Search Results (121)

Tobacco use disorder remains a leading cause of preventable mortality, with nicotine playing a central role in the development and maintenance of dependence, mainly through its action on α4β2 nicotinic acetylcholine receptors (nAChRs). Smoking cessation treatments must address both physiological withdrawal and the affective disturbances (such as anxiety, irritability, and mood lability) which often facilitate relapses. This review compares two pharmacotherapies used in smoking cessation, varenicline and cytisinicline (cytisine), with particular focus on their impact on emotional regulation, psychological symptoms, and neuropsychiatric safety. Varenicline, a high-affinity partial agonist at α4β2 nAChRs, has demonstrated superior efficacy in maintaining abstinence and is well-supported by robust clinical data, including in psychiatric populations. However, its use may be limited by adverse effects such as nausea and sleep disorders. Cytisinicline, a structurally similar but less potent partial agonist, has recently gained renewed interest due to its lower cost, favorable tolerability profile, and comparable effectiveness in the general population. Although less extensively studied in patients with serious mental illness, preliminary data suggest cytisinicline may offer a better side effect profile, particularly regarding sleep disturbances and emotional reactivity. Both agents appear to ameliorate withdrawal-related affective symptoms without significantly increasing psychiatric risk. Ultimately, pharmacotherapy choice should be guided by individual clinical features, mental health status, treatment tolerability, and resource availability. Further research is needed to establish cytisinicline’s efficacy and safety across diverse clinical contexts, particularly among individuals with severe psychiatric comorbidities. Full article

Nicotine addiction is a serious global public health problem, so there is an urgent necessity to develop novel effective smoking cessation treatments with fewer adverse effects. Spontaneous behavioral sensitization induced by repeated intermittent exposure to the addictive substance represents a classical animal model of addiction research. A significant contributor to nicotine addiction is its interaction with α6β2* nAChRs located on midbrain dopaminergic neurons, which leads to an increase in dopamine (DA) release. α-Conotoxin (α-CTx) TxIB is a novel potent antagonist of the α6/α3β2β3* nAChRs, with an IC50 value of 28.4 nM developed by our group. In this study, we aimed to investigate the effectiveness of α-CTx TxIB in countering nicotine-induced behavioral sensitization and moderating the impact of nicotine on dopamine accumulation in the midbrain. Our results demonstrated that repeated nicotine administration remarkably elevated the locomotor activity of mice, including the number of entries, average speed, and total distance traveled, which could be effectively attenuated by α-CTx TxIB intervention in a dose-dependent manner (1 nmol and 5 nmol TxIB per mouse). Furthermore, 5 nmol α-CTx TxIB significantly reduced the nicotine-elevated DA and norepinephrine (NE) levels in the ventral tegmental area (VTA) and nucleus accumbens (NAc) of mice. 5 nmol α-CTx TxIB also markedly decreased the expression of critical proteins such as the dopamine transporter (DAT), N-methyl-D-aspartic acid receptor (NMDAR), and c-Fos in the NAc and prefrontal cortex (PFC) of the nicotine-exposed mice. This research provided the first compelling evidence that α-CTx TxIB attenuated nicotine-induced locomotor sensitization and inhibited the nicotine-induced dopamine elevation in mice. These results open up new avenues for exploring the therapeutic potential of α-CTx TxIB in the treatment of nicotine addiction. Full article

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. Full article

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 >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. Full article

α-Conotoxins are disulfide-rich peptides obtained from the venom of cone snails, which are considered potential molecular probes and drug leads for nAChR-related disorders. However, low specificity towards different nAChR subtypes restricts the further application of many α-conotoxins. In this work, a series of loop1 amino acid-substituted mutants of α-conotoxin RegIIA were synthesized, whose potency and selectivity were evaluated by an electrophysiological approach. The results showed that loop1 alanine scanning mutants [H5A]RegIIA and [P6A]RegIIA blocked rα7 nAChR with IC₅₀s of 446 nM and 459 nM, respectively, while their inhibition against rα3β2 and rα3β4 subtypes was negligible, indicating the importance of the fifth and sixth amino acid residues for RegIIA’s potency and selectivity. Then, second-generation mutants were designed and synthesized, among which the analogues [H5V]RegIIA and [H5S]RegIIA showed significantly improved selectivity and comparable potency towards rα7 nAChR compared with the native RegIIA. Overall, these findings provide deep insights into the structure–activity relationship of RegIIA, as well as revealing a unique perspective for the further modification and optimization of α-conotoxins and other active peptides. Full article

Humans are exposed to significant amounts of blue light from computers and smartphones. However, the effects of blue light on ulcerative colitis remain unclear. In this study, we assessed blue-light-irradiation-induced alterations in colonic symptoms using a dextran sodium sulfate (DSS)-induced ulcerative colitis model mice. Both male and female institute of cancer research (ICR) mice were administered DSS (5%) ad libitum for 5 days while irradiated with 40 kJ/m² blue light daily. Additionally, tranexamic acid (TA) was administered daily throughout the study. Male mice treated with DSS/blue light exhibited exacerbated colitis compared to those treated with DSS alone. In contrast, female mice treated with DSS/blue light exhibited enhanced symptoms compared to those treated with DSS alone. Additionally, in male mice exposed to blue light, the clock/brain and muscle Arndt-like 1 (Bma1)/noradrenaline/macrophage or beta2-adrenergic receptor (β2-AR) pathways were activated. In female mice, the Bmal1/acetylcholine/macrophage/nicotinic acetylcholine receptor alpha 7 (α7nAChR) pathway was activated. These findings highlight sex differences in the effects of blue light on DSS-induced ulcerative colitis. Moreover, the worsening of symptoms in males was ameliorated through TA administration. Full article

α-Conotoxins, as selective nAChR antagonists, can be valuable tools for targeted drug delivery and fluorescent labeling, while conotoxin-drug or conotoxin-fluorescent conjugates through the disulfide bond are rarely reported. Herein, we demonstrate the [2,4] disulfide bond of α-conotoxin as a feasible new chemical modification site. In this study, analogs of the α-conotoxin LsIA cysteine[2,4] were synthesized by stapling with five linkers, and their inhibitory activities against human α7 and rat α3β2 nAChRs were maintained. To further apply this method in targeted delivery, the alkynylbenzyl bromide linker was synthesized and conjugated with Coumarin 120 (AMC) and Camptothecin (CPT) by copper-catalyzed click chemistry, and then stapled between cysteine[2,4] of the LsIA to construct a fluorescent probe and two peptide-drug conjugates. The maximum emission wavelength of the LsIA fluorescent probe was 402.2 nm, which was essentially unchanged compared with AMC. The cytotoxic activity of the LsIA peptide-drug conjugates on human A549 was maintained in vitro. The results demonstrate that the stapling of cysteine[2,4] with alkynylbenzyl bromide is a simple and feasible strategy for the exploitation and utilization of the α-conotoxin LsIA. Full article

The venom of cone snails has been proven to be a rich source of bioactive peptides that target a variety of ion channels and receptors. α-Conotoxins (αCtx) interact with nicotinic acetylcholine receptors (nAChRs) and are powerful tools for investigating the structure and function of the various nAChR subtypes. By studying how conotoxins interact with nAChRs, we can improve our understanding of these receptors, leading to new insights into neurological diseases associated with nAChRs. Here, we describe the discovery and characterization of a novel conotoxin from Conus ateralbus, αCtx-AtIA, which has an amino acid sequence homologous to the well-described αCtx-PeIA, but with a different selectivity profile towards nAChRs. We tested the synthetic αCtx-AtIA using the calcium imaging-based Constellation Pharmacology assay on mouse DRG neurons and found that αCtx-AtIA significantly inhibited ACh-induced calcium influx in the presence of an α7 positive allosteric modulator, PNU-120596 (PNU). However, αCtx-AtIA did not display any activity in the absence of PNU. These findings were further validated using two-electrode voltage clamp electrophysiology performed on oocytes overexpressing mouse α3β4, α6/α3β4 and α7 nAChRs subtypes. We observed that αCtx-AtIA displayed no or low potency in blocking α3β4 and α6/α3β4 receptors, respectively, but improved potency and selectivity to block α7 nAChRs when compared with αCtx-PeIA. Through the synthesis of two additional analogs of αCtx-AtIA and subsequent characterization using Constellation Pharmacology, we were able to identify residue Trp18 as a major contributor to the activity of the peptide. Full article

Nicotinic acetylcholine receptors (nAChRs) are involved in various central nervous system functions and have also been implicated in several neurodegenerative disorders. The heteromeric α4β2* and homomeric α7 are two major nAChR subtypes which have been studied in the brain using positron emission tomography (PET). Our comparative autoradiographic studies of the two receptor types in the mouse and rat brains show major differences in the thalamus (α4β2* >> α7), hippocampus (α7 >> α4β2*), and subiculum (α4β2* >> α7). A relatively newer heteromeric α7β2 nAChR subtype has been identified in the brain which may have a greater role in neurodegeneration. We report the development of KS7 (3-(2-(S)-azetidinylmethoxy)-5-(1,4-diaza-bicyclo[3.2.2]nonane)pyridine) which incorporates structural features of Nifzetidine (high affinity for α4β2* nAChR) and ASEM (high affinity for α7 nAChR) in an effort to target α7 and β2 subunits in α7β2 nAChR. KS7 exhibited higher affinities (IC₅₀ = 50 to 172 nM) for [³H]cytisine radiolabeled sites and weaker affinities (IC₅₀ = 10 μM) for [¹²⁵I]-α-bungarotoxin radiolabeled rat brain sites in several brain regions. The weaker affinity of KS7 to α7 nAChR may suggest lack of binding at the α7 subunit of α7β2 nAChR. A radiolabeled derivative of KS7 may be required to identify any specific binding to brain regions suggested to contain α7β2 nAChR. Full article

Pentameric ligand-gated ion channels (pLGICs) are expressed throughout the central and peripheral nervous systems of vertebrates and modulate many aspects of human health and disease. Recent structural and computational data indicate that cation-selective pLGICs contain a long helical extension (MA) of one of the transmembrane helices. The MA helix has been shown to affect both the membrane expression of, and ion conductance levels through, these pLGICs. Here we probe the functional effects of 68 mutations in the MA region of the α4β2 nicotinic acetylcholine receptor (nAChR), using a voltage-sensitive membrane dye and radioligand binding to measure receptor function and expression/assembly. We found seven alanine mutations in a stretch of the MA helix that prevent correct receptor folding and/or assembly, as evidenced by the lack of both function and ligand binding. A further two alanine mutations resulted in receptors that were capable of binding ligand but showed no functional response, and we propose that, in these mutants, ligand binding is insufficient to trigger channel opening. The data clarify the effect of the MA helix, and as the effects of some of our mutations in the α4β2 nAChR differ from the effects of equivalent mutations in other cation-selective pLGICs, we suggest that residues in the MA helix may play subtly different roles in different receptors. Full article

In addition to binding to nicotinic acetylcholine receptors (nAChRs), nicotine is known to regulate the β-adrenergic receptors (β-ARs) promoting oncogenic signaling. Using A549 (p53 wild-type) and H1299 (p53-null) lung cancer cells, we show that nicotine treatment led to: increased adrenaline/noradrenaline levels, an effect blocked by treatment with the α7nAChR inhibitor (α-BTX) but not by the β-blocker (propranolol) or the α4β2nAChR antagonist (DhβE); decreased GABA levels in A549 and H1299 cell media, an effect blocked by treatment with DhβE; increased VEGF levels and PI3K/AKT activities, an effect diminished by cell co-treatment with α-BTX, propranolol, and/or DhβE; and inhibited p53 activity in A549 cells, that was reversed, upon cell co-treatment with α-BTX, propranolol, and/or DhβE or by VEGF immunodepletion. VEGF levels increased upon cell treatment with nicotine, adrenaline/noradrenaline, and decreased with GABA treatment. On the other hand, the p53 activity decreased in A549 cells treated with nicotine, adrenaline/noradrenaline and increased upon cell incubation with GABA. Knockdown of p53 led to increased VEGF levels in the media of A549 cells. The addition of anti-VEGF antibodies to A549 and H1299 cells decreased cell viability and increased apoptosis; blocked the activities of PI3K, AKT, and NFκB in the absence or presence of nicotine; and resulted in increased p53 activation in A549 cells. We conclude that VEGF can be upregulated via α7nAChR and/or β-ARs and downregulated via GABA and/or p53 in response to the nicotine treatment of NSCLC cells. Full article

Alzheimer disease (AD) is a widespread neurodegenerative disease characterized by the accumulation of oligomeric toxic forms of β-amyloid (Aβ1-42) and dysfunction of the cholinergic system in the different brain regions. However, the exact mechanisms of AD pathogenesis and the role of the nicotinic acetylcholine receptors (nAChRs) in the disease progression remain unclear. Here, we revealed a decreased expression of a number of the Ly6/uPAR proteins targeting nAChRs in the cerebellum of 2xTg-AD mice (model of early AD) in comparison with non-transgenic mice both at mRNA and protein levels. We showed that co-localization of one of them, – neuromodulator Lynx1, with α7-nAChR was diminished in the vicinity of cerebellar astrocytes of 2xTg-AD mice, while Aβ1-42 co-localization with this receptor present was increased. Moreover, the expression of anti-inflammatory transcription factor KLF4 regulating transcription of the Ly6/uPAR genes was decreased in the cerebellum of 2xTg-AD mice, while expression of inflammatory cytokine TNF-α was increased. Based on these data together with observed astrocyte degeneration in the cerebellum of 2xTg-AD mice, we suggest the mechanism by which expression of the Ly6/uPAR proteins upon Aβ pathology results in dysregulation of the cholinergic system and particularly of α7-nAChR function in the cerebellum. This leads to enhanced neuroinflammation and cerebellar astrocyte degeneration. Full article

Neuropathic pain affects about 7–8% of the population, and its management still poses challenges with unmet needs. Over the past decades, researchers have explored the cholinergic system (muscarinic and nicotinic acetylcholine receptors: mAChR and nAChR) and compounds targeting these receptors as potential analgesics for neuropathic pain management. This scoping review aims to provide an overview of studies on peripheral neuropathic pain (PNP) in rodent models, exploring compounds targeting cholinergic neurotransmission. The inclusion criteria were original articles on PNP in rodent models that explored the use of compounds directly targeting cholinergic neurotransmission and reported results of nociceptive behavioral assays. The literature search was performed in the PubMed and Web of Science databases (1 January 2000–22 April 2023). The selection process yielded 82 publications, encompassing 62 compounds. The most studied compounds were agonists of α4β2 nAChR and α7 nAChR, and antagonists of α9/α10 nAChR, along with those increasing acetylcholine and targeting mAChRs. Studies mainly reported antinociceptive effects in traumatic PNP models, and to a lesser extent, chemotherapy-induced neuropathy or diabetic models. These preclinical studies underscore the considerable potential of cholinergic compounds in the management of PNP, warranting the initiation of clinical trials. Full article

Neonicotinoid pesticides were initially designed in order to achieve species selectivity on insect nicotinic acetylcholine receptors (nAChRs). However, concerns arose when agonistic effects were also detected in human cells expressing nAChRs. In the context of next-generation risk assessments (NGRAs), new approach methods (NAMs) should replace animal testing where appropriate. Herein, we present a combination of in silico and in vitro methodologies that are used to investigate the potentially toxic effects of neonicotinoids and nicotinoid metabolites on human neurons. First, an ensemble docking study was conducted on the nAChR isoforms α7 and α3β4 to assess potential crucial molecular initiating event (MIE) interactions. Representative docking poses were further refined using molecular dynamics (MD) simulations and binding energy calculations using implicit solvent models. Finally, calcium imaging on LUHMES neurons confirmed a key event (KE) downstream of the MIE. This method was also used to confirm the predicted agonistic effect of the metabolite descyano-thiacloprid (DCNT). Full article

Anxiety is a serious mental disorder, and recent statistics have determined that 35.12% of the global population had an anxiety disorder during the COVID-19 pandemic. A mechanism associated with anxiolytic effects is related to nicotinic acetylcholine receptor (nAChR) agonists, principally acting on the α4β2 nAChR subtype. nAChRs are present in different animal models, including murine and teleosteos ones. Zebrafish has become an ideal animal model due to its high human genetic similarities (70%), giving it high versatility in different areas of study, among them in behavioral studies related to anxiety. The novel tank diving test (NTT) is one of the many paradigms used for studies on new drugs related to their anxiolytic effect. In this work, an adult zebrafish was used to determine the behavioral effects of 3- and 5-halocytisine derivatives, using the NTT at different doses. Our results show that substitution at position 3 by chlorine or bromine decreases the time spent by the fish at the bottom compared to the control. However, the 3-chloro derivative at higher doses increases the bottom dwelling time. In contrast, substitution at the 5 position increases bottom dwelling at all concentrations showing no anxiolytic effects in this model. Unexpected results were observed with the 5-chlorocytisine derivative, which at a concentration of 10 mg/L produced a significant decrease in bottom dwelling and showed high times of freezing. In conclusion, the 3-chloro and 3-bromo derivatives show an anxiolytic effect, the 3-chlorocytisine derivative being more potent than the 3-bromo derivative, with the lowest time at the bottom of the tank at 1mg/L. On the other hand, chlorine, and bromine at position 5 produce an opposite effect. Full article