Α7 Nicotinic Research Articles

BNC210, an α7 Nicotinic Receptor Modulator, in Post-Traumatic Stress Disorder.

Post-traumatic stress disorder (PTSD) is a serious, debilitating, and prevalent psychiatric condition occurring in people who are traumatized and experience intense, disturbing thoughts and feelings that persist. BNC210 is a novel α7 nicotinic acetylcholine receptor-negative allosteric modulator developed to treat PTSD. ATTUNE was a randomized, double-blind, phase 2b, placebo-controlled trial. Patients between 18 and 75 years of age with a current PTSD diagnosis and a Clinician-Administered PTSD Scale for DSM-5 (Diagnostic and Statistical Manual of Mental Disorders, Fifth Edition) (CAPS-5) total symptom severity score of 30 or more were eligible (range: 0 to 80; in all scales used in this trial a higher score indicates a more severe condition). We randomly assigned patients 1:1 to a BNC210 dose of 900 mg twice daily or placebo for 12 weeks. The primary end point was a change from baseline to week 12 in CAPS-5 total score for BNC210 versus placebo. In the modified intent-to-treat population (n=182), an improvement in the CAPS-5 total score was observed with BNC210 compared with placebo (least squares [LS] mean difference: -4.03; Cohen's d effect size: 0.40; P=0.048) at week 12. A LS mean difference in CAPS-5 score of -4.11 was observed as early as week 4. The LS mean difference to week 12 for depressive symptoms measured on the Montgomery-Åsberg Depression Rating Scale (range: 0 to 60; minimal clinically important difference [MCID] ≥2]) was -3.19 and for sleep measured on the Insomnia Severity Index (range: 0 to 28; MCID 6) was -2.19. Treatment-emergent adverse events (AEs) occurred in 70 (66.7%) patients in the BNC210 group and 56 (53.8%) in the placebo group, most commonly headache, nausea, fatigue, and hepatic enzyme elevations. In the BNC210 treatment group, 21 patients withdrew from treatment for AEs, while 10 did so in the placebo group. There were no serious AEs or deaths reported for the BNC210 group. BNC210 improved PTSD symptom severity at week 12 with indications of effect as early as week 4. Our trial establishes equipoise for additional larger trials that are needed to determine the clinical utility of BNC210 for the treatment of PTSD. (Funded by Bionomics Limited; ClinicalTrials.gov number, NCT04951076.).

Delayed Magnetic Resonance Imaging of Alzheimer's Disease by Using Poly(2-(methacryloyloxy)ethyl phosphorylcholine)-Functionalized Nanoprobes.

Alzheimer's disease (AD) is one of the most common neurodegenerative diseases, commonly affecting the aged, with pathophysiological changes presenting 15 to 20 years before clinical symptoms. Early diagnosis and intervention are crucial in effectively slowing the progression of AD. In the current study, poly(2-(methacryloyloxy)ethyl phosphorylcholine) (PMPC)-functionalized NaGdF4 nanoparticles (NaGdF4-PMPC) were developed as magnetic resonance imaging (MRI) contrast agents for targeting alpha 7 nicotinic acetylcholine receptors (α7 nAChRs) in AD mice. NaGdF4-PMPC showed excellent biocompatibility, targeting ability, and MRI performance, with the longitudinal molar relaxivity (r1) and transverse molar relaxivity (r2) being 1.21-fold and 1.33-fold higher than those of the clinical contrast agent Gd-DTPA, respectively, resulting in higher-sensitive MR angiography. After intravenous injection, 3D dynamic contrast-enhanced (DCE) MR images with high-resolution vasculature of the mouse brain were obtained. In addition, by using NaGdF4-PMPC, susceptibility-weighted imaging (SWI) signals in AD mouse brains were greatly retained compared to those in healthy mice for 24 h, emphasizing the excellent targeting ability of NaGdF4-PMPC. Furthermore, the CD31, α7 nAChRs, and Thioflavin S staining were also utilized to investigate the relationship among vascular inflammation, α7 nAChRs, and amyloid-β (Aβ) deposition in AD mice. This work highlights a promising targeted imaging strategy for the timely diagnosis of AD.

565 The role of the α7 nicotinic acetylcholine receptor in fibrotic endothelial to mesenchymal transition

565 The role of the α7 nicotinic acetylcholine receptor in fibrotic endothelial to mesenchymal transition

Differential participation of CaMKII/ROCK and NOS pathways in the cholinergic inhibitory drive operated by nicotinic α7 receptors in perisynaptic Schwann cells

Nicotinic α7 receptors (α7 nAChRs) present in perisynaptic Schwann cells (PSCs) control acetylcholine (ACh) spillover from the neuromuscular synapse by transiently increasing intracellular Ca2+, which fosters adenosine release via type 1 equilibrative nucleoside transporters (ENT1) and retrograde activation of presynaptic A1 inhibitory receptors. The putative Ca2+-dependent pathways downstream α7 nAChRs involved in the sensing inhibitory drive operated by PSCs is unknown. Herein, we used phrenic nerve-hemidiaphragm preparations from Wistar rats. Time-lapse video-microscopy was instrumental to assess nerve-evoked (50-Hz bursts) transmitter exocytosis and intracellular NO oscillations in nerve terminals and PSCs loaded with FM4-64 and DAF-FM diacetate fluorescent dyes, respectively. Selective activation of α7 nAChRs with PNU 282987 reduced transmitter exocytosis (FM4-64 dye unloading) during 50-Hz bursts. Inhibition of calmodulin activity (with W-7), Ca2+/calmodulin-dependent protein kinase II (CaMKII; with KN-62) and Rho-kinase (ROCK; with H1152) all prevented the release inhibitory effect of PNU 282987. The α7 nAChR agonist transiently increased NO inside PSCs; the same occurred during phrenic nerve stimulation with 50-Hz bursts in the presence of the cholinesterase inhibitor, neostigmine. The nitric oxide synthase (NOS) inhibitor, L-NOARG, but not with the guanylylcyclase (GC) inhibitor, ODQ, prevented inhibition of transmitter exocytosis by PNU 282987. Inhibition of adenosine kinase with ABT 702 favors the intracellular accumulation and translocation of the nucleoside to the synaptic cleft, thus overcoming prevention of the PNU 282987 effect caused by H1152, but not by L-NOARG. In conclusion, the α7nAChR-mediated cholinergic inhibitory drive operated by PSCs involves two distinct Ca2+-dependent intracellular pathways: a CaMKII/ROCK cascade along with a GC-independent NO pathway with divergent end-effects concerning ADK inhibition.

Mechanism of Pyroptosis in Postoperative Cognitive Dysfunction Rats and α7-nicotinic Receptor Agonist Regulating NLRP3 Inflammasome Activation

Background: This study investigates the efficacy of α7-nicotinic acetylcholine receptor (α7nAChR) agonists in mitigating postoperative cognitive dysfunction (POCD) in a rat model. This investigation aimed to elucidate the therapeutic potential of α7nAChR agonists in modulating neuroinflammatory pathways to improve cognitive outcomes post-surgery. Objective: Serum levels of pro-inflammatory cytokines IL-1β and IL-18 were measured as markers of systemic inflammation, while the expression levels of NLRP3 mRNA were quantified to evaluate pyroptosis and NLRP3 inflammasome activation. Methods: Adult male Sprague-Dawley rats were divided into control (no surgery), POCD (surgeryinduced), and POCD treated with an α7nAChR agonist. Cognitive function was assessed using the Morris Water Maze (MWM) and Novel Object Recognition (NOR) tests. Results: The results showed a notable cognitive impairment in the group with POCD, as shown by increased escape latency noted in the MWM test and decreased discrimination index in the NOR test, while controls had no change. However, treatment with the α7nAChR agonist led to a significant improvement in cognitive performance among the POCD rats such that it closely matched those of the controls. Furthermore, the POCD group exhibited elevated serum levels of IL-1β and IL-18 and increased expression of pyroptosis-related markers, indicating enhanced neuroinflammation and inflammasome activation. Conclusion: These findings highlighted the therapeutic efficacy of α7nAChR agonists in mitigating neuroinflammation and improving cognitive outcomes post-surgery. Our study supports the potential of targeting the cholinergic anti-inflammatory pathway, emphasizing clinical evaluation of α7nAChR agonists in postoperative patients.

Salivary peptidomic profiling of chronic gingivostomatitis in cats by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry and nanoscale liquid chromatography-tandem mass spectrometry.

Chronic gingivostomatitis in cats (FCGS) is a moderately to severely painful condition, potentially caused by inadequate immune response to oral antigenic stimulation. Salivary peptidome analysis can identify inflammatory protein mediators and pathways involved in oral mucosal immune activation and may indicate potential therapeutic options for FCGS. Evaluate the diversity and abundance of salivary peptides in cats with FCGS using matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) and nanoscale liquid chromatography-tandem mass spectrometry (nano LC-MS/MS). Thirty-two cats with FCGS and 18 healthy controls. Case-control cross-sectional study. We compared the salivary peptide profiles of diseased and healthy cats. The diagnosis of FCGS was confirmed by histopathology. Saliva samples were analyzed for viral infections using polymerase chain reaction (PCR), peptide mass fingerprint (PMF) using MALDI-TOF MS, and peptide identification using nano LC-MS/MS. Distinct clusters of peptide profiles were observed between groups. In FCGS, 26 salivary peptides were altered, including apolipoprotein A1, nuclear receptor subfamily 1 group I member 3, fibrinogen alpha chain, interleukin 2 receptor gamma, interleukin 23 receptor, hemoglobin subunit alpha, and serpin peptidase inhibitor clade A (alpha-1 antiproteinase, antitrypsin) member 12, protein-tyrosine-phosphatase, and cholinergic receptor nicotinic alpha 10 subunit. Protein-anti-inflammatory drug interaction networks were observed. Peptide mass fingerprint and peptide profiles identified distinct clusters between FCGS and healthy cats. The 9 novel salivary peptide markers were associated with the JAK/STAT and PI3K/Akt pathways and immune responses. These potentially noninvasive biomarkers may facilitate understanding of FCGS pathophysiology and guide future therapeutic research.

Targeting the cholinergic anti-inflammatory pathway for type 2 diabetes prevention: A retrospective cohort study.

Chronic inflammation is a key factor in type 2 diabetes mellitus (T2DM) development. The cholinergic anti-inflammatory pathway (CAP) reduces inflammation by activating α7 nicotinic acetylcholine receptors (α7nAChRs) on macrophages, suppressing proinflammatory cytokines. Acetylcholinesterase inhibitors (AChEis), primarily used for Alzheimer's disease (AD), may exert anti-inflammatory effects through the CAP. One AChEi, galantamine, also directly agonizes α7nAChRs, potentially enhancing its anti-inflammatory properties. This study aimed to investigate the association between AChEi use, particularly galantamine, and T2DM risk in AD patients. We conducted a retrospective analysis of Veterans Health Administration (VA) data, examining early- and late-onset AD patients receiving galantamine or other AD medications. Propensity score matching was used to balance groups and minimize confounding. Cox proportional hazard models assessed T2DM risk for galantamine, other AChEis and memantine. A total of 40 065 AD patients were included in the study. Among early-onset AD patients, galantamine use significantly reduced T2DM risk (hazard ratio [HR] = 0.80, 95% confidence interval [CI]: 0.66-0.98). Memantine also showed a protective effect (HR = 0.82, 95% CI: 0.69-1) in this group. Neither galantamine nor memantine influenced T2DM risk in late-onset AD. Other AD medications showed no association with T2DM risk. Galantamine use was associated with a lower risk of T2DM in early-onset AD patients, potentially due to enhanced anti-inflammatory effects through both AChE inhibition and direct α7nAChR agonism. Memantine also demonstrated a protective effect. These findings suggest potential new applications for existing AD medications in T2DM prevention, particularly in early-onset AD patients. Further research, including randomized controlled trials with diverse populations, is needed to confirm these results and the underlying mechanisms.

Activation of α7 nicotinic acetylcholine receptor augments nerve growth factor action on PCtrk cells

Although cigarette smoking is known to be a critical risk factor for various organ systems and cancers, accumulating evidence indicates that nicotine – a main constituent of cigarette smoking – can exert neuroprotective effects on neuronal cells through nicotinic acetylcholine receptors (nAChRs). However, the precise molecular mechanisms for nicotinic neuroprotective actions remain to be fully elucidated. In this study, we examine the effects of agonists, such as nicotine and PNU282987, on tropomyosin-related kinase (Trk)-dependent neuroprotective pathways in PC12 cells overexpressing a Trk neurotrophin receptor (PCtrk cells). We found that even considerably higher concentrations (mM range for nicotine and µM range for PN282987) of nAChR agonists exert favorable effects, such as the augmentation of nerve growth factor (NGF)-induced Trk neurotrophin receptor autophosphorylation of tyrosine residues and NGF-induced neurite extension. Moreover, nicotine upregulated reactive oxygen species (ROS) levels in the cells. ROS production was completely cancelled by pretreatment with Mito-Tempo, a mitochondria-targeted antioxidant, indicating that the main source of ROS production by nicotine was mitochondria. Furthermore, treatment with nAChR agonists appeared to induce autophagic flux, as evidenced by the upregulation of LC3-II expression in cells. Furthermore, sucrose density ultracentrifugation of nicotine-treated cells clearly disclosed the augmented recruitment of α7nAChR protein into the lipid rafts fraction of the membrane. Intriguingly, a pull-down assay of anti-Trk antibody immunoprecipitates clearly included α7nAChR protein, indicating that Trk and α7nAChR proteins form a complex. These results reveal a new molecular interaction between activated α7nAChR and Trk protein that may serve as a new molecular basis of nicotine-induced neuroprotective action.

Dampened α7 nAChR activity contributes to audiogenic seizures and hyperactivity in a mouse model of Fragile X Syndrome.

Fragile X Syndrome (FXS) is the most common form of inherited intellectual disability and often accompanied with debilitating pathologies including seizures and hyperactivity. FXS arises from a trinucleotide repeat expansion in the 5' UTR of the FMR1 gene that silences expression of the RNA-binding protein FMRP. Despite progress in understanding FMRP functions, the identification of effective therapeutic targets has lagged and at present there are no viable treatment options. Here we identify the α7 nicotinic acetylcholine receptor (nAChR) as candidate target for intervention in FXS. In the early postnatal hippocampus of Fmr1 knockout (KO) mice, an established pre-clinical model of FXS, the α7 nAChR accessory protein Ly6H is abnormally enriched at the neuronal surface and mislocalized in dendrites. Ly6H, a GPI-anchored protein, binds α7 nAChRs with high affinity and can limit α7 nAChR surface expression and signaling. We find that α7 nAChR-evoked Ca2+ responses are dampened in immature glutamatergic and GABAergic Fmr1 KO neurons compared to wild type. Knockdown of endogenous Ly6H in Fmr1 KO neurons is sufficient to rescue dampened α7 nAChR Ca2+ responses in vitro, providing evidence of a cell-autonomous role for Ly6H aberrant expression in α7 nAChR hypofunction. In line with intrinsic deficits in α7 nAChR activity in Fmr1 KO neurons, in vivo administration of the α7 nAChR-selective positive allosteric modulator PNU-120596 reduced hyperactivity and seizure severity in adolescent Fmr1 KO mice. Our mechanistic studies together with evidence of the in vivo efficacy of α7 nAChR augmentation implicate α7 nAChR hypofunction in FXS pathology.

Nicotine promotes M2 macrophage polarization through α5-nAChR/SOX2/CSF-1 axis in lung adenocarcinoma

α5-nicotinic acetylcholine receptor (α5-nAChR) plays a vital part in lung adenocarcinoma (LUAD). However, it is not comprehensively understood that how the α5-nAChR affects LUAD. Through diverse bioinformatics analyses and immunohistochemistry, the expressions of α5-nAChR and SOX2 as well as their relations were dissected. α5-nAChR regulated the differentiation of monocytes into M2 macrophages by targeting the STAT3/SOX2/CSF-1 signaling in the coculture system by western blotting and ChIP. α5-nAChR-mediated macrophage-mediated LUAD cell migration via SOX2/CSF-1 signaling in the cocultured medium. Correlations of α5-nAChR, SOX2 and M2 phenotype tumor-associated macrophages (TAMs) were validated in mouse LUAD models and clinical samples. α5-nAChR expression was connected to SOX2 expression, smoking and bad prognosis of LUAD among clinical samples. Nicotine-induced SOX2 expression was mediated by α5-nAChR via STAT3. Additionally, SOX2-mediated macrophage colony-stimulating factor (CSF-1) expression contributed to LUAD progression in vitro. Furthermore, α5-nAChR expression was strongly linked to pSTAT3, SOX2 and M2 macrophage marker CD206 expression and negatively correlated with M1 macrophage marker CD86 expression in vivo. It is indicated that M2 macrophages are mediated by the new α5-nAChR /SOX2/CSF-1 axis in nicotine-related LUAD, which is a potential therapeutic strategy for cancer.

The angiogenic role of the alpha 9-nicotinic acetylcholine receptor in triple-negative breast cancers.

Nicotine acts as an angiogenic factor by stimulating endogenous cholinergic pathways. Several subtypes of nicotinic acetylcholine receptors (nAChRs) have been demonstrated to be closely correlated to the formation and progression of different types of cancers. Recently, several studies have found that nicotinic acetylcholine receptors α9 (α9-nAChRs) are highly expressed in breast tumors, especially in tumors derived from patients diagnosed at advanced stages. In vitro studies have demonstrated that activation of α9-nAChRs is associated with increased proliferation and migration of breast cancer. To study the tumor-promoting role of α9-nAChRs in breast cancers, we generated a novel anti-α9-nAChR and methoxy-polyethylene glycol (mPEG) bispecific antibody (α9 BsAb) for dissecting the molecular mechanism on α9-nAChR-mediated tumor progression. Unexpectedly, we discovered the angiogenic role of α9-nAChR in nicotine-induced neovascularization of tumors. It revealed α9 BsAbs reduced nicotine-induced endothelial cell tube formation, blood vessel development in Matrigel plug assay and angiogenesis in microtube array membrane murine model (MTAMs). To unbraid the molecular mechanism of α9-nAChR in nicotine-mediated angiogenesis, the α9 BsAbs were applied and revealed the inhibitory roles in nicotine-induced production of hypoxia-inducible factor-2 alpha (HIF-2α), vascular endothelial growth factor A (VEGF-A), phosphorylated vascular endothelial growth factor receptor 2 (p-VEGFR2), vascular endothelial growth factor receptor 2 (VEGFR2) and matrix metalloproteinase-9 (MMP9) from triple-negative breast cancer cells (MDA-MB-231), suggesting α9-nAChRs played an important role in nicotine-induced angiogenesis. To confirm our results, the shRNA targeting α9-nAChRs was designed and used to silence α9-nAChR expression and then evaluated the angiogenic role of α9-nAChRs. The results showed α9 shRNA also played an inhibitory effect in blocking the nicotine-induced angiogenic signaling. Taken together, α9-nAChR played a critical role in nicotine-induced angiogenesis and this bispecific antibody (α9 BsAb) may serve as a potential therapeutic candidate for treatments of the α9 positive cancers.

Ethyl gallate ameliorates diabetes-induced Alzheimer's disease-like phenotype in rats via activation of α7 nicotinic receptors and mitigation of oxidative stress

Cognitive decline, an important comorbidity of type 2 diabetes (T2D), is attributed to oxidative stress and impaired cholinergic signaling in the brain. The α7 nicotinic acetylcholine receptor (α7nAChR) is densely distributed in the hippocampus and cortex, and exerts neuroprotective and procognitive actions. Ethyl gallate (EG), a natural phenolic antioxidant compound, showed high in-silico binding affinity towards α7nAChR and brain penetrability. Therefore, the present study aimed to evaluate the involvement of α7nAChR in the potential of EG to ameliorate T2D-induced Alzheimer's disease-like condition. T2D was induced by intraperitoneal (i.p.) injection of streptozotocin (35 mg/kg) in rats on high-fat diet. Diabetic animals were treated with EG (10 and 20 mg/kg, i.p.) for four weeks, and their learning and memory performance was evaluated by the Morris water maze (MWM). Further, the brains were subjected to biochemical analysis of antioxidants like glutathione (GSH), superoxide dismutase (SOD), and catalase (CAT), and oxidative stress marker malonaldehyde (MDA). While diabetic rats showed a significant decline in cognitive performance in the MWM, a substantial improvement was noticed following EG treatment. Further, the diabetes-associated reductions in GSH, SOD, and CAT levels, along with increased MDA contents in the brain, were effectively restored by EG. Interestingly, pre-treatment with α7nAChR antagonist methyllycaconitine (1 mg/kg, i.p.) attenuated the effects of EG on behavioral and biochemical parameters. The results suggest that EG may augment cholinergic signaling in the brain via α7nAChR to mitigate oxidative stress, consequently alleviating T2D-associated dementia. Therefore, EG could be a potential candidate for addressing cognitive impairment comorbid with T2D.

Proteome of Clothianidin Exposed Honey Bees Reveals a Possible Mechanism Behind Impairment of Sucrose Responsiveness

ABSTRACTNeonicotinoids (NNIs) are the most commonly used insecticides in the world and clothianidin, a type of NNI, is commonly found in honey bee collected pollen. Clothianidin has several negative effects on honey bee health and behaviour, but whether and how it might impact learning and memory remains unclear. Therefore, we exposed honey bee workers to a field‐realistic oral dose of clothianidin for 7 days to assess their sugar responsiveness, and olfactory learning and memory using the proboscis extension response paradigm. Sugar responsiveness impacts important colony‐level traits, such as onset of foraging, that help maintain colony homeostasis as well as impacts learning and memory. We then measured how clothianidin alters protein expression in the brain in an effort to understand the mechanism(s) of clothianidin's effects. Clothianidin‐exposed bees had impaired sugar responsiveness, but no effect was seen on learning and memory. We identified 5069 protein groups and showed that the exposed bees had fewer detectable proteins. The exposed bees also had fewer mitochondrial respiration proteins, as well as reduced levels of nicotinic acetylcholine receptor α7 (nAChRα7) subunit (one of the targets of clothianidin) and reduced levels of acetylcholinesterase and acetylcholinesterase‐2 levels. In the exposed bees, the levels of nAChRα7 subunit positively correlated with sugar responsiveness, providing evidence for a possible mechanical explanation.

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.

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.

Neuroprotection by 4R-cembranoid against Gulf War Illness-related Chemicals is mediated by ERK, PI3K, and CaMKII pathways

Gulf War Illness (GWI) has been consistently linked to exposure to pyridostigmine (PB), N,N-Diethyl-meta-toluamide (DEET), permethrin (PER), and traces of sarin. In this study, diisopropylfluorophosphate (DFP, sarin surrogate) and the GWI-related chemicals were found to reduce the number of functionally active neurons in rat hippocampal slices. These findings confirm a link between GWI neurotoxicants and N-Methyl-D-Aspartate (NMDA)-mediated excitotoxicity, which was successfully reversed by Edelfosine (a phospholipase Cβ (PLCβ3) inhibitor) and Flupirtine (a Kv7 channel agonist).To test whether 4R-cembranoid (4R), a nicotinic α7 acetylcholinesterase receptor (α7AChR) modulator known for its neuroprotective properties, can restore hippocampal neurons from glutamate-induced neurotoxicity, we exposed rat hippocampal slices with DFP for 10 min followed by 60 min treatment with 4R. We investigated the 4R mechanisms of neuroprotection after preincubation with LY294002, PD98059, and KN-62. The inhibition of the phosphatidylinositol 3-kinase (PI3K), mitogen-activated protein kinase (MEK1/2), and calcium/calmodulin-dependent protein kinase (CaMKII) abrogated the protective effect of 4R against DFP-induced neurotoxicity. In separate experiments, after incubation with DFP, followed by 4R for 1 h, cellular extracts were prepared for Western blotting of phospho-Akt, phospho-GSK3β, phosphorylated extracellular signal-regulated kinase (ERK)1/2, CaMKII and cAMP response element-binding protein (CREB). Our results show that DFP induces neuronal dysfunction by dephosphorylation, while 4R restores the phosphorylation of Akt, GSK3, ERK1/2, CREB, and CaMKII. Moreover, our proteomics analysis supported the notion that 4R activates additional signaling pathways related to enhancing neuronal signaling, synaptic plasticity, and apoptotic inhibition to promote cell survival against DFP, offering biomarkers for developing treatment against GWI.

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.