Interactions of Nereistoxin and Its Analogs with Vertebrate Nicotinic Acetylcholine Receptors and Molluscan ACh Binding Proteins

Nereistoxin (NTX) is a marine toxin isolated from an annelid worm that lives along the coasts of Japan. Its insecticidal properties were discovered decades ago and this stimulated the development of a variety of insecticides such as Cartap that are readily transformed into NTX. One unusual feature of NTX is that it is a small cyclic molecule that contains a disulfide bond. In spite of its size, it acts as an antagonist at insect and mammalian nicotinic acetylcholine receptors (nAChRs). The functional importance of the disulfide bond was assessed by determining the effects of inserting a methylene group between the two sulfur atoms, creating dimethylaminodithiane (DMA-DT). We also assessed the effect of methylating the NTX and DMA-DT dimethylamino groups on binding to three vertebrate nAChRs. Radioligand receptor binding experiments were carried out using washed membranes from rat brain and fish (Torpedo) electric organ; [3H]-cytisine displacement was used to assess binding to the predominantly high affinity alpha4beta2 nAChRs and [125I]-alpha-bungarotoxin displacement was used to measure binding of NTX and analogs to the alpha7 and skeletal muscle type nAChRs. While the two quaternary nitrogen analogs, relative to their respective tertiary amines, displayed lower α4β2 nAChR binding affinities, both displayed much higher affinities for the Torpedo muscle nAChR and rat alpha7 brain receptors than their respective tertiary amine forms. The binding affinities of DMA-DT for the three nAChRs were lower than those of NTX and MeNTX. An AChBP mutant lacking the C loop disulfide bond that would potentially react with the NTX disulfide bond displayed an NTX affinity very similar to the parent AChBP. Inhibition of [3H]-epibatidine binding to the AChBPs was not affected by exposure to NTX or MeNTX for up to 24 hr prior to addition of the radioligand. Thus, the disulfide bond of NTX is not required to react with the vicinal disulfide in the AChBP C loop for inhibition of [3H]-epibatidine binding. However, a reversible disulfide interchange reaction of NTX with nAChRs might still occur, especially under reducing conditions. Labeled MeNTX, because it can be readily prepared with high specific radioactivity and possesses relatively high affinity for the nAChR-rich Torpedo nAChR, would be a useful probe to detect and identify any nereistoxin adducts.

Cytisine Exerts an Anti-Epileptic Effect via α7nAChRs in a Rat Model of Temporal Lobe Epilepsy

Background and Purpose: Temporal lobe epilepsy (TLE) is a common chronic neurological disease that is often invulnerable to anti-epileptic drugs. Increasing data have demonstrated that acetylcholine (ACh) and cholinergic neurotransmission are involved in the pathophysiology of epilepsy. Cytisine, a full agonist of α7 nicotinic acetylcholine receptors (α7nAChRs) and a partial agonist of α4β2nAChRs, has been widely applied for smoking cessation and has shown neuroprotection in neurological diseases. However, whether cytisine plays a role in treating TLE has not yet been determined.Experimental Approach: In this study, cytisine was injected intraperitoneally into pilocarpine-induced epileptic rats for three weeks. Alpha-bungarotoxin (α-bgt), a specific α7nAChR antagonist, was used to evaluate the mechanism of action of cytisine. Rats were assayed for the occurrence of seizures and cognitive function by video surveillance and Morris water maze. Hippocampal injuries and synaptic structure were assessed by Nissl staining and Golgi staining. Furthermore, levels of glutamate, γ-aminobutyric acid (GABA), ACh, and α7nAChRs were measured.Results: Cytisine significantly reduced seizures and hippocampal damage while improving cognition and inhibiting synaptic remodeling in TLE rats. Additionally, cytisine decreased glutamate levels without altering GABA levels, and increased ACh levels and α7nAChR expression in the hippocampi of TLE rats. α-bgt antagonized the above-mentioned effects of cytisine treatment.Conclusion and Implications: Taken together, these findings indicate that cytisine exerted an anti-epileptic and neuroprotective effect in TLE rats via activation of α7nAChRs, which was associated with a decrease in glutamate levels, inhibition of synaptic remodeling, and improvement of cholinergic transmission in the hippocampus. Hence, our findings not only suggest that cytisine represents a promising anti-epileptic drug, but provides evidence of α7nAChRs as a novel therapeutic target for TLE.

Trifluoro-Icaritin Ameliorates Complete Freund’s Adjuvant-Induced Inflammatory Pain by α7nAChR-Mediated Inhibition of HMGB1/NF-κB Signaling in the Spinal Cord of Rats

Objectives: To explore whether Trifluoro-icaritin (ICTF) has anti-nociceptive effect on CFA-induced inflammatory pain and its potential mechanisms. Methods: Intraperitoneal injection (0.3, 1.0, and 3.0 mg/kg, i.p.) of ICTF to complete Freund’s adjuvant (CFA)-induced inflammatory pain rats once daily for 21 consecutive days. Pain-related behaviors were evaluated with paw withdrawal threshold (PWT), paw withdrawal latency (PWL), and CatWalk gait analysis. Hematoxylin eosin (HE) staining was applied to determine the morphological alterations in inflamed paw. Molecular docking was conducted to assess the possible targets for ICTF. Expression of pain-related signaling molecules in the spinal cord were detected using qRT-PCR, western blot assay, and immunofluorescence staining. Results:This results showed that ICTF(3.0 mg/kg) effectively alleviated mechanical allodynia and thermal hyperalgesiabut not 0.3 and 1.0 mg/kg in CFA rats. Both paw edema volume and paw tissue inflammatory response were obviously reduced by ICTF. Subsequently, we further observed that ICTF dramatically decreased the mRNA and protein levels of HMGB1, NF-κB p65, and IL-1β but markedly enhanced α7nAChR and IL-10 expression in the spinal cord of CFA rats, and inhibitedthe co-expression of spinal α7nAChR with IBA-1 in double immunofluorescence staining,along with suppressing the alterations of gait parameters induced by CFA. Moreover, Intrathecal injection (i.t.) of α7nAChR antagonist alpha-bungarotoxin (α-Bgtx, 1.0 μg/kg) not only reversed the anti-nociceptive effect of ICTF on pain hypersensitivity, but also inhibited the down-regulation of HMGB1, NF-κB p65, and IL-1β as well as the up-regulation of α7nAChR and IL-10 protein expression induced by ICTF treatment.Conclusion: Our results illustrate that ICTF enables to alleviate CFA-induced inflammatory pain through α7nAChR-mediated inhibition of HMGB1/NF-κB signaling pathway in the spinal cord of rats, suggesting that ICTF may be exploited as a potential painkiller against chronic inflammatory pain.

Nicotine Regulates Autophagy of Human Periodontal Ligament Cells Through α7 nAchR That Promotes Secretion of Inflammatory Factors IL-1β and IL-8

Backgroud: Nicotine is an important risk factor and the main toxic component associated with periodontitis. However, the mechanism of nicotine induced periodontitis is not clear. To investigated the mechanism through which nicotine regulates autophagy of human periodontal ligament cells (hPDLCs) through the alpha7 nicotinic acetylcholine receptor (α7 nAChR) and how autophagy further regulates the release of IL-1β and IL-8 secretion in hPDLCs. Methods: HPDLCs were obtained from root of extracted teeth and pre-incubated in alpha-bungarotoxin (α-BTX) or 3-Methyladenine (3-MA), followed by culturing in nicotine. We used a variety of experimental detection techniques including western blotting, immunofluorescence, enzyme-linked immunosorbent assay (ELISA), transmission electron microscopy (TEM) and RT-qPCR to assess the expression of the LC3 protein, autolysosome, and release of IL-1β and IL-8 from hPDLCs. Results: Western blots, immunofluorescence and TEM results found that the nicotine significantly increased the autophagy expression in hPDLCs that was time and concentration dependent and reversed by α-BTX treatment (p﹤0.05). RT-qPCR and ELISA results revealed a noticeable rise in the release of inflammatory factors IL–1β and IL-8 from hPDLCs in response to nicotine. RT-qPCR and ELISA results showed that nicotine can significantly up-regulate the release of inflammatory factors IL-1β and IL-8 in hPDLCs, and this effect can be inhibited by 3-MA (p﹤0.05).Conclusions: Nicotine regulated autophagy of hPDLCs through α7 nAChR and in turn the regulation of the release of inflammatory factors 1L-1β and 1L-8 by hPDLCs.

Street RABV Induces the Cholinergic Anti-inflammatory Pathway in Human Monocyte-Derived Macrophages by Binding to nAChr α7

Rabies virus (RABV) is able to reach the central nervous system (CNS) without triggering a strong immune response, using multiple mechanisms to evade and suppress the host immune system. After infection via a bite or scratch from a rabid animal, RABV comes into contact with macrophages, which are the first antigen-presenting cells (APCs) that are recruited to the area and play an essential role in the onset of a specific immune response. It is poorly understood how RABV affects macrophages, and if the interaction contributes to the observed immune suppression. This study was undertaken to characterize the interactions between RABV and human monocyte-derived macrophages (MDMs). We showed that street RABV does not replicate in human MDMs. Using a recombinant trimeric RABV glycoprotein (rRABV-tG) we showed binding to the nicotinic acetylcholine receptor alpha 7 (nAChr α7) on MDMs, and confirmed the specificity using the nAChr α7 antagonist alpha-bungarotoxin (α-BTX). We found that this binding induced the cholinergic anti-inflammatory pathway (CAP), characterized by a significant decrease in tumor necrosis factor α (TNF-α) upon LPS challenge. Using confocal microscopy we found that induction of the CAP is associated with significant cytoplasmic retention of nuclear factor κB (NF-κB). Co-cultures of human MDMs exposed to street RABV and autologous T cells further revealed that the observed suppression of MDMs might affect their function as T cell activators as well, as we found a significant decrease in proliferation of CD8+ T cells and an increased production of the anti-inflammatory cytokine IL-10. Lastly, using flow cytometric analysis we observed a significant increase in expression of the M2-c surface marker CD163, hinting that street RABV might be able to affect macrophage polarization. Taken together, these results show that street RABV is capable of inducing an anti-inflammatory state in human macrophages, possibly affecting T cell functioning.

Molecular Modelling and Docking Experiments Examining the Interaction between SARS-CoV-2 Spike Glycoprotein and Neuronal Nicotinic Acetylcholine Receptors

While SARS-CoV-2 uses angiotensin converting enzyme 2 (ACE2) as the receptor for cell entry, it is important to examine for other potential interactions between the virus and other cell receptors. Based on the clinical observation of low smoking prevalence among hospitalized COVID-19 patients, we recently identified a “toxin-like” amino acid (aa) sequence on the receptor binding domain of the spike glycoprotein of SARS-CoV-2 (aa 375-390) with homology to a sequence of a snake venom toxin, which could interact with nicotinic acetylcholine receptors (nAChRs). We now present computational molecular modelling and docking experiments using 3D structures of the SARS-CoV-2 spike glycoprotein and the extracellular domain of the nAChR alpha9 subunit. We identified an interaction between the aa381-386 of the SARS-CoV-2 spike glycoprotein and the aa189-192 of the extracellular domain of the nAChR alpha9 subunit, a region which forms the core of the “toxin-binding site” of the nAChRs. The mode of interaction is very similar to the interaction between the alpha9 nAChR and alpha-bungarotoxin. A similar interaction was observed between the pentameric alpha7 AChR and the SARS-CoV-2 spike glycoprotein. Our findings support the hypothesis that severe COVID-19 may be associated with disruption of the nicotinic cholinergic system which could be caused by an interaction between SARS-CoV-2 and nAChRs.

Why Does Knocking Out NACHO, But Not RIC3, Completely Block Expression of α7 Nicotinic Receptors in Mouse Brain?

Alpha7 nicotinic acetylcholine receptors (α7nAChRs) are interesting not only because of their physiological effects, but because this receptor requires chaperones to traffic to cell surfaces (measured by alpha-bungarotoxin [αBGT] binding). While knockout (KO) animals and antibodies that react across species exist for tmem35a encoding the protein chaperone NACHO, commercially available antibodies against the chaperone RIC3 that allow Western blots across species have not been generally available. Further, no effects of deleting RIC3 function (ric3 KO) on α7nAChR expression are reported. Finally, antibodies against α7nAChRs have shown various deficiencies. We find mouse macrophages bind αBGT but lack NACHO. We also report on a new α7nAChR antibody and testing commercially available anti-RIC3 antibodies that react across species allowing Western blot analysis of in vitro cultures. These antibodies also react to specific RIC3 splice variants and single-nucleotide polymorphisms. Preliminary autoradiographic analysis reveals that ric3 KOs show subtle αBGT binding changes across different mouse brain regions, while tmem35a KOs show a complete loss of αBGT binding. These findings are inconsistent with effects observed in vitro, as RIC3 promotes αBGT binding to α7nAChRs expressed in HEK cells, even in the absence of NACHO. Collectively, additional regulatory factors are likely involved in the in vivo expression of α7nAChRs.

Mechanical Stress Modulates the RANKL/OPG System of Periodontal Ligament Stem Cells via α7 nAChR in Human Deciduous Teeth: An In Vitro Study

The aim of this study was to investigate the mechanism by which periodontal ligament stem cells (PDLSCs) modulate root resorption of human deciduous teeth under mechanical stress. In this investigation, the PDLSCs were derived from deciduous and permanent teeth at different stages of root resorption. A cyclic hydraulic pressure was applied on the PDLSCs to mimic chewing forces in the oral environment. The cultured cells were characterized using osteogenic and adipogenic differentiation assays, quantitative real-time polymerase chain reaction (qRT-PCR), and Western blotting analysis. The PDLSCs exhibited the ability to induce osteoclast differentiation under certain mechanical stresses. As the expressions of RUNX2, alkaline phosphatase (ALP), and osteoprotegerin (OPG) were significantly reduced, the receptor activator of the nuclear factor kappa-B ligand (RANKL) was upregulated increasing the RANKL/OPG ratio. Under hydrodynamic pressure at 0-135 kPa, the expressions of alpha 7 nicotinic acetylcholine receptors (α7 nAChR), p-GSK-3β, and active-β-catenin were markedly upregulated in PDLSCs from unresorbed deciduous teeth. Treatment with the α7 nAChR inhibitor alpha-bungarotoxin (α-BTX) and the Wnt pathway inhibitor DKK1 may reverse the mechanical stress inducing upregulation of RANKL and reduction of RUNX2, ALP, and OPG. Alizarin red staining confirmed these results. The mechanical stress applied on the deciduous tooth PDLSCs can induce osteoclastic effects through upregulation of α7 nAChR and activation of the canonical Wnt pathway. It can be suggested that chewing forces may play a major role at the beginning of the physiological root resorption of deciduous teeth.

Atrophy, inducible satellite cell activation, and possible denervation of supraspinatus muscle in injured human rotator-cuff muscle

The high frequency of poor outcome and chronic pain after surgical repair of shoulder rotator-cuff injury (RCI) prompted this study to explore the potential to amplify muscle regeneration using nitric oxide (NO)-based treatment. After preoperative magnetic resonance imaging (MRI), biopsies of supraspinatus and ipsilateral deltoid (as a control) were collected during reparative surgery for RCI. Muscle fiber diameter, the pattern of neuromuscular junctions observed with alpha-bungarotoxin staining, and the γ:ε subunit ratio of acetylcholine receptors in Western blots were examined in tandem with experiments to determine the in vitro responsiveness of muscle satellite cells to activation (indicated by uptake of bromodeoxyuridine, BrdU) by the NO-donor drug, isosorbide dinitrate (ISDN). Consistent with MRI findings of supraspinatus atrophy (reduced occupation ratio and tangent sign), fiber diameter was lower in supraspinatus than in deltoid. ISDN induced a significant increase over baseline (up to 1.8-fold), in the proportion of BrdU+ (activated) Pax7+ satellite cells in supraspinatus, but not in deltoid, after 40 h in culture. The novel application of denervation indices revealed a trend for supraspinatus muscle to have a higher γ:ε subunit ratio than deltoid ( P = 0.13); this ratio inversely with both occupancy ratio ( P < 0.05) and the proportion of clusters at neuromuscular junctions ( P = 0.05). Results implicate possible supraspinatus denervation in RCI and suggest NO-donor treatment has potential to promote growth in atrophic supraspinatus muscle after RCI and improve functional outcome.