reliable drought resistance genes and then utilised to validate gene function.

Characterization of two novel pentose-fermenting and GABA-producing species: Levilactobacillus tujiorum sp. nov. and Secundilactobacillus angelensis sp. nov. Isolated from a solid-state fermented zha-chili.

Lactobacilli are dominant in zha-chili. This study provides a taxonomic characterization of five bacterial strains isolated from zha-chili in China. The cells were Gram-positive, facultative anaerobic, non-spore-forming, flagella-free, catalase-negative, heterofermentative, pentose-fermenting, and gamma-aminobutyric acid (GABA)-producing rods. For HBUAS51241T, HBUAS51329, and HBUAS51416, C16:0, C18:1ω9c and C19:0 iso were the predominant cellular fatty acids; diphosphatidylglycerol (DPG), phosphatidylglycerol (DP), glycolipids (GL), and glycolipids (AL) were the major phospholipids. While for HBUAS51383T and HBUAS58055, C16:0, C18:1ω9c, C19:0 cyclo ω8c were the predominant cellular fatty acids; DPG, DP, GL, and AL were the major phospholipids. Strains HBUAS51241T, HBUAS51329, and HBUAS51416 showed 98.1-99.1% 16S rRNA gene sequence similarity, 80.2-81.4% ANI, 87.7-90.0% AAI, and 23.8-32.8% digital DDH to their closest related type strains Levilactobacillus hammesii DSM 16381T, Levilactobacillus parabrevis ATCC 53295T, and Levilactobacillus fuyuanensis 244-4T. Strains HBUAS51383T and HBUAS58055 showed 98.7-99.5% 16S rRNA gene sequence similarity, 75.4-81.4% ANI, 75.5-89.1% AAI, and 19.7-24.0% digital DDH to their closest related type strains Secundilactobacillus silagincola IWT5T, Secundilactobacillus silagei JCM 19001T, Secundilactobacillus pentosiphilus IWT25T, Secundilactobacillus mixtipabuli IWT30T, Secundilactobacillus odoratitofui DSM 19909T, and Secundilactobacillus similis DSM 23365T. The central carbon metabolism pathways for the five strains were summarizeded. Based on the phenotypic, chemotaxonomic, and genomic data, we propose two novel species Levilactobacillus tujiorum sp. nov. whose type strain is HBUAS51241T (=GDMCC 1.3022T = JCM 35241T), and Secundilactobacillus angelensis sp. nov. whose type strain is HBUAS51383T (=GDMCC 1.3021T = JCM 35209T).

Watercore Pear Fruit Respiration Changed and Accumulated γ-Aminobutyric Acid (GABA) in Response to Inner Hypoxia Stress.

Watercore is a physiological disorder which often occurs on the pear fruit and the excessive accumulation of sorbitol in fruit intercellular space is considered to be an important cause of watercore. Our previous studies found that the metabolic disorder of sugars may lead to hypoxia stress and disturb respiration, resulting in aggravated fruit rot and the formation of bitter substances. However, the further changes of respiration and the fruit response mechanism are not well understood. A comprehensive transcriptome analysis of 'Akibae' pear watercore fruit was performed in this study. The transcriptome results revealed the hypoxia stress significantly induced the expression of several key enzymes in the TCA cycle and may lead to the accumulation of succinic acid in watercore fruit. The glycolytic pathway was also significantly enhanced in watercore fruit. Moreover, the γ-aminobutyric acid (GABA) synthesis related genes, glutamate decarboxylase (GAD) genes and polyamine oxidase (PAO) genes, which associated with the GABA shunt and the polyamine degradation pathway were significantly upregulated. In addition, the PpGAD1 transcript level increased significantly along with the increase of GAD activity and GABA content in the watercore fruit. Above all, these findings suggested that the hypoxic response was marked by a significant increase of the hypoxia-inducible metabolites succinic acid and GABA and that PpGAD1 may play a key role in response to watercore by controlling the GABA synthesis.

Transcriptomic analysis supports collective endometrial cell migration in the pathogenesis of adenomyosis.

RESEARCH QUESTION: Adenomyosis is a common uterine disorder of uncertain causes. Can transcriptomic analyses of the endometrium and myometrium reveal potential mechanisms underlying adenomyosis pathogenesis? DESIGN: Transcriptomic profiles of eutopic endometrium and myometrium from women with and without diffuse adenomyosis and with symptomatic FIGO type 2-5 fibroids in the proliferative phase of the menstrual cycle were assessed using RNA sequencing and bioinformatic analysis. Differentially expressed genes (DEG) and potential pathways were validated by quantitative reverse transcription polymerase chain reaction, immunoblotting and Masson staining, using additional clinical samples. RESULTS: Top biological processes in the endometrium of women with versus without adenomyosis, enriched from DEG, comprised inflammation, extracellular matrix (ECM) organization, collagen degradation and hyaluronan synthesis, which are key in cell migration and cell movement. Top biological processes enriched from DEG in the myometrium of women with versus without adenomyosis revealed ECM organization dysfunction, abnormal sensory pain perception and gamma aminobutyric acid (GABA) synaptic transmission. Dysregulation of prolactin signalling was also enriched in eutopic endometrium and in the myometrium of women with adenomyosis. CONCLUSIONS: Overall, our results support the invasive endometrium theory in the pathogenesis of adenomyosis, in which inflammation induces ECM remodelling resulting in a track for subsequent endometrial collective cell migration and onset of adenomyosis. Moreover, abnormal myometrial GABA synaptic transmission may contribute to dysmenorrhoea in women with adenomyosis and is a possible target for novel therapeutic development. Prolactin signalling abnormalities may serve as another opportunity for therapeutic intervention.

Cell-Selective Adeno-Associated Virus-Mediated SCN1A Gene Regulation Therapy Rescues Mortality and Seizure Phenotypes in a Dravet Syndrome Mouse Model and Is Well Tolerated in Nonhuman Primates.

Dravet syndrome (DS) is a developmental and epileptic encephalopathy caused by monoallelic loss-of-function variants in the SCN1A gene. SCN1A encodes for the alpha subunit of the voltage-gated type I sodium channel (NaV1.1), the primary voltage-gated sodium channel responsible for generation of action potentials in GABAergic inhibitory interneurons. In these studies, we tested the efficacy of an adeno-associated virus serotype 9 (AAV9) SCN1A gene regulation therapy, AAV9-REGABA-eTFSCN1A, designed to target transgene expression to GABAergic inhibitory neurons and reduce off-target expression within excitatory cells, in the Scn1a+/- mouse model of DS. Biodistribution and preliminary safety were evaluated in nonhuman primates (NHPs). AAV9-REGABA-eTFSCN1A was engineered to upregulate SCN1A expression levels within GABAergic inhibitory interneurons to correct the underlying haploinsufficiency and circuit dysfunction. A single bilateral intracerebroventricular (ICV) injection of AAV9-REGABA-eTFSCN1A in Scn1a+/- postnatal day 1 mice led to increased SCN1A mRNA transcripts, specifically within GABAergic inhibitory interneurons, and NaV1.1 protein levels in the brain. This was associated with a significant decrease in the occurrence of spontaneous and hyperthermia-induced seizures, and prolonged survival for over a year. In NHPs, delivery of AAV9-REGABA-eTFSCN1A by unilateral ICV injection led to widespread vector biodistribution and transgene expression throughout the brain, including key structures involved in epilepsy and cognitive behaviors, such as hippocampus and cortex. AAV9-REGABA-eTFSCN1A was well tolerated, with no adverse events during administration, no detectable changes in clinical observations, no adverse findings in histopathology, and no dorsal root ganglion-related toxicity. Our results support the clinical development of AAV9-REGABA-eTFSCN1A (ETX101) as an effective and targeted disease-modifying approach to SCN1A+ DS.

Characterization of GABA-Transaminase Gene from Mulberry (Morus multicaulis) and Its Role in Salt Stress Tolerance.

Gamma-aminobutyric acid (GABA) has been reported to accumulate in plants when subjected to salt stress, and GABA-transaminase (GABA-T) is the main GABA-degrading enzyme in the GABA shunt pathway. So far, the salt tolerance mechanism of the GABA-T gene behind the GABA metabolism remains unclear. In this study, the cDNA (designated MuGABA-T) of GABA-T gene was cloned from mulberry, and our data showed that MuGABA-T protein shares some conserved characteristics with its homologs from several plant species. MuGABA-T gene was constitutively expressed at different levels in mulberry tissues, and was induced substantially by NaCl, ABA and SA. In addition, our results demonstrated that exogenous application of GABA significantly reduced the salt damage index and increased plant resistance to NaCl stress. We further performed a functional analysis of MuGABA-T gene and demonstrated that the content of GABA was reduced in the transgenic MuGABA-T Arabidopsis plants, which accumulated more ROS and exhibited more sensitivity to salt stress than wild-type plants. However, exogenous application of GABA significantly increased the activities of antioxidant enzymes and alleviated the active oxygen-related injury of the transgenic plants under NaCl stress. Moreover, the MuGABA-T gene was overexpressed in the mulberry hairy roots, and similar results were obtained for sensitivity to salt stress in the transgenic mulberry plants. Our results suggest that the MuGABA-T gene plays a pivotal role in GABA catabolism and is responsible for a decrease in salt tolerance, and it may be involved in the ROS pathway in the response to salt stress. Taken together, the information provided here is helpful for further analysis of the function of GABA-T genes, and may promote mulberry resistance breeding in the future.