nAChR Agonist Research Articles

Activation of endothelial NO synthase and P2X7 receptor modification mediates the cholinergic control of ATP-induced interleukin-1β release by mononuclear phagocytes.

The pro-inflammatory cytokine interleukin-1β (IL-1β) plays a central role in host defense against infections. High systemic IL-1β levels, however, promote the pathogenesis of inflammatory disorders. Therefore, mechanisms controlling IL-1β release are of substantial clinical interest. Recently, we identified a cholinergic mechanism inhibiting the ATP-mediated IL-1β release by human monocytes via nicotinic acetylcholine receptor (nAChR) subunits α7, α9 and/or α10. We also discovered novel nAChR agonists that trigger this inhibitory function in monocytic cells without eliciting ionotropic functions at conventional nAChRs. Here, we investigate the ion flux-independent signaling pathway that links nAChR activation to the inhibition of the ATP-sensitive P2X7 receptor (P2X7R). Different human and murine mononuclear phagocytes were primed with lipopolysaccharide and stimulated with the P2X7R agonist BzATP in the presence or absence of nAChR agonists, endothelial NO synthase (eNOS) inhibitors, and NO donors. IL-1β was measured in cell culture supernatants. Patch-clamp and intracellular Ca2+ imaging experiments were performed on HEK cells overexpressing human P2X7R or P2X7R with point mutations at cysteine residues in the cytoplasmic C-terminal domain. The inhibitory effect of nAChR agonists on the BzATP-induced IL-1β release was reversed in the presence of eNOS inhibitors (L-NIO, L-NAME) as well as in U937 cells after silencing of eNOS expression. In peripheral blood mononuclear leukocytes from eNOS gene-deficient mice, the inhibitory effect of nAChR agonists was absent, suggesting that nAChRs signal via eNOS to inhibit the BzATP-induced IL-1β release. Moreover, NO donors (SNAP, S-nitroso-N-acetyl-DL-penicillamine; SIN-1) inhibited the BzATP-induced IL-1β release by mononuclear phagocytes. The BzATP-induced ionotropic activity of the P2X7R was abolished in the presence of SIN-1 in both, Xenopus laevis oocytes and HEK cells over-expressing the human P2X7R. This inhibitory effect of SIN-1 was absent in HEK cells expressing P2X7R, in which C377 was mutated to alanine, indicating the importance of C377 for the regulation of the P2X7R function by protein modification. We provide first evidence that ion flux-independent, metabotropic signaling of monocytic nAChRs involves eNOS activation and P2X7R modification, resulting in an inhibition of ATP signaling and ATP-mediated IL-1β release. This signaling pathway might be an interesting target for the treatment of inflammatory disorders.

A consensus phosphoserine within the large cytoplasmic loop of insect nAChR α8 subunits modulated interaction between 14-3-3ε and nAChRs to regulate neonicotinoid efficacy

Neonicotinoids are insect-selective nicotinic acetylcholine receptors (nAChRs) agonists that are used extensively for plant protection and animal health care. Some chaperone proteins, such as 14-3-3 proteins, importantly modulate nAChRs to display the physiological and pharmacological properties. Here we found that there is a 14-3-3 binding motif RSPSTH within the cytoplasmic loop of most insect α8 subunits. In the motif, a potential phosphorylated serine residue, serine 337, was a putative protein kinase A (PKA) substrate. Using Locusta migratoria α8 subunit as a representative, here we demonstrated that Loc14‐3-3ε interacted with the unique phosphoserine (α8S337) of Locα8 subunit to regulate agonist efficacy on hybrid Locα8/β2 nAChRs in Xenopus oocytes. Co-expression of Loc14-3-3ε caused a dramatic rise of maximal inward currents (Imax) of Locα8/β2 for acetylcholine and imidacloprid to 2.9-fold and 3.1-fold of that of Locα8/β2 alone. The S337A substitution of Locα8 reduced the Imax rise when Locα8S337A/β2 and Loc14-3-3ε were co-expressed. The increased agonist currents by exogenous Loc14-3-3ε on Locα8/β2 could be almost abolished by either PKA inhibitor KT5720 or 14-3-3 inhibitor difopein. The findings revealed that serine 337 within motif RSPSTH was important for the interaction between insect nAChRs and 14-3-3ε, and inhibiting the interaction would change the pharmacological property of insect nAChRs to agonist such as neonicotinoids which may provide insights to develop new targets for insecticide design.

Synthesis and Antagonist Activity of Methyllycaconitine Analogues on Human α7 Nicotinic Acetylcholine Receptors.

Methyllycaconitine (MLA), 1, is a naturally occurring norditerpenoid alkaloid that is a highly potent (IC50 = 2 nM) selective antagonist of α7 nicotinic acetylcholine receptors (nAChRs). Several structural factors affect its activity such as the neopentyl ester side-chain and the piperidine ring N-side-chain. The synthesis of simplified AE-bicyclic analogues 14-21 possessing different ester and nitrogen side-chains was achieved in three steps. The antagonist effects of synthetic analogues were examined on human α7 nAChRs and compared to that of MLA 1. The most efficacious analogue (16) reduced α7 nAChR agonist responses [1 nM acetylcholine (ACh)] to 53.2 ± 1.9% compared to 3.4 ± 0.2% for MLA 1. This demonstrates that simpler analogues of MLA 1 possess antagonist effects on human α7 nAChRs but also indicates that further optimization may be possible to achieve antagonist activity comparable to that of MLA 1.

Human-specific duplicate CHRFAM7A gene is associated with more severe osteoarthritis and amplifies pain behaviours

ObjectivesCHRFAM7A is a uniquely human fusion gene that functions as a dominant negative regulator of alpha 7 acetylcholine nicotinic receptor (α7nAChR) in vitro. This study determined the impact of CHRFAM7A...

Functional Changes in the Adult Mouse Retina using an Alpha7 Nicotinic Acetylcholine Receptor Agonist after Blast Exposure

Currently, there is a lack of treatments for retinal neurotrauma. To address this issue, this study uses an alpha7 nAChR agonist, PNU-282987, to determine it effects on functional activity in the retina shortly after a traumatic blast exposure. The objectives of this research include: (1) examination of the cellular and functional damage associated with ocular blast exposure, and (2) evaluation of structural and functional changes that occur post PNU-282987 treatment. Significant ocular blast damage was induced in adult mice after exposure to a single blast of 35 psi to the left eye. Blast-exposed transgenic mice expressing tdTomato Müller glia were treated daily with eyedrops containing PNU-282987 for 4 weeks following the blast exposure. Antibody staining studies in these transgenic mice was conducted to examine lineage tracing and electroretinograms (ERGs) were obtained to examine functional changes. Blast exposure caused a significant loss of cells in all retinal layers after 4 weeks. Immunohistochemical analysis demonstrated tdTomato-positive labeled photoreceptors and retinal ganglion cells in blast-exposed mice treated with PNU-282987. ERG recordings were taken from control animals, from blast-damaged animals and from animals exposed to blast followed by 4 weeks of PNU-282987 treatment. Scotopic ERG recordings from blast-exposed mice had significantly decreased amplitudes of a-wave, b-wave, oscillatory potentials and flicker frequencies, which were prevented after PNU-282987 treatment. In photopic experiments, the PhNR response was reduced significantly after blast exposure but the decrease was prevented after treatment with PNU-282987. These are the first experiments that demonstrate preservation of retinal function after blast exposure using an alpha7 nAChR agonist.

Cholinergic dysfunction in COVID-19: frantic search and hoping for the best.

A novel coronavirus known as severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2) is a potential cause of acute respiratory infection called coronavirus disease 2019 (COVID-19). The binding of SARS-CoV-2 with angiotensin-converting enzyme 2 (ACE2) induces a series of inflammatory cellular events with cytopathic effects leading to cell injury and hyperinflammation. Severe SARS-CoV-2 infection may lead to dysautonomia and sympathetic storm due to dysfunction of the autonomic nervous system (ANS). Therefore, this review aimed to elucidate the critical role of the cholinergic system (CS) in SARS-CoV-2 infection. The CS forms a multi-faceted network performing diverse functions in the body due to its distribution in the neuronal and non-neuronal cells. Acetylcholine (ACh) acts on two main types of receptors which are nicotinic receptors (NRs) and muscarinic receptors (MRs). NRs induce T cell anergy with impairment of antigen-mediated signal transduction. Nicotine through activation of T cell NRs inhibits the expression and release of the pro-inflammatory cytokines. NRs play important anti-inflammatory effects while MRs promote inflammation by inducing the release of pro-inflammatory cytokines. SARS-CoV-2 infection can affect the morphological and functional stability of CS through the disruption of cholinergic receptors. SARS-CoV-2 spike protein is similar to neurotoxins, which can bind to nicotinic acetylcholine receptors (nAChR) in the ANS and brain. Therefore, cholinergic receptors mainly nAChR and related cholinergic agonists may affect the pathogenesis of SARS-CoV-2 infection. Cholinergic dysfunction in COVID-19 is due to dysregulation of nAChR by SARS-CoV-2 promoting the central sympathetic drive with the development of the sympathetic storm. As well, nAChR activators through interaction with diverse signaling pathways can reduce the risk of inflammatory disorders in COVID-19. In addition, nAChR activators may mitigate endothelial dysfunction (ED), oxidative stress (OS), and associated coagulopathy in COVID-19. Similarly, nAChR activators may improve OS, inflammatory changes, and cytokine storm in COVID-19. Therefore, nAChR activators like varenicline in virtue of its anti-inflammatory and anti-oxidant effects with direct anti-SARS-CoV-2 effect could be effective in the management of COVID-19.

Molecular docking and dynamic simulation studies of α4β2 and α7 nicotinic acetylcholine receptors with tobacco smoke constituents nicotine, NNK and NNN

Smoking constitutes a major global health problem. As it triggers various health hazards including cancers, cardiac and pulmonary illness, it is imperative to understand the mechanism of action of various smoke constituents on our cellular processes. Various in vitro studies have compiled the affinity of cigarette smoke constituents on various nicotinic acetylcholine receptors (nAChRs). But the nature of the intermolecular interactions contributing to this affinity and the key amino acids in the receptor active sites involved in this are not investigated so far. Here, we are examining the interaction of α7nAChR and α4β2nAChR on nicotine, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and N-nitrosornicotine (NNN), the physiologically significant constituents in smoke, through molecular docking and dynamics simulations study. The docking of α4β2nAChR structure with the ligands nicotine, NNK and NNN yielded docking scores of −41.45 kcal/mol, −59.28 kcal/mol and −54.60 kcal/mol, respectively, and that of α7nAChR receptor molecule with the ligands yielded docking scores of −59.54 kcal/mol, −71.06 kcal/mol and −70.86 kcal/mol, respectively. The study showed that NNK exhibited the highest affinity with the ligands which was confirmed by dynamics simulation. But higher stability of interactions as surmised from Molecular dynamics simulations was found for nicotine with α4β2nAChR and NNN with α7nAChR. The findings validate the in vitro studies comparing the affinities of these compounds. The study will be useful in formulating effective nAChR agonists to treat neurological disorders and antagonists for smoke deaddiction and improve health standards. Communicated by Ramaswamy H. Sarma

Targeting α7 nicotinic acetylcholine receptors for chronic pain.

Despite rapid advances in the field of chronic pain, it remains extremely challenging in the clinic. Pain treatment strategies have not improved for decades as opioids remain the main prescribed drugs for chronic pain management. However, long-term use of opioids often leads to detrimental side effects. Therefore, uncovering the mechanisms underlying the development and maintenance of chronic pain may aid the discovery of novel therapeutics to benefit patients with chronic pain. Substantial evidence indicates downregulation of α7 nicotinic acetylcholine receptors (α7 nAChR) in the sciatic nerve, dorsal root ganglia, and spinal cord dorsal horn in rodent models of chronic pain. Moreover, our recent study and results from other laboratories demonstrate that potentiation of α7 nAChR attenuates pain behaviors in various murine models of chronic pain. This review summarized and discussed the preclinical evidence demonstrating the therapeutic potential of α7 nAChR agonists and allosteric modulators in chronic pain. This evidence indicates that potentiation of α7 nAChR is beneficial in chronic pain, mostly by alleviating neuroinflammation. Overall, α7 nAChR-based therapy for chronic pain is an area with great promise, but more research regarding its detailed mechanisms is warranted.

The UV-induced uptake of melanosome by skin keratinocyte is triggered by α7 nicotinic acetylcholine receptor-mediated phagocytosis.

The melanosome is an organelle that produces melanin for skin pigmentation, which is synthesized by epidermal melanocytes, subsequently transported and internalized by epidermal keratinocytes. Exposure to ultraviolet (UV) from sunlight radiation is a major stimulator of melanosome uptake by keratinocytes. Acetylcholine (ACh) is known to be released by keratinocytes under UV exposure, which regulates melanin production in melanocytes by participating in which has been named as 'skin synapse'. Here, the role of cholinergic molecules, i.e. ACh and α7 nicotinic acetylcholine receptor (nAChR), in regulating melanosome uptake through phagocytosis by keratinocytes was illustrated. In cultured keratinocytes (HaCaT cells), the fluorescent beads at different sizes imitating melanosomes, or melanosomes, were phagocytosed under UV exposure. The UV-induced phagocytosis in keratinocytes was markedly increased by applied ACh, an acetylcholinesterase (AChE) inhibitor or an α7 nAChR agonist. By contrast, the antagonist of α7 nAChR was able to fully block the UV-induced phagocytosis, suggesting the role of α7 nAChR in this event. The intracellular Ca++ mobilization was triggered by UV exposure, accounting for the initiation of phagocytosis. The blockage of UV-mediated Ca++ mobilization, triggered by BAPTA-AM or α7 nAChR antagonist, resulted in a complete termination of phagocytosis. Besides, the phosphorylation of cofilin, as well as expression and activation of RhoA, accounting for phagocytosis was induced by UV exposure: the phosphorylation was blocked by BAPTA-AM or α7 nAChR antagonist. The result suggests that the cholinergic system, especially α7 nAChR, is playing a regulatory role in modulating melanosome uptake in keratinocytes being induced by UV exposure.

Mesenchymal stromal cells alleviate acute respiratory distress syndrome through the cholinergic anti-inflammatory pathway

Mesenchymal stromal cells (MSCs) have been considered a promising alternative for treatment of acute respiratory distress syndrome (ARDS). However, there is significant heterogeneity in their therapeutic efficacy, largely owing to the incomplete understanding of the mechanisms underlying the therapeutic activities of MSCs. Here, we hypothesize that the cholinergic anti-inflammatory pathway (CAP), which is recognized as a neuroimmunological pathway, may be involved in the therapeutic mechanisms by which MSCs mitigate ARDS. Using lipopolysaccharide (LPS) and bacterial lung inflammation models, we found that inflammatory cell infiltration and Evans blue leakage were reduced and that the expression levels of choline acetyltransferase (ChAT) and vesicular acetylcholine transporter (VAChT) in lung tissue were significantly increased 6 hours after MSC infusion. When the vagus nerve was blocked or α7 nicotinic acetylcholine (ACh) receptor (α7nAChR)-knockout mice were used, the therapeutic effects of MSCs were significantly reduced, suggesting that the CAP may play an important role in the effects of MSCs in ARDS treatment. Our results further showed that MSC-derived prostaglandin E2 (PGE2) likely promoted ACh synthesis and release. Additionally, based on the efficacy of nAChR and α7nAChR agonists, we found that lobeline, the nicotinic cholinergic receptor excitation stimulant, may attenuate pulmonary inflammation and alleviate respiratory symptoms of ARDS patients in a clinical study (ChiCTR2100047403). In summary, we reveal a previously unrecognized MSC-mediated mechanism of CAP activation as the means by which MSCs alleviate ARDS-like syndrome, providing insight into the clinical translation of MSCs or CAP-related strategies for the treatment of patients with ARDS.

Varenicline Solution Nasal Spray: A Review in Dry Eye Disease.

Increasing endogenous tear film production via pharmacological neuroactivation of the nasolacrimal reflex [NLR; also known as the trigeminal parasympathetic pathway (TPP)] is a novel therapeutic approach to treating dry eye disease (DED). An intranasal formulation of the water-soluble, small-molecule, nicotinic acetylcholine receptor (nAChR) agonist varenicline (Tyrvaya™) has been approved in the USA for the treatment of DED. Twice-daily administration of varenicline solution nasal spray resulted in rapid, statistically significant and clinically meaningful improvements in the signs and symptoms of DED over a period of 4 weeks in two pivotal studies (ONSET-1 and -2). The efficacy of varenicline solution was maintained over a longer-term period of 12 weeks in a third study (MYSTIC). Consistent with the nasal route of delivery, the most common adverse events reported by varenicline solution recipients were non-ocular in nature (mild and transient sneezing and cough). Thus, varenicline solution nasal spray is a rapidly-acting, effective and generally well tolerated treatment for DED that offers several potentially useful advantages over existing topical ocular therapies in terms of increasing endogenous tear secretion and reducing ophthalmic treatment burden.Supplementary InformationThe online version contains supplementary material available at 10.1007/s40265-022-01782-4.

Efficacy and Safety of Single-dose OC-02 (Simpinicline Solution) Nasal Spray on Signs and Symptoms of Dry Eye Disease: The PEARL Phase II Randomized Trial

PurposeDry eye disease is a multifactorial disorder that affects the ocular surface, with symptoms including ocular irritation, impaired vision, and pain. Nicotinic acetylcholine receptor (nAChR) agonists are novel treatments for dry eye disease; this study investigates the nAChR agonist OC-02 (simpinicline solution) as an aqueous nasal spray. MethodsPEARL (Clinical Trial to Evaluate the Efficacy of OC-02 Nasal Spray on Signs and Symptoms of Dry Eye Disease) was a Phase II study that evaluated the efficacy and safety of OC-02 (simpinicline solution) nasal spray (OC-02 SNS) in adult patients with dry eye disease. Patients ≥22 years of age were eligible if they had an Ocular Surface Disease Index score ≥23, corneal fluorescein staining score ≥2 in >1 region or ≥4 for all regions, or Schirmer test score (STS) ≤10 mm; there were no restrictions on eye dryness score (EDS). Patients (N = 165) were randomly assigned 1:1:1:1 to vehicle (control; n = 42) or OC-02 SNS (0.11 mg, 0.55 mg, or 1.1 mg; n = 41 per group) and received a single dose of study drug (100 µL using a nasal spray atomizer) at visit 1 and visit 2 (15–19 days after visit 1). Primary efficacy outcomes were change in the STS from baseline to immediately after treatment administration (visit 1) and change in the EDS from before to 5 minutes after treatment during controlled adverse environment exposure (visit 2). FindingsBaseline demographic and ocular clinical characteristics were similar across all groups. Single-dose OC-02 SNS improved the signs and symptoms of dry eye disease. For the STS, statistically significant and dose-dependent improvements were found from before to after treatment with OC-02 SNS versus vehicle (least-squares mean change from baseline: vehicle, 3.0 mm; 0.11 mg OC-02 SNS, 9.0 mm; 0.55 mg, 17.5 mm; and 1.1 mg, 19.6 mm). For EDS, statistically significant and dose-dependent improvements were found from before to 5 minutes after treatment with higher doses of OC-02 SNS versus vehicle (least-squares mean change from baseline: vehicle, –6.5; 0.11 mg OC-02 SNS, –9.4; 0.55 mg, –17.4; and 1.1 mg, –20.7). OC-02 SNS was well tolerated: only 2 ocular adverse events were reported (eye pruritis and keratitis), and the most common nonocular events were cough and throat irritation. ImplicationsSingle-dose OC-02 SNS over a range of doses immediately and significantly increased tear production and improved eye dryness. Together with previous studies of OC-01 (varenicline solution) nasal spray, our findings suggest that agonist stimulation of nAChRs in the nasal cavity is a valid and effective mechanism to elicit natural tear production in patients with dry eye disease. ClinicalTrials.gov identifier: NCT03452397.

Delineating the activity of the potent nicotinic acetylcholine receptor agonists (+)-anatoxin-a and (-)-hosieine-A.

The affinity and thermodynamic parameters for the interactions of two naturally occurring neurotoxins, (+)-anatoxin-a and (-)-hosieine-A, with acetylcholine-binding protein were investigated using a fluorescence-quenching assay and isothermal titration calorimetry. The crystal structures of their complexes with acetylcholine-binding protein from Aplysia californica (AcAChBP) were determined and reveal details of molecular recognition in the orthosteric binding site. Comparisons treating AcAChBP as a surrogate for human α4β2 and α7 nicotinic acetylcholine receptors (nAChRs) suggest that the molecular features involved in ligand recognition and affinity for the protein targets are conserved. The ligands exploit interactions with similar residues as the archetypal nAChR agonist nicotine, but with greater affinity. (-)-Hosieine-A in particular has a high affinity for AcAChBP driven by a favorable entropic contribution to binding. The ligand affinities help to rationalize the potent biological activity of these alkaloids. The structural data, together with comparisons with related molecules, suggest that there may be opportunities to extend the hosieine-A scaffold to incorporate new interactions with the complementary side of the orthosteric binding site. Such a strategy may guide the design of new entities to target human α4β2 nAChR that may have therapeutic benefit.

A Functional Interaction Between Y674-R685 Region of the SARS-CoV-2 Spike Protein and the Human α7 Nicotinic Receptor.

The α7 nicotinic acetylcholine receptor (nAChR) is present in neuronal and non-neuronal cells and has anti-inflammatory actions. Molecular dynamics simulations suggested that α7 nAChR interacts with a region of the SARS-CoV-2 spike protein (S), and a potential contribution of nAChRs to COVID-19 pathophysiology has been proposed. We applied whole-cell and single-channel recordings to determine whether a peptide corresponding to the Y674-R685 region of the S protein can directly affect α7 nAChR function. The S fragment exerts a dual effect on α7. It activates α7 nAChRs in the presence of positive allosteric modulators, in line with our previous molecular dynamics simulations showing favourable binding of this accessible region of the S protein to the nAChR agonist binding site. The S fragment also exerts a negative modulation of α7, which is evidenced by a profound concentration-dependent decrease in the durations of openings and activation episodes of potentiated channels and in the amplitude of macroscopic responses elicited by ACh. Our study identifies a potential functional interaction between α7 nAChR and a region of the S protein, thus providing molecular foundations for further exploring the involvement of nAChRs in COVID-19 pathophysiology.Supplementary InformationThe online version contains supplementary material available at 10.1007/s12035-022-02947-8.

N-Hetaryl-[2(1H)-pyridinyliden]cyanamides: A New Class of Systemic Insecticides.

The new chemical class N-hetaryl-[2(1H)-pyridinylidene]cyanamides were inspired by the long known five-ring structure 2-chloro-5-[2-(nitro-methylene)-1-imidazolidinyl]-pyridine (Shell) and the current development candidate flupyrimin (Meiji Seika Pharma) via scaffold hopping and the concept for designing "shortened structures" by omitting the "methylene link" as a structural feature. The most active N-hetaryl-[2(1H)-pyridinylidene]cyanamides can be synthesized on a technical scale by a simple manufacturing procedure. As full nicotinic acetylcholine receptor (nAChR) agonists, the compounds bind with low affinity at the orthosteric binding site of nAChR. In molecular modeling studies, structural differences are visible in the superposition of active N-[6'-(trifluoromethyl)[1(2H),3'-bipyridin]-2-ylidene]cyanamide onto imidacloprid (IMD) and sulfoxaflor (SXF) in bound conformation. On the basis of their physicochemical properties, the most active xylem systemic candidates offer excellent aphicidal activity in vegetables and cotton, when applied as a foliar spray, by soil drench application, or, in particular, as seed dressing for seed treatment uses. Selected candidates show good plant compatibility and reveal a better risk profile with respect to bee pollinators than the majority of currently registered nAChR competitive modulators for seed treatment uses. Applied as a seed dressing in greenhouse profiling, good to excellent control of different aphid species has been observed. In field trials, an interesting level of activity potential against cereal grain aphids (inclusive virus vector control), corn rootworm, and wireworm could be demonstrated. According to molecular modeling investigations (Fukui functions, dipole moments, and electrostatic potentials), there is a broad scope for structure optimization of the chemical class leading to proposals for novel bicyclic insecticides.

Potency matters: Impacts of embryonic exposure to nAChR agonists thiamethoxam and nicotine on hatching success, growth, and neurobehavior in larval zebrafish

ABSTRACT Thiamethoxam (TM) is a neonicotinoid insecticide that acts as a nicotinic acetylcholine receptor (nAChR) agonist. While designed to specifically target invertebrate nAChRs, recent studies have reported adverse effects of neonicotinoid exposure in early life-stage fish. This study examined the health and neurobehavioral impacts of chronic exposure to various concentrations of TM or nicotine (NIC) in early life zebrafish (Danio rerio) in conjunction with in-silico molecular docking to compare their ligand–receptor interactions with vertebrate nAChR. Chronic exposure to both reduced survival by approximately 20% (163 µg TM/l) and 25–100% (≥0.49 µg NIC/l). Hatching and growth were impaired following exposure to ≥0.21 µg TM/l or 4.9 µg NIC/l. Both TM and NIC produced morphological and behavioral indicators of neurotoxicity, with more potent effects following NIC exposure. NIC impaired embryonic motor activity by 40% (49 µg NIC/l), while both TM and NIC significantly altered predator escape response in larvae, specifically the latency and the initial burst movement of the response were impacted. Molecular docking predicted variations in the type and strength of interactions that occur between NIC or TM and vertebrate nAChR. These findings demonstrate that chronic exposure to TM might impact general health and neurobehavior of early-stage zebrafish. Our data support hypotheses that TM presents low affinity for vertebrate nAChR but may still pose an adverse risk to larval fish growth and neurobehavior.

Activation of α7 nicotinic acetylcholine receptor promotes HIV-1 transcription.

Alpha7 nicotinic acetylcholine receptor (α7 nAChR), a hub of the cholinergic anti-inflammatory pathway (CAP), is required for the treatment of inflammatory diseases. HIV-1 infection can upregulate the expression of α7 nAChR in T lymphocytes and affect the role of CAP. However, whether α7 nAChR regulates HIV-1 infection in CD4+ T cells is unclear. In this study, we first found that activation of α7 nAChR by GTS-21 (an α7 nAChR agonist) can promote the transcription of HIV-1 proviral DNA. Then, through transcriptome sequencing analysis, we found that p38 MAPK signaling was enriched in GTS-21 treated HIV-latent T cells. Mechanistically, activation of α7 nAChR could increase reactive oxygen species (ROS), reduce DUSP1 and DUSP6, and consequently enhance the phosphorylation of p38 MAPK. By co-immunoprecipitation and liquid chromatography tandem mass spectrometry, we found that p-p38 MAPK interacted with Lamin B1 (LMNB1). Activation of α7 nAChR increased the binding between p-p38 MAPK and LMNB1. We confirmed that knockdown of MAPK14 significantly downregulated NFATC4, a key activator of HIV-1 transcription. Taken together, activation of the α7 nAChR could trigger ROS/p-p38 MAPK/LMNB1/NFATC4 signaling pathway enhancing HIV-1 transcription. We have revealed an unrecognized mechanism of α7 nAChR-mediated neuroimmune regulation of HIV infection.

Sex‐specific cholinergic regulation of dopamine release mechanisms through nicotinic receptors in the nucleus accumbens

Biological sex has been pinpointed as significant biological factor impacting the prevalence and prognosis of Substance Use Disorder (SUD). Despite the overwhelming utilization of male subjects in SUD research, epidemiological evidence shows that women are the most susceptible population. At the hub of female vulnerability to SUD is significant dysfunction in the mesolimbic dopamine system connecting the ventral tegmental area (VTA) to the nucleus accumbens (NAc). Importantly, an important characteristic of dopamine release from axon terminals in the NAc is that it is rapidly modulated by local regulatory microcircuit mechanisms independent of somatic activity in the VTA. In the NAc, dopamine is released in tonic (slow and regular) and phasic (short, burst/spikes) patterns that are subject to heavy modulation by cholinergic (ChAT) interneurons signaling through α4β2*‐containing nicotinic acetylcholine receptors (nAChRs) located directly on dopamine terminals. Previous work suggests that ChAT regulation of dopamine release through nAChRs is fundamentally different between males and females, yet the processes and mechanisms that underlie these sex differences are largely unknown. In this project, we took a multifaceted approach to determine sex‐dependent neurochemical mechanisms that underlie ChAT regulation of dopamine release through nAChRs in male, naturally cycling and ovariectomized (OVX) female mice. Using ex vivo fast scan cyclic voltammetry (FSCV) paired with pharmacological applications, we found that ChAT regulation of dopamine release through α4β2*‐nAChRs is not present in female mice under most conditions. Deficits in nAChR modulation of dopamine release in intact females were not affected by the estrous cycle; however, they were rescued by ovariectomy – indicating that ovarian hormones play significant a role in this process. Critically, we find that 17β−Estradiol (E2) increases dopamine release acutely, an effect that is blocked by antagonism of α4β2*‐nAChRs. Additionally, we observed reduced nAChR agonist effects on dopamine release in females; which is what would be expected for desensitized receptors. Finally, Gq‐DREADD behavior studies revealed that male mice learned at a faster rate than intact females when ChAT interneurons were activated. Overall, we show that circulating ovarian hormones, regardless of the hormone cycle, alter the ability of α4β2*‐nAChRs on dopamine terminals to modulate dopamine release in the NAc. This suggests that sex differences in ChAT regulation of dopamine neurotransmission underlies sex‐dependent differentiation in reward learning. Moving forward, it will be critical to directly link these sex differences to reward learning for the development of sex‐specific pharmacotherapies to treat SUD.

Anti-Inflammatory and Neuroprotective Mechanisms of GTS-21, an α7 Nicotinic Acetylcholine Receptor Agonist, in Neuroinflammation and Parkinson’s Disease Mouse Models

Neuroinflammation is crucial in the progression of neurodegenerative diseases. Thus, controlling neuroinflammation has been proposed as an important therapeutic strategy for neurodegenerative disease. In the present study, we examined the anti-inflammatory and neuroprotective effects of GTS-21, a selective α7 nicotinic acetylcholine receptor (α7 nAChR) agonist, in neuroinflammation and Parkinson’s disease (PD) mouse models. GTS-21 inhibited the expression of inducible nitric oxide synthase (iNOS) and proinflammatory cytokines in lipopolysaccharide (LPS)-stimulated BV2 microglial cells and primary microglia. Further research revealed that GTS-21 has anti-inflammatory properties by inhibiting PI3K/Akt, NF-κB, and upregulating AMPK, Nrf2, CREB, and PPARγ signals. The effects of GTS-21 on these pro-/anti-inflammatory signaling molecules were reversed by treatment with an α7 nAChR antagonist, suggesting that the anti-inflammatory effects of GTS-21 are mediated through α7 nAChR activation. The anti-inflammatory and neuroprotective properties of GTS-21 were then confirmed in LPS-induced systemic inflammation and MPTP-induced PD model mice. In LPS-injected mouse brains, GTS-21 reduced microglial activation and production of proinflammatory markers. Furthermore, in the brains of MPTP-injected mice, GTS-21 restored locomotor activity and dopaminergic neuronal cell death while inhibiting microglial activation and pro-inflammatory gene expression. These findings suggest that GTS-21 has therapeutic potential in neuroinflammatory and neurodegenerative diseases such as PD.

Synthetic Analogs of 6-Bromohypaphorine, a Natural Agonist of Nicotinic Acetylcholine Receptors, Reduce Cardiac Reperfusion Injury in a Rat Model of Myocardial Ischemia

The data available to date indicate that the activation of nicotinic acetylcholine receptors (nAChR) of α7 type can reduce heart damage resulting from ischemia and subsequent reperfusion. We have studied two new synthetic D-analogs of 6-bromohypaphorine, which are selective agonists of α7 nAChR, in a rat model of myocardial ischemia. Acute myocardial infarction in animals was induced by occlusion of the left coronary artery with its subsequent reperfusion under mechanical lung ventilation. It was found that one of the analogs was more active, and treatment with it at the onset of reperfusion statistically reduced infarct size. This analog also prevented changes in the concentration of potassium and sodium ions in the blood, occurring during occlusion/reperfusion injury. The data obtained indicate that hypaphorine analogs are promising for the development of drugs that reduce the adverse effects of myocardial infarction.