Α1 Subunits Research Articles

The regional and cellular distribution of GABAA receptor subunits in the human amygdala

GABAergic neurotransmission in the amygdala plays a crucial role in mediating emotional learning, fear, and memory. In this study, expression of five major GABAA receptor subunits (α1, α2, α3, β2,3, and γ2) was investigated in the normal human amygdala using immunohistochemistry. At the regional level, the amygdala contains a highly heterogeneous distribution of all the subunits investigated. The most intense staining for α1, α2, β2,3, and γ2 subunits was present in the lateral nucleus (LA), and α3 in the intercalated nuclei (ICM). Six distinct cell populations that express GABAA receptor subunits were identified throughout the amygdala: type 1 aspiny cells in the basolateral nuclear group (BLNG) and superficial cortical-like nuclear region (SCLR) express α1, β2,3, and γ2; type 2 larger aspiny cells in the paralaminar nucleus (PL) express α1, β2,3, and γ2; type 3 aspiny cells in the BLNG express α1, β2,3, and γ2 as well as calcium-binding proteins including parvalbumin (PV), calbindin (CB), and calretinin (CR); type 4 pyramidal cells in the BLNG and SCLR express α2, α3, β2,3, and γ2 subunits at high levels on proximal specialised spines; type 5 cells in the central nucleus (CE) express α2, α3, and β2,3; type 6 cells are found closely packed in the intercalated cell masses (ICM) and express α3 and β2,3. The α1 subunit rarely co-labelled with α2 and α3 in the same cell population, while the α2 and α3 were often expressed within the same type 4 or 5 cell though not at always at the same puncta. The predominant GABAA receptor subunit combinations expressed in the human amygdala are the α1β2,3γ2 and α2β2,3γ2. Cells classified as interneuron types (types 1–3) contained GAD and principally expressed α1β2,3γ2. The major projection neurons of the BLNG (type 4) are non-GABAergic and mainly express α2β2,3γ2. The α3 subunit was found intracellularly in type 5 cells and decorating the surface of type 6 cells but rarely co-labelled with the subunits investigated. The results reveal a complex and heterogeneous distribution of GABAA receptor subtypes throughout the amygdala as well as on a variety of cell types through which inhibitory processing is carried out to maintain emotional responses, and control anxiety and fear responses in the brain.

Postsynaptic plasticity of cholinergic synapses underlies the induction and expression of appetitive and familiarity memories in Drosophila.

In vertebrates, several forms of memory-relevant synaptic plasticity involve postsynaptic rearrangements of glutamate receptors. In contrast, previous work indicates that Drosophila and other invertebrates store memories using presynaptic plasticity of cholinergic synapses. Here, we provide evidence for postsynaptic plasticity at cholinergic output synapses from the Drosophila mushroom bodies (MBs). We find that the nicotinic acetylcholine receptor (nAChR) subunit α5 is required within specific MB output neurons for appetitive memory induction but is dispensable for aversive memories. In addition, nAChR α2 subunits mediate memory expression and likely function downstream of α5 and the postsynaptic scaffold protein discs large (Dlg). We show that postsynaptic plasticity traces can be induced independently of the presynapse, and that in vivo dynamics of α2 nAChR subunits are changed both in the context of associative and non-associative (familiarity) memory formation, underlying different plasticity rules. Therefore, regardless of neurotransmitter identity, key principles of postsynaptic plasticity support memory storage across phyla.

Control of cell surface expression of GABAA receptors by a conserved region at the end of the N-terminal extracellular domain of receptor subunits

Type A γ-aminobutyric acid receptors (GABAARs) represent a family of pentameric GABA-gated Cl-/HCO3- ion channels which mediate inhibitory transmission in the central nervous system. Cell surface expression of GABAARs, a prerequisite for their function, is dependent on the appropriate assembly of the receptor subunits and their transient interactions with molecular chaperones within the endoplasmic reticulum (ER) and Golgi apparatus. Here, we describe a highly conserved amino acid sequence within the extracellular N-terminal domain of the receptor subunits adjoining the first transmembrane domain as a region important for GABAAR processing within the ER. Modifications of this region in the α1, β3, and γ2 subunits using insertion or site-directed mutagenesis impaired GABAAR trafficking to the cell surface in heterologous cell systems although they had no effect on the subunit assembly. We found that mutated receptors accumulated in the ER where they were shown to associate with chaperones calnexin, BiP, and Grp94. However, their surface expression was increased when ER-associated degradation or proteosome function was inhibited, while modulation of ER calcium stores had little effect. When compared to the wt, mutated receptors showed decreased interaction with calnexin, similar binding to BiP, and increased association with Grp94. Structural modeling of calnexin interaction with the wt or mutated GABAAR revealed that disruption in structure caused by mutations in the conserved region adjoining the first transmembrane domain may impair calnexin binding. Thus, this previously uncharacterized region plays an important role in intracellular processing of GABAARs at least in part by stabilizing their interaction with calnexin.

Genetic Variant in Nicotinic Receptor α4-Subunit Causes Sleep-Related Hyperkinetic Epilepsy via Increased Channel Opening.

We describe genetic and molecular-level functional alterations in the α4β2 neuronal nicotinic acetylcholine receptor (nAChR) from a patient with sleep-related hyperkinetic epilepsy and a family history of epilepsy. Genetic sequencing revealed a heterozygous variant c.851C>G in the CHRNA4 gene encoding the α4 subunit, resulting in the missense mutation p.Ser284Trp. Patch clamp recordings from genetically engineered nAChRs incorporating the α4-Ser284Trp subunit revealed aberrant channel openings in the absence of agonist and markedly prolonged openings in its presence. Measurements of single channel current amplitude distinguished two pentameric stoichiometries of the variant nAChR containing either two or three copies of the α4-Ser284Trp subunit, each exhibiting aberrant spontaneous and prolonged agonist-elicited channel openings. The α4-Ser284 residue is highly conserved and located within the M2 transmembrane α-helix that lines the ion channel. When mapped onto the receptor’s three-dimensional structure, the larger Trp substitution sterically clashes with the M2 α-helix from the neighboring subunit, promoting expansion of the pore and stabilizing the open relative to the closed conformation of the channel. Together, the clinical, genetic, functional, and structural observations demonstrate that α4-Ser284Trp enhances channel opening, predicting increased membrane excitability and a pathogenic seizure phenotype.

Effects of nicotine and lipopolysaccharide stimulation on adhesion molecules in human gingival endothelial cells.

Smoking is a risk factor for periodontitis, and the immune response of periodontal tissues in patients with periodontitis may be strongly affected by smoking. The purpose of this study was to elucidate the bioactivity and signal transduction of human gingival endothelial cells (HGECs) due to nicotinic stimulation using a cultured medium supplemented with lipopolysaccharide (LPS) as a model of periodontitis. HGECs were cultured in medium supplemented with LPS, nicotine, nicotine + LPS, and medium supplemented without nicotine or LPS (control). Cell proliferation was assessed using Alamar blue. Cytotoxicity was assessed by lactate dehydrogenase leakage. The expression of adhesion molecule-1 (ICAM-1, VCAM-1) was assessed by reverse transcription quantitative polymerase chain reaction (RT-qPCR) and enzyme-linked immunosorbent assay. The expression of nicotinic acetylcholine receptor (nAChR) subunits (α3, α5, α7, β2 and β4) was evaluated by RT-PCR. The involvement of p38 mitogen-activated protein kinase (p38MAPK) and protein kinase C (PKC) cell signaling pathways in ICAM-1 and VCAM-1 expression was investigated by RT-qPCR with specific inhibitors. HGECs stimulated with LPS, nicotine and nicotine + LPS showed inhibition of cell proliferation, increase of cell death, and increase of gene and protein expression of ICAM-1. Moreover, HGECs showed the presence of α5 and α7 nAChR subunits. The expression of ICAM-1 in HGECs stimulated with LPS, nicotine, and nicotine + LPS was significantly suppressed by p38MAPK inhibitor, but not by a PKC inhibitor. The nAChR subunits of HGECs are α5 and α7, and that HGECs stimulated with nicotine and LPS express ICAM-1 via p38MAPK pathway.

Enhanced BDNF and TrkB Activation Enhance GABA Neurotransmission in Cerebellum in Hyperammonemia.

Background: Hyperammonemia is a main contributor to minimal hepatic encephalopathy (MHE) in cirrhotic patients. Hyperammonemic rats reproduce the motor incoordination of MHE patients, which is due to enhanced GABAergic neurotransmission in the cerebellum as a consequence of neuroinflammation. In hyperammonemic rats, neuroinflammation increases BDNF by activating the TNFR1–S1PR2–CCR2 pathway. (1) Identify mechanisms enhancing GABAergic neurotransmission in hyperammonemia; (2) assess the role of enhanced activation of TrkB; and (3) assess the role of the TNFR1–S1PR2–CCR2–BDNF pathway. In the cerebellum of hyperammonemic rats, increased BDNF levels enhance TrkB activation in Purkinje neurons, leading to increased GAD65, GAD67 and GABA levels. Enhanced TrkB activation also increases the membrane expression of the γ2, α2 and β3 subunits of GABAA receptors and of KCC2. Moreover, enhanced TrkB activation in activated astrocytes increases the membrane expression of GAT3 and NKCC1. These changes are reversed by blocking TrkB or the TNFR1–SP1PR2–CCL2–CCR2–BDNF–TrkB pathway. Hyperammonemia-induced neuroinflammation increases BDNF and TrkB activation, leading to increased synthesis and extracellular GABA, and the amount of GABAA receptors in the membrane and chloride gradient. These factors enhance GABAergic neurotransmission in the cerebellum. Blocking TrkB or the TNFR1–SP1PR2–CCL2–CCR2–BDNF–TrkB pathway would improve motor function in patients with hepatic encephalopathy and likely with other pathologies associated with neuroinflammation.

Analyzing the mechanisms that facilitate the subtype-specific assembly of γ-aminobutyric acid type A receptors.

Impaired inhibitory signaling underlies the pathophysiology of many neuropsychiatric and neurodevelopmental disorders including autism spectrum disorders and epilepsy. Neuronal inhibition is regulated by synaptic and extrasynaptic γ-aminobutyric acid type A receptors (GABAARs), which mediate phasic and tonic inhibition, respectively. These two GABAAR subtypes differ in their function, ligand sensitivity, and physiological properties. Importantly, they contain different α subunit isoforms: synaptic GABAARs contain the α1–3 subunits whereas extrasynaptic GABAARs contain the α4–6 subunits. While the subunit composition is critical for the distinct roles of synaptic and extrasynaptic GABAAR subtypes in inhibition, the molecular mechanism of the subtype-specific assembly has not been elucidated. To address this issue, we purified endogenous α1- and α4-containing GABAARs from adult murine forebrains and examined their subunit composition and interacting proteins using liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) and quantitative analysis. We found that the α1 and α4 subunits form separate populations of GABAARs and interact with distinct sets of binding proteins. We also discovered that the β3 subunit, which co-purifies with both the α1 and α4 subunits, has different levels of phosphorylation on serines 408 and 409 (S408/9) between the two receptor subtypes. To understand the role S408/9 plays in the assembly of α1- and α4-containing GABAARs, we examined the effects of S408/9A (alanine) knock-in mutation on the subunit composition of the two receptor subtypes using LC-MS/MS and quantitative analysis. We discovered that the S408/9A mutation results in the formation of novel α1α4-containing GABAARs. Moreover, in S408/9A mutants, the plasma membrane expression of the α4 subunit is increased whereas its retention in the endoplasmic reticulum is reduced. These findings suggest that S408/9 play a critical role in determining the subtype-specific assembly of GABAARs, and thus the efficacy of neuronal inhibition.

Subunit-dependent interaction of propoxazepam and its metabolite with the -aminobuturic acid type A receptor

Benzodiazepines (BDZ) are widely used in clinics in the treatment of psychiatric disorders, and their main action is considered to be determined by more selective binding with α1, α2, α3 or α5 subunits of GABA receptor.
 The aim of this work was studying of the molecular mechanism of action of new analgesic – propoxazepam and its metabolite (3-hydroxypropoxazepam) on α1, α2, α3, α4 or α5 subunits containing GABAA channels.
 Materials and methods GABA ha1b3g2, ha2b3g2, ha3b3g2, ha4b3g2 and ha5b3g2 ionotropic GABAARs expressed in HEK293 were used on the automated SP384PE Patch Clamp system. In addition, Propoxazepam, 3-hydroxypropoxazepam, diazepam (positive allosteric modulator) and GABA (positive control) were administered at concentrations 0.001–300 nM to determine the EC50 and Emax for corresponding substances.
 Results The α subunit plays a significantl role in determining the receptor’s affinity for propoxazepam and 3-hydroxypropoxazepam. The rank order of decreasing EC50 are α1 = α5> α2 > α3 > α4 (propoxazepam) and α1> α2> α5 > α3 > α4 (3-hydroxypropoxazepam), and for Emax α3 > α2 >α5 > α1 > α4 (propoxazepam), α3 > α1 > α2> α5 > α4 (3-hydroxypropoxazepam).
 The data, transformed to Emax/EC50, show that propoxazepam exhibits tenfold (compared to diazepam) activity (taking into account the magnitude of the maximum effect) to the α3 subunit, which distinguishes it from 3-hydroxypropoxazepam.
 Conclusion Due to the determined selectivity of propoxazepam for binding with different α subunit-containing GABAA-receptors (mostly α3 and α2 types), it has the potential to provide analgesia with less sedation than non-selective BDZ.

Overexpression of wild type glycine alpha 1 subunit rescues ethanol sensitivity in accumbal receptors and reduces binge drinking in mice.

The nucleus accumbens (nAc) is a critical region in the brain reward system since it integrates abundant synaptic inputs contributing to the control of neuronal excitability in the circuit. The presence of inhibitory α1 glycine receptor (GlyRs) subunits, sensitive to ethanol, has been recently reported in accumbal neurons suggesting that they are protective against excessive binge consumption. In the present study, we used viral vectors (AAV) to overexpress mutant and WT α1 subunits in accumbal neurons in D1 Cre and α1 KI mice. Injection of a Cre-inducible AAV carrying an ethanol insensitive α1 subunit in D1 Cre neurons was unable to affect sensitivity to ethanol in GlyRs or affect ethanol drinking. On the other hand, using an AAV that transduced WT α1 GlyRs in GABAergic neurons in the nAc of high-ethanol consuming mice caused a reduction in ethanol intake as reflected by lowered drinking in the dark and reduced blood ethanol concentration. As expected, the AAV increased the glycine current density by 5-fold without changing the expression of GABAA receptors. Examination of the ethanol sensitivity in isolated accumbal neurons indicated that the GlyRs phenotype changed from an ethanol resistant to an ethanol sensitive type. These results support the conclusion that increased inhibition in the nAc can control excessive ethanol consumption and that selective targeting of GlyRs by pharmacotherapy might provide a mechanistic procedure to reduce ethanol binge.

Dipeptidyl Peptidase-4 Stabilizes Integrin α4β1 Complex to Promote Thyroid Cancer Cell Metastasis by Activating Transforming Growth Factor-Beta Signaling Pathway.

Background: Metastatic disease is a major cause of thyroid cancer-related death. However, the mechanisms responsible for thyroid cancer metastasis are unclear. Dipeptidyl peptidase-4 (DPP4) is a multifunctional cell surface glycoprotein that has been reported to be a negative prognostic factor in thyroid cancer. We explored the molecular mechanism of the role of DPP4 in thyroid cancer cell metastasis. Methods: The effects of DPP4 on thyroid cancer cell migration/invasion in vitro were assessed by transwell assays. A lung metastatic mouse model was also established to determine the effect of DPP4 on tumor metastasis in vivo. DPP4 inhibitor sitagliptin was used to test its effect on thyroid cancer cell metastasis. The mechanism of which DPP4 promotes thyroid cancer cell metastasis was explored by a series of molecular and biochemical experiments. Results: We observed that DPP4 was significantly upregulated in papillary thyroid cancers compared with control subjects, and its expression was positively associated with lymph node metastasis and BRAFV600E mutation. Functional studies showed that DPP4 knockdown significantly inhibited metastatic potential of thyroid cancer cells, and vice versa. However, DPP4 inhibitor sitagliptin did not affect the metastatic ability of thyroid cancer cells, indicating that the promoting effect of DPP4 on tumor metastasis was independent of its enzymatic activity. Mechanistically, DPP4 interacted with the α4 and β1 integrin subunits, and stabilized the formation of integrin α4β1 complex. DPP4-mediated integrin signal activation promoted the nuclear localization of c-Jun through the FAK/AKT pathway, thereby inducing the transcription of transforming growth factor-beta 1 (TGFB1 coding for protein TGF-β1). TGF-β1 then facilitated tumor metastasis by inducing the epithelial-mesenchymal transition. Conclusions: DPP4 promotes thyroid cancer cell metastasis through the integrins/FAK/AKT/c-Jun/TGF-β1 signaling axis. These findings may have implications for an alternative therapeutic strategy for thyroid cancer.

Alkaloid ligands enable function of homomeric human α10 nicotinic acetylcholine receptors.

In the nervous system, nicotinic acetylcholine receptors (nAChRs) rapidly transduce a chemical signal into one that is electrical via ligand-gated ion flux through the central channel of the receptor. However, some nAChR subunits are expressed by non-excitable cells where signal transduction apparently occurs through non-ionic mechanisms. One such nAChR subunit, α10, is present in a discreet subset of immune cells and has been implicated in pathologies including cancer, neuropathic pain, and chronic inflammation. Longstanding convention holds that human α10 subunits require co-assembly with α9 subunits for function. Here we assessed whether cholinergic ligands can enable or uncover ionic functions from homomeric α10 nAChRs. Xenopus laevis oocytes expressing human α10 subunits were exposed to a panel of ligands and examined for receptor activation using voltage-clamp electrophysiology. Functional expression of human α10 nAChRs was achieved by exposing the oocytes to the alkaloids strychnine, brucine, or methyllycaconitine. Furthermore, acute exposure to the alkaloid ligands significantly enhanced ionic responses. Acetylcholine-gated currents mediated by α10 nAChRs were potently inhibited by the snake toxins α-bungarotoxin and α-cobratoxin but not by α-conotoxins that target α9 and α9α10 nAChRs. Our findings indicate that human α10 homomers are expressed in oocytes and exposure to certain ligands can enable ionic functions. To our knowledge, this is the first demonstration that human α10 subunits can assemble as functional homomeric nAChRs. These findings have potential implications for receptor regulatory-mechanisms and will enable structural, functional, and further pharmacological characterization of human α10 nAChRs.

Voltage-clamp evidence of GABAA receptor subunit-specific effects: pharmacodynamic fingerprint of chlornordiazepam, the major active metabolite of mexazolam, as compared to alprazolam, bromazepam, and zolpidem.

Anxiolytic benzodiazepines, due to their clinical effectiveness, are one of the most prescribed drugs worldwide, despite being associated with sedative effects and impaired psychomotor and cognitive performance. Not every GABAA receptor functions in the same manner. Those containing α1 subunits are associated with sleep regulation and have a greater effect on the sedative-hypnotic benzodiazepines, whereas those containing α2 and/or α3 subunits are associated with anxiety phenomena and have a greater effect on the anxiolytic benzodiazepines. Therefore, characterization of the selectivity profile of anxiolytic drugs could translate into a significant clinical impact. The present study pharmacodynamically evaluated chlornordiazepam, the main active metabolite of mexazolam, upon GABAA receptors containing α2 and/or α3, anxiety-related, and those containing an α1 subunit, associated with sleep modulation. As shown by whole-cell patch-clamp data, chlornordiazepam potentiated GABA-evoked current amplitude in α2 and α3 containing receptors without changing the current amplitude in α1 containing receptors. However, current decay time increased, particularly in GABAA receptors containing α1 subunits. In contrast, other anxiolytic benzodiazepines such as alprazolam, bromazepam, and zolpidem, all increased currents associated with GABAA receptors containing the α1 subunit. This novel evidence demonstrates that mexazolam (through its main metabolite chlornordiazepam) has a "pharmacodynamic fingerprint" that correlates better with an anxiolytic profile and fewer sedative effects, when compared to alprazolam, bromazepam and zolpidem, explaining clinical trial outcomes with these drugs. This also highlights the relevance of the pharmacological selectivity over GABAA receptor subtypes in the selection of benzodiazepines, in addition to their clinical performance and pharmacokinetic characteristics.

A Sampling of Highlights from the Literature.

IRF8 maintains commitment to a cDC1 phenotype (from CCCOnline via Creative Commons).Conventional type 1 dendritic cells (cDC1) have a critical role in antitumor immunity. The transcription factor interferon regulatory factor 8 (IRF8) is known to be involved in cDC1 development, but now Lança et al. show that it also is required to maintain cDC1 identity and function. Irf8 deletion in committed cDC1 leads to transcriptional and functional reprogramming of the cells to a conventional type 2 dendritic cell (cDC2)-like state. Mechanistically, this is associated with chromatin remodeling and loss of the cDC1 epigenetic landscape. The data provide new insight into the biology of cDC1, which directly impacts understanding of cancer immunology.Lança T, …, Agace WW. Immunity 2022 July 12;55:1431–47.E11.α1 homotrimeric collagen promotes an immunosuppressive tumor microenvironment (from Wikimedia Commons).In contrast to fibroblast-derived type I collagen (Col1), the structure and role of tumor-derived Col1 is largely unknown. Chen et al. find that human and mouse pancreatic cancer cells exclusively produce homotrimeric Col1 composed of α1 subunits, whereas fibroblasts produce heterotrimeric Col1 composed of both α1 and α2 subunits. Genetic suppression of Col1 production reduces tumor progression, induces a shift in gut- and tumor-resident microbial composition, promotes microbiome-dependent and -independent proinflammatory tumor immune infiltration, and increases sensitivity to anti–PD-1. These results implicate homotrimeric Col1 in generating a broadly immunosuppressive tumor microenvironment.Chen Y, …, Kalluri R. Cancer Cell 2022 July 21;40:818–34.E9.γδ T-cell subsets can have opposite roles in CRC (by Gregory Maxwell via Wikimedia Commons).The role of γδ T cells in the tumor microenvironment (TME) is not fully elucidated. Reis et al. find that γδ T cells in colorectal cancer (CRC) have distinct phenotypes depending on localization. The authors identify both tumor-promoting and antitumor γδ T-cell subsets, each with distinct Vγδ gene usage. Alterations that occur in these subsets as a result of treatment are demonstrated to affect CRC progression, emphasizing that broadly targeting γδ T cells should be performed with caution and that fine-tuning targeted therapy to specific subsets may be more efficacious.Reis B, …, Mucida D. Science 2022 July 14;377:276–84.TLSs could be a biomarker of response to pembrolizumab (by Fvasconcellos via Wikimedia Commons).Efficacy of immune checkpoint inhibitors (ICIs) is limited. This phase 2 study (PEMBROSARC) tested pembrolizumab (anti–PD-1) combined with low-dose cyclophosphamide in patients with advanced soft-tissue sarcoma (STS). By evaluating data from patients based on tertiary lymphoid structure (TLS) presence, authors found that TLS-enriched tumors, as well as those having high infiltration of plasma cells and dendritic cells into the tumor microenvironment, associate with improved outcome. These data highlight TLSs as predictive biomarkers that could potentially be used for selecting STS patients to be treated with anti–PD-1.Italiano A, …, Fridman WH. Nat Med 2022 May 26;28:1199–1206.A new computational model identifies tumor-resilient T cells (from rawpixel).To answer the question, “How can we identify CD8+ T cells able to elicit efficient antitumor responses?” Zhang et al. developed a computational model called Tres, for tumor-resilient T cell. Tres, which uses single-cell RNA sequencing data, reliably predicts patient response to immune checkpoint inhibitors and adoptive-cell therapies. FIBP, which encodes FGF1 intracellular binding protein, is the top negative marker of T-cell antitumor efficacy identified using Tres. Knocking out Fibp in CD8+ T cells enhances their ability to kill tumor cells in vitro and limits tumor growth in an in vivo model of adoptive-cell therapy. The data highlight how Tres can identify biomarkers of response to immunotherapy and new targets for therapeutic intervention.Zhang Y, …, Jiang P. Nat Med 2022 May 2;28:1421–31.A novel implantable device allows for multiplexed testing of therapeutic agents in the TME (by Bb3cxv from Wikimedia Commons).Evaluating novel chemotherapy/immunotherapy combination treatments traditionally relies on tumor-bearing mouse models, which can be time-intensive, laborious, and challenging to apply to broad patient populations. Tatarova et al. developed an intratumoral implantable device that releases up to 18 drug formulations separated spatially, allowing for multiplexed in vivo tumor microenvironment (TME) analyses. A panel of treatments delivered to a murine breast cancer model identifies a triple combination therapy (panobinostat, venetoclax, and CD40-agonist) that targets cancer stem cells and promotes dendritic cell activity in the TME. The work highlights the implantable drug-delivery system as a method to rapidly design and evaluate TME-targeting drug combinations in situ.Tatarova Z, …, Gray JW. Nat Biotechnol 2022 July 4. DOI:10.1038/s41587-022-01379-y.

P53 positively regulates the proliferation of hepatic progenitor cells promoted by laminin-521

Hepatic progenitor cells (HPCs) hold tremendous potential for liver regeneration, but their well-known limitation of proliferation hampers their broader use. There is evidence that laminin is required for the proliferation of HPCs, but the laminin isoform that plays the dominant role and the key intracellular downstream targets that mediate the regulation of HPC proliferation have yet to be determined. Here we showed that p53 expression increased gradually and reached maximal levels around 8 days when laminin α4, α5, β2, β1, and γ1 subunit levels also reached a maximum during HPC activation and expansion. Laminin-521 (LN-521) promoted greater proliferation of HPCs than do laminin, matrigel or other laminin isoforms. Inactivation of p53 by PFT-α or Ad-p53V143A inhibited the promotion of proliferation by LN-521. Further complementary MRI and bioluminescence imaging analysis showed that p53 inactivation decreased the proliferation of transplanted HPCs in vivo. p53 was activated by LN-521 through the Integrin α6β1/FAK-Src-Paxillin/Akt axis. Activated p53 was involved in the nuclear translocation of CDK4 and inactivation of Rb by inducing p27Kip1. Taken together, this study identifies LN-521 as an ideal candidate substrate for HPC culture and uncovers an unexpected positive role for p53 in regulating proliferation of HPCs, which makes it a potential target for HPC-based regenerative medicine.

Gabra5 plays a sexually dimorphic role in POMC neuron activity and glucose balance.

Pro-opiomelanocortin (POMC) neurons are important for the regulation of body weight and glucose balance. The inhibitory tone to POMC neurons is mediated primarily by the GABA receptors. However, the detailed mechanisms and functions of GABA receptors are not well understood. The α5 subunit of GABAA receptor, Gabra5, is reported to regulate feeding, and we found that Gabra5 is highly expressed in POMC neurons. To explore the function of Gabra5 in POMC neurons, we knocked down Gabra5 specifically from mature hypothalamic POMC neurons using the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 strategy. This POMC-specific knock-down of Gabra5 did not affect body weight or food intake in either male or female mice. Interestingly, the loss of Gabra5 caused significant increases in the firing frequency and resting membrane potential, and a decrease in the amplitude of the miniature inhibitory postsynaptic current (mIPSC) in male POMC neurons. However, the loss of Gabra5 only modestly decreased the frequency of mIPSC in female POMC neurons. Consistently, POMC-specific knock-down of Gabra5 significantly improved glucose tolerance in male mice but not in female mice. These results revealed a sexually dimorphic role of Gabra5 in POMC neuron activity and glucose balance, independent of body weight control.

Synthesis, insecticidal activity, and mode of action of novel imidazopyridine mesoionic derivatives containing an amido group

In our previous work, we applied a new synthetic strategy to design and synthesize a series of imidazopyridine mesoionic derivatives with an ester group. The newly synthesized compounds had excellent insecticidal activity against aphids; however, insecticidal activity against planthoppers was less than satisfactory. In the present study, we designed and synthesized a series of novel imidazopyridine mesoionic compounds, containing an amido group, and these compounds were found to have improved insecticidal activity against planthoppers. The bioassay results demonstrated that most of the target compounds had moderate-to-good insecticidal activity against Sogatella furcifera, and some exhibited good-to-excellent insecticidal activity against Aphis craccivora. Among them, compound C6 had the highest insecticidal activity against S.furcifera and A.craccivora, with LC50 values of 10.5 and 2.09 μg mL-1 , respectively. Proteomic results suggested that the differentially expressed proteins mainly were enriched in the nervous system-related pathways after compound C6 treatment. Enzymatic assay results showed that compound C6 and triflumezopyrim had a certain inhibitory effect on acetylcholinesterase. Molecular docking and real-time quantitative PCR results indicated that compound C6 not only may act on the nicotinic acetylcholine receptor, but also may interact with the α4 and β1 subunits of this receptor. The results reported here contribute to the development of new mesoionic insecticides and further our understanding of the mode-of-action of imidazopyridine mesoionic derivatives. © 2022 Society of Chemical Industry.

Quantitative interactome proteomics identifies a proteostasis network for GABAA receptors

Gamma-aminobutyric acid type A (GABAA) receptors are the primary inhibitory neurotransmitter-gated ion channels in the mammalian central nervous system. Maintenance of GABAA receptor protein homeostasis (proteostasis) in cells utilizing its interacting proteins is essential for the function of GABAA receptors. However, how the proteostasis network orchestrates GABAA receptor biogenesis in the endoplasmic reticulum is not well understood. Here, we employed a proteomics-based approach to systematically identify the interactomes of GABAA receptors. We carried out a quantitative immunoprecipitation-tandem mass spectrometry analysis utilizing stable isotope labeling by amino acids in cell culture. Furthermore, we performed comparative proteomics by using both WT α1 subunit and a misfolding-prone α1 subunit carrying the A322D variant as the bait proteins. We identified 125 interactors for WT α1-containing receptors, 105 proteins for α1(A322D)-containing receptors, and 54 overlapping proteins within these two interactomes. Our bioinformatics analysis identified potential GABAA receptor proteostasis network components, including chaperones, folding enzymes, trafficking factors, and degradation factors, and we assembled a model of their potential involvement in the cellular folding, degradation, and trafficking pathways for GABAA receptors. In addition, we verified endogenous interactions between α1 subunits and selected interactors by using coimmunoprecipitation in mouse brain homogenates. Moreover, we showed that TRIM21 (tripartite motif containing-21), an E3 ubiquitin ligase, positively regulated the degradation of misfolding-prone α1(A322D) subunits selectively. This study paves the way for understanding the molecular mechanisms as well as fine-tuning of GABAA receptor proteostasis to ameliorate related neurological diseases such as epilepsy.

Insights into the molecular mechanism of triazolopyrimidinone derivatives effects on the modulation of α1β2γ2 subtype of GABAA receptor: An in silico approach

Due to the importance of benzodiazepine drugs in clinical practice, such as the treatment of anxiety disorders, depression, and insomnia and the side effects of classical benzodiazepines, the study of new benzodiazepine agonists has received much attentions. In this work, we used in silico methods to explore the molecular mechanism of 1,2,4-triazolo [1,5-a] pyrimidinone derivatives in the modulation of α1β2γ2 subtype of GABAA receptor. To this aim, molecular docking, molecular dynamics simulation (MD), post-MD analysis, binding free energy calculation, and prediction of ADME properties were performed. Results showed that all new compounds have a better binding affinity for the Benzodiazepine (BZD) site of the receptor than diazepam and compound 4c had the highest affinity among them. Moreover, a good agreement was observed between the calculated ΔGbinding and experimental IC50 values. Also, we noticed that residues in loop regions (particularly loop C and D-F in α1 and γ2 subunits, respectively) forming BZD binding site, take part in forming several H-bonds between the agonists and the receptor. Ser205, Thr207, Tyr160, and His102 of α1 subunit and Thr207 of γ2 subunit are mainly involved in forming H-bonds. Also, the orientation of agonists in the BZD binding site leads to π-π interactions with hydrophobic residues in loops A-F. Based on the DCCM analysis, the correlated motions in the γ2 subunit residues are greater than those of α1 subunit residues. Further, predicted ADME results indicated that all agonists meet the criteria. The triplicate MD simulation showed the reproducibility of the results and strengthened the study. Our results provide a comprehensive insight into the receptor-agonist interactions and clues for designing future BZD agonists.

Association of Polymorphism CHRNA5 and CHRNA3 Gene in People Addicted to Nicotine.

Smoking is a chronic and relapsing addictive trait that harms public health. Among the many identified genetic variants of nicotine dependence, the variants in the CHRNA5/A3/B4 gene cluster on chromosome 15 that encode the α5, α3, and β4 subunits have recently received a lot of attention. Importantly, variants in this gene cluster have been associated with nicotine addiction. Among the many significant variants in this cluster, the polymorphism SNP rs16969968 seems to be the most interesting factor in nicotine addiction. This polymorphism causes an amino acid change from aspartate to asparagine at position 398 of the α5 nicotinic receptor protein sequence. Our study aimed to analyze three polymorphic variants: the rs16969968 located in the CHRNA5 gene, the rs578776 and rs1051730 located in the CHRNA3 gene in nicotine-addicted subjects, and in controls. Our study encompasses an association analysis of genotypes and haplotypes. A group of 401 volunteers was recruited for the study and divided into two groups: the study group consisted of addicted smokers and a control group of 200 unrelated non-smokers who were not dependent on any substance and healthy. A statistically significant difference was observed in the frequency of genotypes of the rs1051730 polymorphism of the CHRNA3 gene (χ2 = 6.704 p = 0.035). The T/T genotype was statistically significantly more frequent in the group of nicotine-dependent subjects. The haplotypes rs16969968, rs578776, and rs1051730 were distinguished, of which the G-T-T and G-C-T haplotypes were present only in the study group. With differences in frequencies, statistical significance was noted—for the G-T-T haplotype p = 0.01284 and the G-C-T haplotype p = 0.00775. The research stated that novel haplotypes G-T-T and G-C-T, though with very low-frequency variants in CHRNA3, were associated with nicotine addiction.

α3β4 Acetylcholine Nicotinic Receptors Are Components of the Secretory Machinery Clusters in Chromaffin Cells.

The heteromeric assembly of α3 and β4 subunits of acetylcholine nicotinic receptors (nAChRs) seems to mediate the secretory response in bovine chromaffin cells. However, there is no information about the localization of these nAChRs in relationship with the secretory active zones in this cellular model. The present work presents the first evidence that, in fact, a population of these receptors is associated through the F-actin cytoskeleton with exocytotic machinery components, as detected by SNAP-25 labeling. Furthermore, we also prove that, upon stimulation, the probability to find α3β4 nAChRs very close to exocytotic events increases with randomized distributions, thus substantiating the clear dynamic behavior of these receptors during the secretory process. Modeling on secretory dynamics and secretory component distributions supports the idea that α3β4 nAChR cluster mobility could help with improving the efficiency of the secretory response of chromaffin cells. Our study is limited by the use of conventional confocal microscopy; in this sense, a strengthening to our conclusions could come from the use of super-resolution microscopy techniques in the near future.