Nicotinic Research Articles

The alpha 7 nicotinic acetylcholine receptor agonist PHA 568487 dampens inflammation in PBMCs from patients with newly discovered coronary artery disease.

The alpha 7 nicotinic acetylcholine receptor (α7nAChR) regulates inflammation in experimental models and is expressed in human peripheral blood mononuclear cells (PBMCs) and in human atherosclerotic plaques. However, its role in regulating inflammation in patients with cardiovascular disease is unknown. This study aims to investigate whether α7nAChR stimulation can reduce the inflammatory response in PBMCs from patients with newly diagnosed coronary artery disease (CAD). Human PBMCs, extracted from patients with verified CAD (n = 38) and control participants with healthy vessels (n = 38), were challenged in vitro with lipopolysaccharide (LPS) in combination with the α7nAChR agonist PHA 568487. Cytokine levels of the supernatants were analyzed using a multiplex immunoassay. Patients in the CAD group were reexamined after 6 mo. The immune response to LPS did not differ between PBMCs from control and CAD groups. α7nAChR stimulation decreased TNFα in both control and CAD groups. The most pronounced effect of α7nAChR stimulation was observed in patients with CAD at their first visit, where 15 of 17 cytokines were decreased [IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 (p70), IL-17A, G-CSF, GM-CSF, IFN-γ, MCP-1, MIP-1β, and TNFα]. In conclusion, stimulation with α7nAChR agonist PHA 568487 dampens the inflammatory response in human PBMCs. This finding suggests that the anti-inflammatory properties of the α7nAChR may have a role in treating CAD.NEW & NOTEWORTHY The α7nAChR is an important regulator of inflammation; however, its anti-inflammatory function in patients with newly diagnosed coronary artery disease (CAD) remains unclear. We demonstrate that stimulation of α7nAChR with PHA 568487 attenuates the inflammatory response in immune cells extracted from healthy controls and patients with newly diagnosed CAD, with a more pronounced effect observed in patients with CAD. This suggests that the anti-inflammatory properties of α7nAChR may have a role in treating chronic inflammatory diseases.

Disruption of exploratory behavior and olfactory memory in cockroaches exposed to sublethal doses of the neonicotinoid Thiamethoxam

Neonicotinoid insecticides (NNI) are agonists of insect nicotinic acetylcholine receptors (nAChR) that induce non-elucidate mechanisms of abnormal behavior in insects. In this work, we investigated the effects of sublethal doses of the neonicotinoid thiamethoxam (TMX) on neurochemical and physiological parameters in cockroaches. Sublethal doses of TMX (0.01–10 ng.g−1 body mass) caused significant alterations in most of the neurophysiological parameters evaluated. TMX reduced sustained locomotor activity by 19.9–25.8 %, depending on the dose. Leg grooming activity increased by 124.5 ± 3.4 %, 158.7 ± 3.5 %, 168.3 ± 3.4 %, and 160.4 ± 3.4 % (mean ± SEM) with TMX doses of 0.01, 0.1, 1, and 10 ng.g−1, respectively. Exploratory activity was significantly reduced only at the lowest TMX dose (0.01 ng.g−1) – the time spent immobile increased from 30 % to ~45 %, whereas none of the doses affected the walking speed. Treatment with TMX (0.01 ng.g−1) markedly reduced the olfactory sensitivity of the cockroaches and also reduced the mechanosensory action potential amplitude, rise time and decay time by 61.2 ± 19 %, 50 ± 4 %, and 76.8 ± 9.5 %, respectively. In semi-isolated heart preparations, TMX caused positive chronotropism (increases of 34.7 ± 15.9 %, 26.8 ± 7.8 %, 43.0 ± 16.5 %, and 19.0 ± 13.7 % for 0.01, 0.1, 1, and 10 ng of TMX, respectively). TMX attenuated the activity of glutathione-S-transferase by 35.1 ± 6.4 % at the highest dose tested (10 ng.g−1). TMX caused alterations in the metal ion content of cockroach brains that varied with the dose tested and the ion examined. These findings indicate that sublethal doses of TMX can interfere with normal neurological function in cockroaches and disrupt brain metal ion homeostasis.

Inflammation as a shared mechanism of chronic stressrelated disorders with potential novel therapeutic targets.

Stress is a subjective experience that varies across individuals depending on their sensitivity, resilience, and length of exposure to stressors. Stress may be categorised as acute (positive stress) or chronic (negative stress). Acute stress is advantageous for the human body, but chronic stress results in changes in cardiovascular, neuroendocrine, autonomic, and immunological functions, eventually causing different illnesses. The specific process relating stress to chronic stress associated diseases is still a topic of continuing debate. Inflammation has been recognised as a new and fascinating physiological mechanism that connects chronic stress and its associated illnesses. This article explored the relationships between chronic stress, inflammation, and chronic illnesses, including depression, cancer, and cardiovascular disease. This article also emphasises on various possible therapeutic targets for the management of chronic stressrelated illnesses by targeting inflammation, namely lipoxins and alpha7 nicotinic receptors. These therapeutic targets may be useful in developing new and safe therapies for the management of chronic stressrelated dysfunctions.

Transcutaneous auricular vagus nerve stimulation mitigates gouty inflammation by reducing neutrophil infiltration in BALB/c mice

Gouty inflammation, caused by uric acid crystal deposition, primarily affects tissues around the toe joints and triggers potent inflammatory responses. Current treatments focus on alleviating inflammation and pain using pharmaceutical agents, which can lead to side effects and complications. This has generated interest in non-pharmacological interventions, such as non-invasive vagus nerve stimulation (VNS). In this study, we explored the anti-inflammatory mechanisms of transcutaneous auricular vagus nerve stimulation (taVNS) in a mouse model of acute gout. Gouty inflammation was induced by injecting monosodium urate (MSU) crystals into the ankle joints of BALB/c mice. The effects of taVNS on the expression of inflammatory cytokines and chemokines in the ankle joint tissue were assessed using real-time quantitative PCR (qPCR), western blotting, histological assessments (H&E staining), and immunohistochemistry (IHC). The role of α7 nicotinic acetylcholine receptors (α7nAChR) was also evaluated by signal blocking. Our findings revealed that MSU significantly elevated gout-associated inflammatory cascades and mediators in the ankle joint. Notably, taVNS at 200 µA and 25 Hz effectively reduced these inflammatory responses, decreasing neutrophil infiltration and chemoattraction within the tissue. taVNS showed significant anti-inflammatory properties by suppressing neutrophil activity, offering a novel therapeutic approach for gout beyond conventional pharmacological methods. Additionally, taVNS holds potential for managing various chronic joint diseases. These results highlight taVNS as a promising non-pharmacological therapy for chronic inflammation.

Development of an Intravenously Stable Disulfide-Rich Peptide for the Treatment of Chemotherapy-Induced Neuropathic Pain.

α-conotoxins (α-Ctxs), a class of disulfide-rich conopetides, are excellent drug leads due to their small size, high selectivity, and potency for specific membrane receptors and ion channels involved in pain transmission. However, their high susceptibility to proteolytic degradation limits their therapeutic potential. In this study, we designed and synthesized a series of conformationally stable analogues of α-Ctx Mr1.1[S4Dap] using various structural optimization strategies. The Mr1.1[S4Dap, C16Pen] analogue maintained potency at human α9α10 nicotinic acetylcholine receptors, with a half-maximal inhibitory concentration (IC50) of 4 nM. It exhibited over a 5-fold increase in serum stability compared to Mr1.1[S4Dap], without disrupting its overall conformation. Furthermore, intravenous application of Mr1.1[S4Dap, C16Pen] showed potent analgesic activity in oxaliplatin-induced cold allodynia, indicating a high potential for drug development. Overall, the results from this study provide valuable insights for optimizing the serum stability of disulfide-rich peptides in future therapeutic applications.

An endogenous cholinergic system controls electrical conduction in the heart.

The cholinergic system is distributed in the nervous system, mediating electrical conduction through acetylcholine (ACh). This study aims to identify whether the heart possesses an intact endogenous cholinergic system and to explore its electrophysiological functions and relationship with arrhythmias in both humans and animals. The components of the heart's endogenous cholinergic system were identified by a combination of multiple molecular cell biology techniques. The relationship of this system with cardiac electrical conduction and arrhythmias was analysed through electrophysiological techniques. An intact cholinergic system including ACh, ACh transmitter vesicles, ACh transporters, ACh metabolic enzymes, and ACh receptors was identified in both human and mouse ventricular cardiomyocytes (VCs). The key components of the system significantly regulated the conductivity of electrical excitation among VCs. The influence of this system on electrical excitation conduction was further confirmed both in the mice with α4 or α7 nicotinic ACh receptors (nAChRs) knockouts and in the monolayers of human induced pluripotent stem cell-derived cardiomyocytes. Mechanistically, ACh induced an inward current through nAChRs to reduce the minimum threshold current required to generate an action potential in VCs, thereby enhancing the excitability that acts as a prerequisite for electrical conduction. Importantly, defects in this system were associated with fatal ventricular arrhythmias in both patients and mice. This study identifies an integrated cholinergic system inherent to the heart, rather than external nerves that can effectively control cardiac electrical conduction. The discovery reveals arrhythmia mechanisms beyond classical theories and opens new directions for arrhythmia research.

Upregulation of cholinergic modulators Lypd6 and Lypd6b associated with autism drives anxiety and cognitive decline

Intellectual disability and autistic features are associated with chromosome region 2q23.q23.2 duplication carrying LYPD6 and LYPD6B genes. Here, we analyzed LYPD6 and LYPD6B expression in patients with different neuropsychiatric disorders. Increased LYPD6 and LYPD6B expression was revealed in autism and other disorders. To study possible consequences of Lypd6 and Lypd6b overexpression in the brain, we used a mouse model with intracerebroventricular delivery of recombinant analogs of these proteins. A two-week infusion evoked significant memory impairment and acute stress. Both modulators downregulated hippocampal and amygdala dendritic spine density. No changes in synaptic plasticity were observed. Intracerebroventricular administration by both proteins downregulated hippocampal expression of Lypd6, Lypd6b, and α7 nicotinic acetylcholine receptor (nAChR). Similar to Lypd6, Lypd6b targeted different nAChR subtypes in the brain with preferential inhibition of α7- and α4β2-nAChRs. Thus, increased Lypd6 and Lypd6b level in the brain are linked to cholinergic system depression, neuronal atrophy, memory decline, and anxiety.

α4 Nicotinic Acetylcholine Receptors in Lipopolysaccharide-Related Lung Inflammation

Sepsis remains an important healthcare challenge. The lungs are often affected in sepsis, resulting in acute lung injury characterized by inflammation. Mechanisms involving lipopolysaccharide (LPS) stimulation of toll-like receptor (TLR) signaling with induction of proinflammatory pathways have been implicated in this process. To date, however, studies targeting these pathways have failed to improve outcomes. We have found that LPS may also promote lung injury through the activation of α4 nicotinic acetylcholine receptors (α4 nAChRs) in immune cells. We observed increased expression of α4 nAChRs in human THP-1 monocytic cells exposed to LPS (100 ng/mL, 24 h). We also observed that LPS stimulated the expression of other relevant genes, including tumor necrosis factor-α, interleukin-1β, plasminogen activator inhibitor-1, the solute carrier family 7 member 11, extracellular superoxide dismutase, and transforming growth factor-β1. Of interest, dihydro-β-erythroidine hydrobromide (DHβE), a specific chemical inhibitor of α4 nAChRs, inhibited the LPS-induced expression of these genes. We generated mice with a global knockout mutation of the α4 nAChR subunit in the C57BL/6 background using CRISPR/Cas9 technology. The lungs of these LPS-treated animals demonstrated a reduction in the expression of the above-mentioned genes when compared with the lungs of wild-type animals. In support of the role of oxidative stress, we observed that LPS induced expression of the cystine transporter Slc7a11 in both THP-1 cells and in wild-type mouse lungs. The effects of LPS on THP-1 cells were blocked by the thiol antioxidant N-acetylcysteine and mimicked by redox stress. Importantly, the induction of IL-1β by redox stress was inhibited by the α4 nAChR inhibitor DHβE. Finally, we showed that LPS stimulated calcium influx in THP-1 cells, which was blocked by the α4 nAChR inhibitor. Our observations suggest that LPS promotes lung injury by stimulating redox stress, which activates α4 nAChR signaling and drives proinflammatory cytokine expression.

Symmetry-adapted Markov state models of closing, opening, and desensitizing in α 7 nicotinic acetylcholine receptors.

α7 nicotinic acetylcholine receptors (nAChRs) are homopentameric ligand-gated ion channels with critical roles in the nervous system. Recent studies have resolved and functionally annotated closed, open, and desensitized states of these receptors, providing insight into ion permeation and lipid binding. However, the process by which α7 nAChRs transition between states remains unclear. To understand gating and lipid modulation, we generated two ensembles of molecular dynamics simulations of apo α7 nAChRs, with or without cholesterol. Using symmetry-adapted Markov state modeling, we developed a five-state gating model. Free energies recapitulated functional behavior, with the closed state dominating in absence of agonist. Open-to-nonconducting transition rates corresponded to experimental open durations. Cholesterol relatively stabilized the desensitized state, and reduced open-desensitized barriers. These results establish plausible asymmetric transition pathways between states, define lipid modulation effects on the α7 nAChR conformational cycle, and provide an ensemble of structural models applicable to rational design of lipidic pharmaceuticals.

Effects of Different Photoperiods on Growth Performance, Glucose Metabolism, Acetylcholine, and Its Relative Acetylcholine Receptor Modulation in Broiler Chickens.

Photoperiods are crucial environmental factors in the growth and health of modern intensive broiler chicken production. To date, the effects of different photoperiods on glucose metabolism, acetylcholine (ACh), and its relative acetylcholine receptor modulation in broilers remain elusive. Herein, we aimed to identify the effects of different photoperiods on regulating glucose metabolism, ACh, nicotinic acetylcholine receptor alpha 4 (α4 nAChR) mRNA, and M3 muscarinic acetylcholine receptor (M3 mAChR) modulation in broilers. A total of 216 healthy 5-day-old Arbor Acres (AA) male broilers was randomly assigned to 12L:12D, 18L:6D, and 24L:0D photoperiods for 4 weeks. The results show that, compared with the 12L:12D photoperiod, the 18L:6D and 24L:0D photoperiods significantly increase the average daily gain (ADG) and average daily feed intake (ADFI) of broilers (p < 0.05). However, the feed efficiency (FE) of broilers significantly decreased in the 18L:6D and 24L:0D photoperiods (p < 0.05). Moreover, compared with the 12L:12D photoperiod, the ACh concentrations and α4 nAChR mRNA expression levels in the hypothalamus and medulla oblongata of broilers significantly increased (p < 0.05); M3 mAChR mRNA expression levels in cecum significantly reduced in the 18L:6D photoperiod and the 24L:0D photoperiod (p < 0.05). Compared with the 12L:12D photoperiod, the serum glucose (GLU), serum insulin (INS), serum triglyceride (TG) levels, and homeostasis model assessment of insulin resistance (HOMA-IR) of broilers significantly enhanced in the 18L:6D and 24L:0D photoperiods (p < 0.05). Our results indicate that extending the photoperiod can promote the growth rate, ACh expression, and α4 nAChR mRNA expression of broilers while reducing the feed efficiency, inhibiting M3 mAChR mRNA expression, and inducing glucose metabolism disorders in broilers.

Discovery of Antinociceptive α9α10 Nicotinic Acetylcholine Receptor Antagonists by Stable Receptor Expression.

Chronic neuropathic pain is an increasingly prevalent societal issue that responds poorly to existing therapeutic strategies. The α9α10 nicotinic acetylcholine receptor (nAChR) has emerged as a potential target to treat neuropathic pain. However, challenges in expressing functional α9α10 nAChRs in mammalian cell lines have slowed the discovery of α9α10 ligands and studies into the relationship between α9α10 nAChRs and neuropathic pain. Here, we develop a cell line in the HEK293 background that stably expresses functional α9α10 nAChRs. By also developing cell lines expressing only α9 and α10 subunits, we identify distinct receptor pharmacology between homomeric α9 or α10 and heteromeric α9α10 nAChRs. Moreover, we demonstrate that incubation with nAChR ligands differentially regulates the expression of α9- or α10-containing nAChRs, suggesting a possible mechanism by which ligands may modify receptor composition and trafficking in α9- and α10-expressing cells. We then apply our α9α10 cell line in a screen of FDA-approved and investigational drugs to identify α9α10 ligands that provide new tools to probe α9α10 nAChR function. We demonstrate that one compound from this screen, diphenidol, possesses antinociceptive activity in a murine model of neuropathic pain. These results expand our understanding of α9α10 receptor pharmacology and provide new starting points for developing efficacious neuropathic pain treatments.

Insights into Crystallization of Neuronal Nicotinic α4β2 Receptor in Polarized Lipid Matrices

Obtaining high-resolution 3D structures of membrane proteins through X-ray crystallography remains a longstanding bottleneck in the field of structural biology. This challenge has led to the optimization of purification methods to acquire high-yielding, pure proteins suitable for crystallization. In this study, we performed crystallization screenings of purified human α4β2 nAChR using a polarized in meso method. After reconstituting the detergent-solubilized α4β2 nAChR into the LCP matrix, the samples were incubated in a polarized lipid matrix using the RMP@LMx device developed in our laboratory. The results showed that under these conditions, the α4β2-nAChR-LFC 16 complex gave a mobile fraction &gt;0.8, suggesting that its diffusion in the polarized lipid matrix is favorable for crystal nucleation. Voltages above 70 mV restricted crystal formation due to sample dehydration. Furthermore, a lipid analysis using UPLC-ESI MS/MS revealed a profile necessary for preserving protein integrity and promoting diffusion across the LCP. We harvested a single crystal and subjected it to X-ray diffraction, resulting in reflections comparable to previous studies of the muscle-type nAChR from Torpedo californica. X-ray diffraction of a single crystal gave distinct low-resolution diffractions of protein nature. These findings lay the groundwork for further optimization of membrane protein crystallization in polarized in meso phases.

Reliable deep learning in anomalous diffusion against out-of-distribution dynamics.

Anomalous diffusion plays a crucial rule in understanding molecular-level dynamics by offering valuable insights into molecular interactions, mobility states and the physical properties of systems across both biological and materials sciences. Deep-learning techniques have recently outperformed conventional statistical methods in anomalous diffusion recognition. However, deep-learning networks are typically trained by data with limited distribution, which inevitably fail to recognize unknown diffusion models and misinterpret dynamics when confronted with out-of-distribution (OOD) scenarios. In this work, we present a general framework for evaluating deep-learning-based OOD dynamics-detection methods. We further develop a baseline approach that achieves robust OOD dynamics detection as well as accurate recognition of in-distribution anomalous diffusion. We demonstrate that this method enables a reliable characterization of complex behaviors across a wide range of experimentally diverse systems, including nicotinic acetylcholine receptors in membranes, fluorescent beads in dextran solutions and silver nanoparticles undergoing active endocytosis.

Effects of thiacloprid, a neonicotinoid pesticide, on rat reproductive system: Pregnancy hormone disruption and abortion trends

Thiacloprid (TCL), commonly known as Biscaya, is among the most widely used pesticides in agriculture, designed to eliminate insects by targeting their nicotinic receptors. This study explores the effects of orally administering TCL (at a dose of 50 mg/kg) on the hormone secretion crucial for pregnancy and the factors influencing abortion throughout the early, middle, and late stages of pregnancy in female rats.Following TCL exposure, there were significant increases in levels of 17β-Estradiol, prostaglandins F2α and E2, and serum oxytocin hormone in different stages of pregnancy. In contrast, progesterone and endothelin-1 serum levels notably decreased during the initial and final stages of pregnancy. Additionally, TCL led to a substantial rise in lipid peroxidation levels and a decrease in total thiol molecules and total antioxidant capacity, especially in uterine tissue. Although TCL did not significantly affect the morphological characteristics of the delivered fetuses, it notably increased the number of abortions, especially during the second and third stages of pregnancy.In summary, our findings suggest that TCL elevates the risk of abortion in pregnant rats by disrupting the secretion of hormones crucial for fertility (such as 17β-Estradiol/progesterone) and by increasing the secretion of abortion-inducing hormones like prostaglandins and oxytocin. Furthermore, these effects may be associated with disruptions in the oxidant/antioxidant balance within the ovaries and uterus.

Exploring the Benzazoles Derivatives as Pharmacophores for AChE, BACE1, and as Anti-Aβ Aggregation to Find Multitarget Compounds against Alzheimer’s Disease

Despite the great effort that has gone into developing new molecules as multitarget compounds to treat Alzheimer’s disease (AD), none of these have been approved to treat this disease. Therefore, it will be interesting to determine whether benzazoles such as benzimidazole, benzoxazole, and benzothiazole, employed as pharmacophores, could act as multitarget drugs. AD is a multifactorial disease in which several pharmacological targets have been identified—some are involved with amyloid beta (Aβ) production, such as beta secretase (BACE1) and beta amyloid aggregation, while others are involved with the cholinergic system as acetylcholinesterase (AChE) and butirylcholinesterase (BChE) and nicotinic and muscarinic receptors, as well as the hyperphosphorylation of microtubule-associated protein (tau). In this review, we describe the in silico and in vitro evaluation of benzazoles on three important targets in AD: AChE, BACE1, and Aβ. Benzothiazoles and benzimidazoles could be the best benzazoles to act as multitarget drugs for AD because they have been widely evaluated as AChE inhibitors, forming π–π interactions with W286, W86, Y72, and F338, as well as in the AChE gorge and catalytic site. In addition, the sulfur atom from benzothiazol interacts with S286 and the aromatic ring from W84, with these compounds having an IC50 value in the μM range. Also, benzimidazoles and benzothiazoles can inhibit Aβ aggregation. However, even though benzazoles have not been widely evaluated on BACE1, benzimidazoles evaluated in vitro showed an IC50 value in the nM range. Therefore, important chemical modifications could be considered to improve multitarget benzazoles’ activity, such as substitutions in the aromatic ring with electron withdrawal at position five, or a linker 3 or 4 carbons in length, which would allow for better interaction with targets.

Genetically engineered human induced pluripotent stem cells for the production of brain-targeting extracellular vesicles

BackgroundExtracellular vesicles (EVs) are cell-secreted membrane vesicles that have become a promising, natural nanoparticle system for delivering either naturally carried or exogenously loaded therapeutic molecules. Among reported cell sources for EV manufacture, human induced pluripotent stem cells (hiPSCs) offer numerous advantages. However, hiPSC-EVs only have a moderate ability for brain delivery. Herein, we sought to develop a stable hiPSC line for producing EVs with substantially enhanced brain targeting by genetic engineering to overexpress rabies viral glycoprotein (RVG) peptide fused to the N terminus of lysosomal associated membrane protein 2B (RVG-Lamp2B) which has been shown capable of boosting the brain delivery of EVs via the nicotinic acetylcholine receptor.MethodsAn RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. Western blot was used to detect the expression of RVG-Lamp2B-HA in RVG-edited hiPSCs as well as EVs derived from RVG-edited hiPSCs. Uptake of EVs by SH-SY5Y cells in the presence of various endocytic inhibitors was analyzed using flow cytometry. Biodistribution and brain delivery of intravenously injected control and RVG-modified EVs in wild-type mice were examined using ex vivo fluorescent imaging.ResultsHere we report that an RVG-Lamp2B-HA expression cassette was knocked into the AAVS1 safe harbor locus of a control hiPSC line using the CRISPR/Cas9-assisted homologous recombination. The RVG-edited iPSCs have normal karyotype, express pluripotency markers, and have differentiation potential. Expression of RVG-Lamp2B-HA was detected in total cell extracts as well as EVs derived from RVG-edited (vs. control) hiPSCs. The RVG-modified EVs enter neuronal cells via distinct endocytic pathways, compared with control EVs. The biodistribution study confirmed that EVs derived from RVG-edited hiPSCs possess higher brain delivery efficiency.ConclusionTaken together, we have established stable, genetically engineered hiPSCs for producing EVs with RVG expression, offering the improved ability for brain-targeted drug delivery.

Exploring Synaptic Pathways in Traumatic Brain Injury: A Cross-Phenotype Genomics Approach.

Traumatic brain injury (TBI), a global leading cause of mortality and disability, lacks effective treatments to enhance recovery. Synaptic remodeling has been postulated as one mechanism that influences outcomes after TBI. We sought to investigate whether common mechanisms affecting synapse maintenance are shared between TBI and other neuropsychiatric conditions using pathway enrichment tools and genome-wide genotype data, with the goal of highlighting novel treatment targets. We leveraged an integrative approach, combining data from genome-wide association studies with pathway and gene-set enrichment analyses. Literature review-based and Reactome database-driven approaches were combined to identify synapse-related pathways of interest in TBI outcome and to assess for shared associations with conditions in which synapse-related pathobiological mechanisms have been implicated, including Alzheimer's disease, schizophrenia (SCZ), major depressive disorder, post-traumatic stress disorder, attention-deficit hyperactivity disorder, and autism spectrum disorder. Gene and pathway-level enrichment analyses were conducted using MAGMA and its extensions, e- and H-MAGMA, followed by Mendelian randomization to investigate potential causal associations. Of the 98 pathways tested, 32 were significantly enriched in the included conditions. In TBI outcome, we identified significant enrichment in five pathways: "Serotonin clearance from the synaptic cleft" (p = 0.0001), "Presynaptic nicotinic acetylcholine receptors" (p = 0.0003), "Postsynaptic nicotinic acetylcholine receptors" (p = 0.0003), "Highly sodium permeable postsynaptic acetylcholine nicotinic receptors" (p = 0.0001), and "Acetylcholine binding and downstream events" pathways (p = 0.0003). These associations highlight potential involvement of the cholinergic and serotonergic systems in post-TBI recovery. Three of those pathways were shared between TBI and SCZ, suggesting possible pathophysiologic commonalities. In this study, we utilize comparative and integrative genomic approaches across brain conditions that share synaptic mechanisms to explore the pathophysiology of TBI outcomes. Our results implicate associations between TBI outcome and synaptic pathways as well as pathobiological overlap with other neuropsychiatric diseases.

Berberine and palmatine, acting as allosteric potential ligands of α7 nAChR, synergistically regulate inflammation and phagocytosis of microglial cells.

Berberine and palmatine are isoquinoline quaternary alkaloids derived from Chinese medicinal herbs. These alkaloids have shown promising synergy in inhibiting acetylcholinesterase (AChE), indicating their potential in treating Alzheimer's disease (AD). Besides, the anti-inflammatory effects of berberine and palmatine have been widely reported, although the underlying mechanism remains unclear. Here, we found that berberine and palmatine could induce calcium ion (Ca2+) influx via activating α7 nicotinic acetylcholine receptor (α7 nAChR) in cultured microglial cells, possibly serving as its allosteric potential ligands. Furthermore, we examined the synergistic anti-inflammatory effects of berberine and palmatine in the LPS-induced microglia, that significantly suppressed the production of TNF-α and iNOS. Notably, this suppression was reversed by co-treatment with a selective antagonist of α7 nAChR. Moreover, the alkaloid-induced microglial phagocytosis was shown to be mediated by the induction of Ca2+ influx through α7 nAChR and subsequent CaMKII-Rac1-dependent pathway. Additionally, the combination of berberine and palmatine, at low concentration, protected against the LPS-induced endoplasmic reticulum stress and mitochondrial dysfunction in microglia. These findings indicate the potential of berberine and palmatine, either individually or in combination, in contributing to anti-AD drug development, which provide valuable insights into the mechanisms by which natural products, such as plant alkaloids, exert their anti-AD effects.

N-Terminal Capping of the αO-Conotoxin Analogue GeX-2 Improves the Serum Stability and Selectivity toward the Human α9α10 Nicotinic Acetylcholine Receptor.

α9α10 nicotinic acetylcholine receptors (nAChRs) are a promising nonopioid analgesic target, with α9α10 nAChR antagonists showing efficacy against chemotherapy-induced hyperalgesia and allodynia. GeX-2, a potent analgesic conotoxin antagonist of α9α10 nAChRs, has limited serum stability. This study improved GeX-2 stability by capping its N-terminal with fatty acids or polyethylene glycol chains, which enhanced its serum stability but eliminated activity at G protein-coupled γ-aminobutyric acid type B (GABAB) receptor-coupled CaV2.2 channels while preserving activity at α9α10 nAChRs. In vivo, α9α10 nAChRs antagonism alone did not alleviate neuropathic pain, highlighting the importance of GABAB receptor-coupled CaV2.2 channels in GeX-2's antinociceptive effects in the chronic constriction injury rat model. The GeX-2 analogue, with an N-terminal methyl group, showed improved activity and selectivity for α9α10 nAChRs, increased serum half-life, and strong analgesic effects in oxaliplatin-induced cold allodynia models. AlphaFold3 and molecular dynamics simulations provided insights into the binding modes and the effects of N-terminal capping, which informed future peptide therapeutic developments.

A Deficiency in Glutamine-Fructose-6-Phosphate Transaminase 1 (Gfpt1) in Skeletal Muscle Results in Reduced Glycosylation of the Delta Subunit of the Nicotinic Acetylcholine Receptor (AChRδ).

The neuromuscular junction (NMJ) is the site where the motor neuron innervates skeletal muscle, enabling muscular contraction. Congenital myasthenic syndromes (CMS) arise when mutations in any of the approximately 35 known causative genes cause impaired neuromuscular transmission at the NMJ, resulting in fatigable muscle weakness. A subset of five of these CMS-causative genes are associated with protein glycosylation. Glutamine-fructose-6-phosphate transaminase 1 (Gfpt1) is the rate-limiting enzyme within the hexosamine biosynthetic pathway (HBP), a metabolic pathway that produces the precursors for glycosylation. We hypothesized that deficiency in Gfpt1 expression results in aberrant or reduced glycosylation, impairing the proper assembly and stability of key NMJ-associated proteins. Using both in vitro and in vivo Gfpt1-deficient models, we determined that the acetylcholine receptor delta subunit (AChRδ) has reduced expression and is hypo-glycosylated. Using laser capture microdissection, NMJs were harvested from Gfpt1 knockout mouse muscle. A lower-molecular-weight species of AChRδ was identified at the NMJ that was not detected in controls. Furthermore, Gfpt1-deficient muscle lysates showed impairment in protein O-GlcNAcylation and sialylation, suggesting that multiple glycan chains are impacted. Other key NMJ-associated proteins, in addition to AChRδ, may also be differentially glycosylated in Gfpt1-deficient muscle.