Γ-aminobutyric Acid Receptor Research Articles

Review on anti-alzheimer drug development: approaches, challenges and perspectives.

Alzheimer is an irreversible progressive neurodegenerative disease that causes failure of cerebral neurons and disability of the affected person to practice normal daily life activities. There is no concrete evidence to identify the exact reason behind the disease, so several relevant hypotheses emerged, highlighting many possible therapeutic targets, such as acetylcholinesterase, cholinergic receptors, N-methyl d-aspartate receptors, phosphodiesterase, amyloid β protein, protein phosphatase 2A, glycogen synthase kinase-3 beta, β-secretase, γ-secretase, α-secretase, serotonergic receptors, glutaminyl cyclase, tumor necrosis factor-α, γ-aminobutyric acid receptors, and mitochondria. All of these targets have been involved in the design of new potential drugs. An extensive number of these drugs have been studied in clinical trials. However, only galantamine, donepezil, and rivastigmine (ChEIs), memantine (NMDA antagonist), and aducanumab and lecanemab (selective anti-Aβ monoclonal antibodies) have been approved for AD treatment. Many drugs failed in the clinical trials to such an extent that questions have been posed about the significance of some of the aforementioned targets. On the contrary, the data of other drugs were promising and shed light on the significance of their targets for the development of new potent anti-alzheimer drugs.

γ-Aminobutyric acid transporter and GABAA receptor mechanisms in Slc6a1+/A288V and Slc6a1+/S295L mice associated with developmental and epileptic encephalopathies.

We have previously characterized the molecular mechanisms for variants in γ-aminobutyric acid transporter 1-encoding solute carrier family 6-member 1 (SLC6A1) in vitro and concluded that a partial or complete loss of γ-aminobutyric acid uptake due to impaired protein trafficking is the primary aetiology. Impairment of γ-aminobutyric acid transporter 1 function could cause compensatory changes in the expression of γ-aminobutyric acid receptors, which, in turn, modify disease pathophysiology and phenotype. Here we used different approaches including radioactive 3H γ-aminobutyric acid uptake in cells and synaptosomes, immunohistochemistry and confocal microscopy as well as brain slice surface protein biotinylation to characterize Slc6a1+/A288V and Slc6a1+/S295L mice, representative of a partial or a complete loss of function of SLC6A1 mutations, respectively. We employed the γ-aminobutyric acid transporter 1-specific inhibitor [3H]tiagabine binding and GABAA receptor subunit-specific radioligand binding to profile the γ-aminobutyric acid transporter 1 and GABAA receptor expression in major brain regions such as cortex, cerebellum, hippocampus and thalamus. We also determined the total and surface expression of γ-aminobutyric acid transporter 1, γ-aminobutyric acid transporter 3 and expression of GABAA receptor in the major brain regions in the knockin mice. We found that γ-aminobutyric acid transporter 1 protein was markedly reduced in cortex, hippocampus, thalamus and cerebellum in both mutant mouse lines. Consistent with the findings of reduced γ-aminobutyric acid uptake for both γ-aminobutyric acid transporter 1(A288V) and γ-aminobutyric acid transporter 1(S295L), both the total and the γ-aminobutyric acid transporter 1-mediated 3H γ-aminobutyric acid reuptake was reduced. We found that γ-aminobutyric acid transporter 3 is only abundantly expressed in the thalamus and there was no compensatory increase of γ-aminobutyric acid transporter 3 in either of the mutant mouse lines. γ-Aminobutyric acid transporter 1 was reduced in both somatic regions and nonsomatic regions in both mouse models, in which a ring-like structure was identified only in the Slc6a1+/A288V mouse, suggesting more γ-aminobutyric acid transporter 1 retention inside endoplasmic reticulum in the Slc6a1+/A288V mouse. The [3H]tiagabine binding was similar in both mouse models despite the difference in γ-aminobutyric acid uptake function and γ-aminobutyric acid transporter 1 protein expression for both mutations. There were no differences in GABAA receptor subtype expression, except for a small increase in the expression of α5 subunits of GABAA receptor in the hippocampus of Slc6a1S295L homozygous mice, suggesting a potential interaction between the expression of this GABAA receptor subtype and the mutant γ-aminobutyric acid transporter 1. The study provides the first comprehensive characterization of the SLC6A1 mutations in vivo in two representative mouse models. Because both γ-aminobutyric acid transporter 1 and GABAA receptors are targets for anti-seizure medications, the findings from this study can help guide tailored treatment options based on the expression and function of γ-aminobutyric acid transporter 1 and GABAA receptor in SLC6A1 mutation-mediated neurodevelopmental and epileptic encephalopathies.

GABAA Receptor Benzodiazepine Binding Sites and Motor Impairments in Parkinson's Disease.

Flumazenil is an allosteric modulator of the γ-aminobutyric acid-A receptor (GABAAR) benzodiazepine binding site that could normalize neuronal signaling and improve motor impairments in Parkinson's disease (PD). Little is known about how regional GABAAR availability affects motor symptoms. We investigated the relationship between regional availability of GABAAR benzodiazepine binding sites and motor impairments in PD. Methods: A total of 11 Patients with PD (males; mean age 69.0 ± 4.6 years; Hoehn and Yahr stages 2-3) underwent [11C]flumazenil GABAAR benzodiazepine binding site and [11C]dihydrotetrabenazine vesicular monoamine transporter type-2 (VMAT2) PET imaging and clinical assessment. Stepwise regression analysis was used to predict regional cerebral correlates of the four cardinal UPDRS motor scores using cortical, striatal, thalamic, and cerebellar flumazenil binding estimates. Thalamic GABAAR availability was selectively associated with axial motor scores (R2 = 0.55, F = 11.0, β = -6.4, p = 0.0009). Multi-ligand analysis demonstrated significant axial motor predictor effects by both thalamic GABAAR availability (R2 = 0.47, β = -5.2, F = 7.2, p = 0.028) and striatal VMAT2 binding (R2 = 0.30, β = -3.9, F = 9.1, p = 0.019; total model: R2 = 0.77, F = 11.9, p = 0.0056). Post hoc analysis demonstrated that thalamic [11C]methyl-4-piperidinyl propionate cholinesterase PET and K1 flow delivery findings were not significant confounders. Findings suggest that reduced thalamic GABAAR availability correlates with worsened axial motor impairments in PD, independent of nigrostriatal degeneration. These findings may augur novel non-dopaminergic approaches to treating axial motor impairments in PD.

GluD1 binds GABA and controls inhibitory plasticity.

Fast synaptic neurotransmission in the vertebrate central nervous system relies primarily on ionotropic glutamate receptors (iGluRs), which drive neuronal excitation, and type A γ-aminobutyric acid receptors (GABAARs), which are responsible for neuronal inhibition. However, the GluD1 receptor, an iGluR family member, is present at both excitatory and inhibitory synapses. Whether and how GluD1 activation may affect inhibitory neurotransmission is unknown. In this work, by using a combination of biochemical, structural, and functional analyses, we demonstrate that GluD1 binds GABA, a previously unknown feature of iGluRs. GluD1 activation produces long-lasting enhancement of GABAergic synaptic currents in the adult mouse hippocampus through a non-ionotropic mechanism that is dependent on trans-synaptic anchoring. The identification of GluD1 as a GABA receptor that controls inhibitory synaptic plasticity challenges the classical dichotomy between glutamatergic and GABAergic receptors.

Mechanistic insights into the selectivity of bicyclophosphorothionate antagonists for housefly versus rat GABA receptors.

γ-Aminobutyric acid (GABA) receptors (GABARs) are validated targets of insecticides. Bicyclophosphorothionates are a group of insecticidal compounds that act as noncompetitive antagonists of GABARs. We previously reported that the analogs exhibit various degrees of selectivity for housefly versus rat GABARs, depending on substitutions at the 3- and 4-positions. We here sought to elucidate the unsolved mechanisms of the receptor selectivity using quantitative structure-activity relationship (QSAR), molecular docking, and molecular dynamics approaches. Three-dimensional (3D)-QSAR studies using Topomer comparative molecular field analysis quantitatively demonstrated how the introduction of a small alkyl group at the 3-position of bicyclophosphorothionates contributes to the housefly versus rat GABAR selectivity. To investigate the molecular mechanisms of the selective action, bicyclophosphorothionates were docked into housefly Resistance to dieldrin (RDL) GABAR and rat α1β2γ2 GABAR homology models built using the published 3D-structures of human GABARs as templates. The results of molecular docking and molecular dynamics simulations revealed that the 2'Ala and 6'Thr residues of the RDL subunit within the channel are the key amino acids for binding to the housefly GABARs, whereas the 2'Ser residue of γ2 subunit plays a crucial role in binding to rat GABARs. We revealed the molecular mechanisms underlying the selective antagonistic action of bicyclophosphorothionates on housefly versus rat GABARs. The information presented should help design and develop novel, safe GABAR-targeting insecticides. © 2023 Society of Chemical Industry.

Characterization of a de novo GABBR2 variant linked to autism spectrum disorder

GABAB receptors (GABABRs) are G protein-coupled receptors for γ-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system. Pathogenic variants in the GABBR1 and GABBR2 genes, which encode the GB1 and GB2 subunits of GABABRs, are implicated in several neurological and developmental disorders, including epilepsy and autism. Here we present a 7-year-old boy with Level 3 Autism Spectrum Disorder who carries a de novo heterozygous missense GABBR2 p.Arg212Gln variant. This variant was identified through whole exome sequencing and classified as variant of unknown significance (VUS). Treatment with the GABABR agonist baclofen showed no clinical improvement, raising the question whether this VUS is responsible for the patient’s phenotype. We conducted a study to investigate the impact of the GABBR2 p.Arg212Gln and the previously reported GABBR2 p.Arg212Trp variants on protein structure and receptor activity. This study utilized a combination of molecular dynamics (MD) simulations, and in vitro experiments. Our simulations demonstrate that both amino acid substitutions locally alter amino acid interactions in the extracellular domain of GB2. Most importantly, the substitutions influence the positioning of transmembrane helices, shifting the conformation towards an active state with GABBR2 p.Arg212Gln and an inactive state with GABBR2 p.Arg212Trp. Functional assays confirmed the MD predictions, as evidenced by increased constitutive activity and enhanced potency of GABA for GABBR2 p.Arg212Gln, and a decreased constitutive activity with a loss of GABA potency for GABBR2 p.Arg212Trp. Our findings demonstrate the utility of MD simulations in predicting the functional consequences of VUS. Clarifying the pathogenic mechanisms associated with gene variants will aid in the identification of personalized treatment approaches.

Epiberberine induced p53/p21-dependent G2/M cell cycle arrest and cell apoptosis in gastric cancer cells by activating γ-aminobutyric acid receptor- β3

Background and PurposeEpiberberine (EPI) is one of the most important bioalkaloid found in the rhizome of Coptis chinensis, which has been observed to exhibit pharmaceutical effects against gastric cancer (GC). Nevertheless, the potential mechanism of EPI against GC cells still remains unclear. This study aimed to identify the core receptor on GC cells through which EPI inhibited the growth of GC cells and to explore the underlying inhibitory mechanisms. MethodsTo identify hub receptor targets that respond to EPI treatment, RNA sequencing (RNA-Seq) data from a tumor-bearing mouse model were analyzed using bioinformatics method and molecular docking. The binding interaction between EPI and GABRB3 was validated through western blotting based-cellular thermal shift assay (WB-CETSA). To further verify the binding region between EPI and GABRB3 through circular dichroism (CD) chromatography, fragments of the extracellular and transmembrane domains of the GABRB3 protein were expressed and purified in vitro. Stable cell lines with the overexpression or knockdown of GABRB3 were established using the recombinant lentivirus system. MTT ((3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide)) assay, colony formation assay, invasion and migration experiments, and flow cytometry were conducted to validate the inhibitory effect of EPI on the GC cells via GABRB3. Additionally, western blotting was utilized to explore the potential inhibitory mechanisms. ResultsThrough the combination of multiple bioinformatics methods and molecular docking, we found that the γ-aminobutyric acid type A receptor subunit -β3 (GABRB3) might be the critical receptor target in response to EPI treatment. The results of WB-CETSA analysis indicated that EPI significantly promoted the thermostability of the GABRB3 protein. Importantly, EPI could directly bind to GABRB3 and alter the secondary structure of GABRB3 fragments similar to the natural agonist, γ-aminobutyric acid (GABA). The EPI-induced suppression of the malignant phenotype of GC cells was dependent on the presence of GABRB3. GABRB3 expression was positively correlated with TP53 in patients with GC. The binding of EPI to GABRB3 stimulated p53 accumulation in GC cells. This activated the p21/CDK1/cyclinB1 pathway, resulting in G2/M cell cycle arrest, and induced the Bcl-2/BAX/Caspase axis-dependent cell apoptosis. ConclusionThis study revealed the target receptor for EPI in GC cells and provided new insights into its anticancer mechanisms.

Reduced evoked cortical beta and gamma activity and neuronal synchronization in succinic semialdehyde dehydrogenase deficiency, a disorder of γ-aminobutyric acid metabolism.

Succinic semialdehyde dehydrogenase deficiency is a rare autosomal recessively inherited metabolic disorder of γ-aminobutyric acid catabolism manifested by intellectual disability, expressive aphasia, movement disorders, psychiatric ailments and epilepsy. Subjects with succinic semialdehyde dehydrogenase deficiency are characterized by elevated γ-aminobutyric acid and related metabolites, such as γ-guanidinobutyric acid, and an age-dependent downregulation of cerebral γ-aminobutyric acid receptors. These findings indicate impaired γ-aminobutyric acid and γ-aminobutyric acid sub-type A (GABAA) receptor signalling as major factors underlying the pathophysiology of this neurometabolic disorder. We studied the cortical oscillation patterns and their relationship with γ-aminobutyric acid metabolism in 18 children affected by this condition and 10 healthy controls. Using high-density EEG, we recorded somatosensory cortical responses and resting-state activity. Using electrical source imaging, we estimated the relative power changes (compared with baseline) in both stimulus-evoked and stimulus-induced responses for physiologically relevant frequency bands and resting-state power. Stimulus-evoked oscillations are phase locked to the stimulus, whereas induced oscillations are not. Power changes for both evoked and induced responses as well as resting-state power were correlated with plasma γ-aminobutyric acid and γ-guanidinobutyric acid concentrations and with cortical γ-aminobutyric acid measured by proton magnetic resonance spectroscopy. Plasma γ-aminobutyric acid, γ-guanidinobutyric acid and cortical γ-aminobutyric acid were higher in patients than in controls (P < 0.001 for both). Beta and gamma relative power were suppressed for evoked responses in patients versus controls (P < 0.01). No group differences were observed for induced activity (P > 0.05). The mean gamma frequency of evoked responses was lower in patients versus controls (P = 0.002). Resting-state activity was suppressed in patients for theta (P = 0.011) and gamma (P < 0.001) bands. Evoked power changes were inversely correlated with plasma γ-aminobutyric acid and with γ-guanidinobutyric acid for beta (P < 0.001) and gamma (P < 0.001) bands. Similar relationships were observed between the evoked power changes and cortical γ-aminobutyric acid for all tested areas in the beta band (P < 0.001) and for the posterior cingulate gyrus in the gamma band (P < 0.001). We also observed a negative correlation between resting-state activity and plasma γ-aminobutyric acid and γ-guanidinobutyric acid for theta (P < 0.001; P = 0.003), alpha (P = 0.003; P = 0.02) and gamma (P = 0.02; P = 0.01) bands. Our findings indicate that increased γ-aminobutyric acid concentration is associated with reduced sensory-evoked beta and gamma activity and impaired neuronal synchronization in patients with succinic semialdehyde dehydrogenase deficiency. This further elucidates the pathophysiology of this neurometabolic disorder and serves as a potential biomarker for therapeutic trials.

OR23-01 The Nonredundant Role Of Beta-Amyloid In Mediating Tonic PTH Secretion In Physiological And Pathological States

Abstract Disclosure: C. Tu: None. Z. Cheng: None. K.A. Pena: None. N. Szeto: None. J.A. Sosa: None. J. Vilardaga: None. J. Koh: None. W. Chang: None. Understanding the mechanisms driving parathyroid hormone (PTH) hypersecretion in primary hyperparathyroidism (PHPT) is essential for better management of this common endocrinopathy. Prior studies showed that reduced Ca2+-sensing receptor (CaSR) expression and the subsequent increases in heterodimerization of the CaSR with the type B γ-aminobutyric acid receptor 1 (GABAB1R) are causally linked to PTH hypersecretion in PHPT mouse models (Nat Metab 2:243-255). We further showed that expression of the putative GABAB1R ligands, amyloid precursor protein (APP) and its proteolytic product, β-amyloid (Aβ1-42), is significantly upregulated in adenomas of PHPT patients associated with vitamin D insufficiency/deficiency when compared to normal donor controls. The current study aims to delineate the actions of Aβ1-42 in promoting tonic PTH secretion and its interactions with vitamin D receptor (VDR) signaling in parathyroid glands (PTGs) in basal and PHPT states. We show that Aβ1-42 (200 nM) stimulated tonic PTH secretion in cultures of normal human PTGs without shifting the calcium/PTH secretion setpoint (Ca2+-setpoint). In contrast, conditional knockout (KO) of the App gene in the parathyroid cell (PTC) of PTCAppΔflox/Δflox mice significantly reduced serum PTH levels (KO: 64±13 pg/ml vs Control: 109±12 pg/ml; p&amp;lt;0.05, n=8) despite hypocalcemia, indicating hypoparathyroidism. In PTGs cultured from the PTCAppΔflox/Δflox mice, supplementation of Aβ1-42 dose-dependently (EC50=5.6 nM, p&amp;lt;0.001) increased PTH secretion without altering the Ca2+-setpoint. However, the stimulatory effects of Aβ1-42 on tonic PTH secretion were completely abrogated in the PTGs with concurrent deletions of App, Casr and Gabbr1 genes, supporting a direct action of Aβ1-42 on CaSR and/or GABAB1R. The latter notion is further supported by the ability of Aβ1-42 to stimulate cAMP production in cells co-expressing CaSR and GABAB1R. PTC-specific deletion of the Vdr gene in the PTCVdrΔflox/Δflox mice led to elevated serum PTH levels in vivo and increased tonic PTH secretion with unaffected Ca2+-setpoint in PTGs in vitro. Concurrent ablation of the App gene completely normalized serum PTH levels in the PTCVdrΔflox/Δflox mice and prevented PTH hypersecretion in their PTGs in culture. These findings support a critical role of the APP-derived Aβ1-42 in mediating tonic PTH secretion in the normal physiological state and suggest a new mechanism driving PTH hypersecretion in PHPT due to vitamin D deficiency. Presentation: Saturday, June 17, 2023

THU396 Digital Spatial Profiling Of Human Parathyroid Tumors Reveals Distinct Cellular And Molecular Alterations Linked To Vitamin D Deficiency

Abstract Disclosure: C. Tu: None. W. Chang: None. J.A. Sosa: Advisory Board Member; Self; Novo Nordisk, AstraZeneca, Eli Lilly &amp; Company. Grant Recipient; Self; Exelixis, Inc., Eli Lilly &amp; Company. J. Koh: None. Primary hyperparathyroidism (PHPT) is a common endocrine neoplastic disorder characterized by disrupted calcium homeostasis secondary to inappropriately elevated parathyroid hormone (PTH) secretion. Low levels of serum 25-hydroxyvitamin D (25OHD) are significantly more prevalent in PHPT patients than in the general population, but the pathophysiological basis for this association remains unclear. We employed a spatially defined in situ whole-transcriptomics and selective proteomics profiling approach to compare gene expression patterns and cellular composition in parathyroid adenomas from 9 vitamin D-deficient or 34 vitamin D-replete PHPT patients with 12 eucalcemic cadaveric donor parathyroid glands as normal tissue controls. Parathyroid oxyphil cell content is markedly higher in tumors from vitamin D-deficient PHPT patients (Def-Ts) relative to tumors from vitamin D-replete PHPT patients (Rep-Ts) (47.8% vs 17.8%, respectively; p &amp;lt;0.0001) and normal donor parathyroid glands (7.7%). Def-Ts display increased expression of genes encoding electron transport chain (corr. coefficient=0.546), proteasomal catabolism (corr. coefficient=0.587), and oxidative phosphorylation (corr. coefficient=0.982) pathway components. In contrast, Rep-Ts are characterized by upregulation of the ras (corr. coefficient=0.632) and myc (corr. coefficient=0.811) signaling pathways, suggestive of an oncogene-driven etiology. Parathyroid oxyphil cells are comparable to chief cells at the transcriptional level, and vitamin D-status is reflected in the transcriptional profiles of both cell types in a similar manner. Among PHPT patients who did not present as vitamin D deficient, three distinct tumor transcriptional profile clusters showed a dose-dependent relationship to pre-operative 25OHD levels (p &amp;lt;0.0001 by ANOVA). Relative to normal tissue, the abundance of calcium sensing receptor (CaSR) protein is reduced in Def-Ts (mean protein count 253.6 vs 492.3, respectively; p=0.039 for difference), while the type B γ-aminobutyric acid receptor 1 (GABAB1R) is increased (mean protein count 114.5 vs 64.65, respectively; p=0.0099 for difference). These expression changes resulted in a vitamin D dose-dependent reduction in the ratio of CaSR to GABAB1R receptors in Def-T tumor cells (r2 = 0.218, p&amp;lt;0.0001), favoring the formation of calcium sensing-attenuated CaSR:GABAB1R heteromers. The abundance of the amyloid precursor protein (APP) peptide derivative Aβ1-42, a putative GABAB1R ligand, is preferentially increased in Def-Ts compared to normal tissue (mean protein count 5797 vs 4500, respectively; p=0.0318). Collectively, these findings suggest a novel mechanism where chronic vitamin D deficiency can contribute to tonic PTH hypersecretion in PHPT patients by increasing GABAB1R-mediated antagonism of CaSR activity in parathyroid adenomas. Presentation: Thursday, June 15, 2023

Cryo-EM structures reveal native GABAA receptor assemblies and pharmacology.

Type A γ-aminobutyric acid receptors (GABAARs) are the principal inhibitory receptors in the brain and the target of a wide range of clinical agents, including anaesthetics, sedatives, hypnotics and antidepressants1-3. However, our understanding of GABAAR pharmacology has been hindered by the vast number of pentameric assemblies that can be derived from 19 different subunits4 and the lack of structural knowledge of clinically relevant receptors. Here, we isolate native murine GABAAR assemblies containing the widely expressed α1 subunit and elucidate their structures in complex with drugs used to treat insomnia (zolpidem(ZOL) and flurazepam) and postpartum depression (the neurosteroid allopregnanolone(APG)). Using cryo-electron microscopy (cryo-EM)analysis and single-molecule photobleaching experiments, we uncover three major structural populations in the brain: the canonical α1β2γ2 receptor containing two α1 subunits, and two assemblies containing one α1 and either an α2 or α3 subunit, in which the single α1-containing receptors feature a more compact arrangement between the transmembrane and extracellular domains. Interestingly, APG is bound at the transmembrane α/β subunit interface, even when not added to the sample, revealing an important role for endogenous neurosteroids in modulating native GABAARs. Together with structurally engaged lipids, neurosteroids produce global conformational changes throughout the receptor that modify the ion channel pore and the binding sites for GABA and insomnia medications. Our data reveal the major α1-containing GABAAR assemblies, bound with endogenous neurosteroid, thus defining a structural landscape from which subtype-specific drugs can be developed.

Involvement of the spinal γ-aminobutyric acid receptor in the analgesic effects of intrathecally injected hypertonic saline in spinal nerve-ligated rats.

: Hypertonic saline is used for treating chronic pain; however, clinical studies that aid in optimizing therapeutic protocols are lacking. We aimed to determine the concentration of intrathecally injected hypertonic saline at which the effect reaches its peak as well as the underlying γ-aminobutyric acid (GABA) receptor-related antinociceptive mechanism. : Spinal nerve ligation (SNL; left L5 and L6) was performed to induce neuropathic pain in rats weighing 250-300 g. Experiment 1: one week after implanting the intrathecal catheter, 60 rats were assigned randomly to intrathecal injection with 0.45%, 0.9%, 2.5%, 5%, 10%, and 20% NaCl, followed by behavioral testing at baseline and after 30 minutes, 2 hours, 1 day, and 1 week to determine the minimal concentration which produced maximal analgesia. Experiment 2: after determining the optimal intrathecal hypertonic saline concentration, 60 rats were randomly divided into four groups: Sham, hypertonic saline without pretreatment, and hypertonic saline after pretreatment with one of two GABA receptor antagonists (GABAA [bicuculline], or GABAB [phaclofen]). Behavioral tests were performed at weeks 1 and 3 following each treatment. : Hypertonic saline at concentrations greater than 5% alleviated SNL-induced mechanical allodynia and had a significant therapeutic effect, while showing a partial time- and dose-dependent antinociceptive effect on thermal and cold hyperalgesia. However, pretreatment with GABA receptor antagonists inhibited the antinociceptive effect of 5% NaCl. : This study indicates that the optimal concentration of hypertonic saline for controlling mechanical allodynia in neuropathic pain is 5%, and that its analgesic effect is related to GABAA and GABAB receptors.

The novel insecticide broflanilide dysregulates transcriptional networks associated with ion channels and induces hyperactivity in zebrafish (Danio rerio) larvae

Broflanilide is a novel insecticide that is classified as a non-competitive γ-aminobutyric acid (GABA) receptor antagonist. However, indiscriminate use can have negative effects on non-target species. The objective of this study was to determine the sub-lethal toxicity potential of broflanilide in early staged zebrafish. Embryos/larvae were assessed for multiple molecular and morphological endpoints following exposure to a range of concentrations of broflanilide. The insecticide did not affect hatch rate, the frequency of deformities, nor did it impact survival of zebrafish at exposure concentrations up to 500 μg/L over a 7-day period from hatch. There was also no effect on oxidative consumption rates in embryos, nor induction of reactive oxygen species in fish exposed up to 100 μg/L broflanilide. As oxidative stress was not prominent as a mechanism, we turned to RNA-seq to identify potential toxicity pathways. Gene networks related to neurotransmitter release and ion channels were altered in zebrafish, consistent with its mechanism of action of modulating GABA receptors, which regulate chloride channels. Noteworthy was that genes related to the circadian clock were induced by 1 μg/L broflanilide exposure. The locomotor activity of larval fish at 7 days was increased (i.e., hyperactivity) by broflanilide exposure based on a visual motor response test, corroborating expression data indicating neurotoxicity and motor dysfunction. This study improves the current understanding of the biological responses in fish to broflanilide exposure and contributes to risk assessment strategies for this novel pesticide.

Sixty years of conjecture over a urinary biomarker: a step closer to understanding the proposed link between anxiety and urinary pyrroles.

For over 60 years there has been conjecture about the identity of an Ehrlich's test positive pyrrole (Mauve Factor) reputed to be a biomarker for psychological disorders, including anxiety. We reviewed studies that attempt to identify Mauve Factor and subjected authentic standards of the 2 main candidates, kryptopyrrole and hydroxypyrrole, to the Ehrlich's reaction. Modified Ehrlich's test for kryptopyrrole and hydroxypyrrole were applied to urine samples from 10 volunteers, anxious and nonanxious. Based on the mechanistic chemistry of Ehrlich's reaction and reactions of the 2 compounds, Mauve Factor cannot be hydroxypyrrole. Analyses of urine samples from volunteers, identified by the Generalized Anxiety Disorder - 7 item scale (GAD-7 ≥10; n = 5) and control urine samples (GAD-7 <10; n = 5) using a kryptopyrrole calibration graph, show that concentrations are similar in both groups. Kryptopyrrole may be the elusive Mauve Factor. Its possible origin from stercobilin via gut microbiome-mediated metabolism, its link to gut-mediated neurological effects via γ-aminobutyric acid (GABA) receptors, and its predicted interaction with Zn2+ and consequent impact on zinc homeostasis are discussed. The GAD-7 scale does not differentiate between state and trait anxiety and as such, the minimal difference in pyrrole levels between volunteer groups requires further study.

The efficacy and safety of some new GABAkines for treatment of depression: A systematic review and meta-analysis from randomized controlled trials

Positive allosteric modulators of γ-aminobutyric acid-A (GABAA) receptors, or GABAkines, play important roles in the treatment of depression, epilepsy, insomnia, and other disorders. Recently, some new GABAkines (zuranolone and brexanolone) have been administrated to patients with major depressive disorder (MDD) or postpartum depression (PPD) in randomized controlled trials (RCTs). This study aims to systematically review and examine the efficacy and safety of zuranolone or brexanolone for treatment of depression. A systematic literature retrieval was conducted through August 20, 2023. RCTs evaluating the efficacy and safety of zuranolone or brexanolone for treatment of depression were included. Eight studies (nine reports) were identified in the study. The percentages of patients with PPD achieving Hamilton Depression Rating Scale (HAM-D) response and remission were significantly higher after brexanolone or zuranolone administration compared with placebo at different points. The percentages of patients with MDD achieving HAM-D response and remission were significantly increased during the zuranolone treatment period compared with placebo. In addition, zuranolone caused more adverse events in patients with MDD compared with placebo. Our findings support the effects of brexanolone on improving the core symptoms of depression in patients with PPD, and the potential of zuranolone in treating patients with MDD or PPD.

Brain nuclei and neural circuits in neuropathic pain and brain modulation mechanisms of acupuncture: a review on animal-based experimental research.

Neuropathic pain (NP) is known to be associated with abnormal changes in specific brain regions, but the complex neural network behind it is vast and complex and lacks a systematic summary. With the help of various animal models of NP, a literature search on NP brain regions and circuits revealed that the related brain nuclei included the periaqueductal gray (PAG), lateral habenula (LHb), medial prefrontal cortex (mPFC), and anterior cingulate cortex (ACC); the related brain circuits included the PAG-LHb and mPFC-ACC. Moreover, acupuncture and injurious information can affect different brain regions and influence brain functions via multiple aspects to play an analgesic role and improve synaptic plasticity by regulating the morphology and structure of brain synapses and the expression of synapse-related proteins; maintain the balance of excitatory and inhibitory neurons by regulating the secretion of glutamate, γ-aminobutyric acid, 5-hydroxytryptamine, and other neurotransmitters and receptors in the brain tissues; inhibit the overactivation of glial cells and reduce the release of pro-inflammatory mediators such as interleukins to reduce neuroinflammation in brain regions; maintain homeostasis of glucose metabolism and regulate the metabolic connections in the brain; and play a role in analgesia through the mediation of signaling pathways and signal transduction molecules. These factors help to deepen the understanding of NP brain circuits and the brain mechanisms of acupuncture analgesia.

Physiological Function of RDL1 and RDL2 Subunits of the Ionotropic GABA Receptor in the Spodoptera litura with the CRISPR/Cas9 System In Vivo.

In insect ionotropic γ-aminobutyric acid receptor (iGABAR) subunits, only resistance to dieldrin (RDL) can be individually and functionally expressed in vitro. In lepidopteran, two to three RDL subtypes are identified; however, their physiological roles have not been distinguished in vivo. In this study, SlRdl1 and SlRdl2 of S. litura were individually knocked out using CRISPR/Cas9, respectively. The mortality and larval and pupal duration of KOSlRdl1 and KOSlRdl2 were increased. The flight time and distance were increased by 43.30%-80.66% and 58.96%-198.22%, respectively, in KOSlRdl1. The GABA-induced current was significantly decreased by 53.57%-74.28% and 46.91%-63.34% in the ventral nerve cord, and the GABA titer was significantly reduced by 17.65%-28.05% and 19.85%-42.46% in KOSlRdl1 and KOSlRdl2, respectively. In conclusion, SlRdl1 and SlRdl2 are necessary for the transmission of GABA-induced neural signals; however, only SlRdl1 could regulate the flight capability of S. litura. Our results provided a new avenue to study lepidopteran iGABARs.

PxRdl2 dsRNA increased the insecticidal activities of GABAR-targeting compounds against Plutella xylostella

The utilization of RNA interference (RNAi) for pest management has garnered global interest. The bioassay results suggested the knockout of the PxRdl2 gene significantly increased the insecticidal activities of the γ-aminobutyric acid receptor (GABAR)-targeting compounds (fipronil, two pyrazoloquinazolines, and two isoxazolines), thereby presenting a viable target gene for RNAi-mediated pest control. Consequently, we suggest enhancing the insecticidal activities of GABAR-targeting compounds by knockdown the transcript level of PxRdl2. Furthermore, PxRdl2 dsRNA was expressed in HT115 Escherichia coli to reduce costs and protect dsRNA against degradation. In comparison to in vitro synthesized dsRNA, the recombinant bacteria (ds-B) exhibited superior interference efficiency and greater stability when exposed to UV irradiation. Collectively, our results provide a strategy for insecticide spray that combines synergistically with insecticidal activities by suppressing PxRdl2 using ds-B and may be beneficial for reducing the usage of insecticide and slowing pest resistance.

GL-II-73, a Positive Allosteric Modulator of α5GABAA Receptors, Reverses Dopamine System Dysfunction Associated with Pilocarpine-Induced Temporal Lobe Epilepsy.

Although seizures are a hallmark feature of temporal lobe epilepsy (TLE), psychiatric comorbidities, including psychosis, are frequently associated with TLE and contribute to decreased quality of life. Currently, there are no defined therapeutic protocols to manage psychosis in TLE patients, as antipsychotic agents may induce epileptic seizures and are associated with severe side effects and pharmacokinetic and pharmacodynamic interactions with antiepileptic drugs. Thus, novel treatment strategies are necessary. Several lines of evidence suggest that hippocampal hyperactivity is central to the pathology of both TLE and psychosis; therefore, restoring hippocampal activity back to normal levels may be a novel therapeutic approach for treating psychosis in TLE. In rodent models, increased activity in the ventral hippocampus (vHipp) results in aberrant dopamine system function, which is thought to underlie symptoms of psychosis. Indeed, we have previously demonstrated that targeting α5-containing γ-aminobutyric acid receptors (α5GABAARs), an inhibitory receptor abundant in the hippocampus, with positive allosteric modulators (PAMs), can restore dopamine system function in rodent models displaying hippocampal hyperactivity. Thus, we posited that α5-PAMs may be beneficial in a model used to study TLE. Here, we demonstrate that pilocarpine-induced TLE is associated with increased VTA dopamine neuron activity, an effect that was completely reversed by intra-vHipp administration of GL-II-73, a selective α5-PAM. Further, pilocarpine did not alter the hippocampal α5GABAAR expression or synaptic localization that may affect the efficacy of α5-PAMs. Taken together, these results suggest augmenting α5GABAAR function as a novel therapeutic modality for the treatment of psychosis in TLE.

Structural optimization based on 4,5-dihydropyrazolo[1,5-a]quinazoline scaffold for improved insecticidal activities

The long-term and irrational application of insecticides has increased the rate of development of pest resistance and caused numerous environmental issues. To address these problems, our previous work reported that 4,5-dihydropyrazolo[1,5-a]quinazoline (DPQ) is a class of gelled heterocyclic compounds that act on insect γ-aminobutyric acid receptors (GABAR). DPQ scaffold has no cross-resistance to existing insecticides, so the development of this scaffold is an interesting task for integrated pest management. In the present study, a novel series of 4,5-dihydropyrazolo[1,5-a]quinazolines (DPQs) were designed and synthesized based on pyraquinil, a highly insecticidal compound discovered in our previous work. Insecticidal activities of the target compounds against diamondback moth (Plutella xylostella), beet armyworm (Spodoptera exigua), fall armyworm (Spodoptera frugiperda), and red imported fire ant (Solenopsis invicta Buren) were evaluated. Compounds 6 and 12 showed the best insecticidal activity against Plutella xylostella (P. xylostella) (LC50 = 1.49 and 0.97 mg/L), better than pyraquinil (LC50 = 1.76 mg/L), indoxacarb and fipronil (LC50 = 1.80 mg/L). Meanwhile, compound 12 showed slow toxicity to Solenopsis invicta Buren (S. invicta), with a 5 d mortality rate of 98.89% at 0.5 mg/L that is similar to fipronil. Moreover, Electrophysiological studies against the PxRDL1 GABAR heterologously expressed in Xenopus oocytes indicated that compound 12 could act as a potent GABA receptor antagonist (2 μΜ, inhibition rate, 68.25%). Molecular docking results showed that Ser285 (chain A) and Thr289 (chain D) of P. xylostella GABAR participated in hydrogen bonding interactions with compound 12, and density functional theory (DFT) calculations suggested the importance of pyrazolo[1,5-a]quinazoline core in potency. This systematic study provides valuable clues for the development of DPQ scaffold in the field of agrochemicals, and compound 12 can be further developed as an insecticide and bait candidate.