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Elevated inflammatory reactions are a significant component in cerebral ischemia-reperfusion injury (CIRI). Activation of α7-Nicotinic Acetylcholine Receptor (α7nAChR) reduces stroke-induced inflammation in rats, but the anti-inflammatory pathway in microglia under CIRI condition remains unclear. This study employed qRT-PCR, protein assays, NanoString analysis, and bioinformatics to examine the effects of PNU282987 treatment (α7nAChR agonist) on BV2 microglial functional differentiation in oxygen-glucose deprivation/reoxygenation (OGDR) condition. OGDR significantly increased the gene expression of pro-inflammatory markers such as TNF-α, IL-6, and IL1β, while α7nAChR agonists reduced these markers. The anti-inflammatory gene marker IL-10 was upregulated by α7nAChR agonist treatment. Downstream pathway marker analysis showed that both gene and protein expression of NFκB was associated with anti-inflammatory effects. Blocking microRNA-21 with antagomir reversed the anti-inflammatory effects. NanoString analysis revealed that microRNA-21 inhibition significantly affected inflammation-related genes, including AL1RAP, TLR9, FLT1, PTGIR, NFκB, TREM2, TNF, SMAD7, FOS, CCL5, IFIT1, CFB, CXCL10, IFI44, DDIT3, IRF7, OASL1, IL1A, IFIT2, C3, CD40, STAT2, IFIT3, IL1RN, OAS1A, CSF1, CCL4, CCL2, CCL3, BCL2L1, and ITGB2. Enrichment analysis of upregulated genes identified Gene Ontology Biological Processes related to cytokine responses and TNF-associated pathways. This study highlights α7nAChR activation as a key regulator of anti-inflammatory responses in BV2 microglia under OGDR conditions, with micro-RNA21 identified as a crucial mediator of receptor-driven neuroprotection via the TNF-α/NFκB signalling pathway., Competing Interests: Declarations. Competing Interests: The authors declare no competing interests., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Tatsuro Mutoh reports financial support was provided by the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan. Tatsuro Mutoh reports a relationship with the Ministry of Education, Culture, Sports, Science, and Technology (MEXT) of Japan that includes: funding grants. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: Acute liver injury is a disease characterized by severe liver dysfunction, caused by significant infiltration of immune cells and extensive cell death with a high mortality. Previous studies demonstrated that the α7 nicotinic acetylcholine receptor (α7nAChR) played a crucial role in various liver diseases. The hypothesis of this study was that activating α7nAChR could alleviate acute liver injury and investigate its possible mechanisms., Methods: Acute liver injury was induced by intraperitoneal injection of lipopolysaccharide (LPS)/D-galactosamine (D-Gal) in wild type, α7nAChR knockout (α7nAChR-/-) and stimulator of interferon gene (STING) mutation (Stinggt/gt) mice in the presence or absence of a pharmacologic selective α7nAChR agonist (PNU-282987). The effects of α7nAChR on hepatic injury, inflammatory response, mitochondrial damage, necroptosis, and infiltration of immune cells during acute liver injury were assessed., Results: The expression of α7nAChR in liver tissue was increased in LPS/D-Gal-induced acute liver injury mice. Compared to the age-matched wild-type mice, α7nAChR deficiency decreased the survival rate, exacerbated the hepatic injury accompanied with enhanced inflammatory response and oxidative stress, and aggravated hepatic mitochondrial damage and necroptosis. Conversely, pharmacologic activation of α7nAChR by PNU-282987 displayed the opposite trends. Furthermore, PNU-282987 significantly reduced the proportion of infiltrating monocyte-derived macrophages (CD45+CD11bhiF4/80int), M1 macrophages (CD45+CD11b+F4/80+CD86hiCD163low), and Ly6Chi monocytes (CD45+CD11b+MHC [major histocompatibility complex] ⅡlowLy6Chi), but increased the resident Kupffer cells (CD45+CD11bintF4/80hiTIM4hi) in the damaged hepatic tissues caused by LPS/D-Gal. Interestingly, α7nAChR deficiency promoted the STING signaling pathway under LPS/D-Gal stimulation, while PNU-282987 treatment significantly prevented its activation. Finally, it was found that Sting mutation abolished the protective effects against hepatic injury by activating α7nAChR., Conclusions: The authors' study revealed that activating α7nAChR could protect against LPS/D-Gal-induced acute liver injury by inhibiting hepatic inflammation and necroptosis possibly via regulating immune cells infiltration and inhibiting STING signaling pathway., (Copyright © 2024 American Society of Anesthesiologists. All Rights Reserved.)

Consumption of palatable food (PF) can alleviate anxiety, and pain in humans. Contrary, spontaneous withdrawal of long-term PF intake produces anxiogenic-like behavior and abnormal pain sensation, causing challenges to weight-loss diet and anti-obesity agents. Thus, we examined α7-nicotinic acetylcholine receptors (α7nAChR) involvement since it plays essential role in nociception and psychological behaviors., Methods: Adult male C57BL/6 mice were placed on a Standard Chow (SC) alone or with PF on intermittent or continuous regimen for 6 weeks. Then, mice were replaced with normal SC (spontaneous withdrawal). Body weight, food intake, and calories intake with and without the obesogenic diet were measured throughout the study. During PF withdrawal, anxiety-like behaviors and pain sensitivity were measured with PNU-282987 (α7nAChR agonist) administration., Results: Six weeks of SC + PF-intermittent and continuous paradigms produced a significant weight gain. PF withdrawal displayed hyperalgesia and anxiety-like behaviors. During withdrawal, PNU-282987 significantly attenuated hyperalgesia and anxiety-like behaviors., Conclusion: The present study shows that a PF can increase food intake and body weight. Also, enhanced pain sensitivity and anxiety-like behavior were observed during PF withdrawal. α7nAChR activation attenuated anxiolytic-like behavior and hyperalgesia in PF abstinent mice. These data suggest potential therapeutic effects of targeting α7 nAChRs for obesity-withdrawal symptoms in obese subjects., Competing Interests: Declaration of competing interest None., (Copyright © 2024 The Authors. Production and hosting by Elsevier B.V. All rights reserved.)

Background: Neonatal necrotizing enterocolitis (NEC) is one of the most prevalent and severe intestinal emergencies in newborns. The inflammatory activation of macrophages is associated with the intestinal injury of NEC. The neuroimmune regulation mediated by α7 nicotinic acetylcholine receptor (α7nAChR) plays an important role in regulating macrophage activation and inflammation progression, but in NEC remains unclear. This study aims to explore the effect of macrophage α7nAChR on NEC., Methods: Mice NEC model were conducted with high-osmolarity formula feeding, hypoxia, and cold stimulation. The α7nAChR agonist PNU-282987 and mTOR inhibitor rapamycin were treated by intraperitoneal injections in mice. The expression and distribution of macrophages, α7nAChR, and phospho-mammalian target of rapamycin (p-mTOR) in the intestines of NEC patients and mice was assessed using immunohistochemistry, immunofluorescence, and flow cytometry. The expression of NLRP3, activated caspase-1 and IL-1β in mice intestines was detected by flow cytometry, western blot or ELISA. In vitro, the mouse RAW264.7 macrophage cell line was also cultured followed by various treatments. Expression of p-mTOR, NLRP3, activated caspase-1, and IL-1β in macrophages was determined., Results: Macrophages accumulated in the intestines and the expression of α7nAChR in the mucosal and submucosal layers of the intestines was increased in both the NEC patients and mice. The p-mTOR and CD68 were increased and co-localized in intestines of NEC patients. In vitro, α7nAChR agonist PNU-282987 significantly reduced the increase of NLRP3, activated caspase-1, and IL-1β in macrophages. PNU-282987 also significantly reduced the increase of p-mTOR. The effect was blocked by AMPK inhibitor compound C. The expression of NLRP3, activated caspase-1, and IL-1β was inhibited after mTOR inhibitor rapamycin treatment. In NEC model mice, PNU-282987 reduced the expression of p-mTOR, NLRP3, activated caspase-1, and IL-1β in the intestine. Meanwhile, rapamycin significantly attenuated NLRP3 activation and the release of IL-1β. Moreover, the proportion of intestinal macrophages and intestinal injury decreased after PNU-282987 treatment., Conclusion: Macrophage α7nAChR activation mitigates NLRP3 inflammasome activation by modulating mTOR phosphorylation, and subsequently alleviates intestinal inflammation and injury in NEC., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier B.V. All rights reserved.)

There are presently no acknowledged therapeutic targets or official drugs for the treatment of muscle fatigue. The alpha7 nicotinic acetylcholine receptor (α7nAChR) is expressed in skeletal muscle, with an unknown role in muscle endurance. Here, we try to explore whether α7nAChR could act as a potential therapeutic target for the treatment of muscle fatigue. Results showed that nicotine and PNU-282987 (PNU), as nonspecific and specific agonists of α7nAChR, respectively, could both significantly increase C57BL6/J mice treadmill-running time in a time- and dose-dependent manner. The improvement effect of PNU on running time and ex vivo muscle fatigue index disappeared when α7nAChR deletion. RNA sequencing revealed that the differential mRNAs affected by PNU were enriched in glycolysis/gluconeogenesis signaling pathways. Further studies found that PNU treatment significantly elevates glycogen content and ATP level in the muscle tissues of α7nAChR +/+ mice but not α7nAChR -/- mice. α7nAChR activation specifically increased endogenous glycogen-targeting protein orosomucoid (ORM) expression both in vivo skeletal muscle tissues and in vitro C2C12 skeletal muscle cells. In ORM1 deficient mice, the positive effects of PNU on running time, glycogen and ATP content, as well as muscle fatigue index, were abolished. Therefore, the activation of α7nAChR could enhance muscle endurance via elevating endogenous anti-fatigue protein ORM and might act as a promising therapeutic strategy for the treatment of muscle fatigue., (© 2024 Wiley Periodicals LLC.)

We investigated the impact of the human-specific gene CHRFAM7A on the function of α7 nicotinic acetylcholine receptors (α7 nAChRs) in two different types of neurons: human-induced pluripotent stem cell (hiPSC)-derived cortical neurons, and superior cervical ganglion (SCG) neurons, taken from transgenic mice expressing CHRFAM7A. dupα7, the gene product of CHRFAM7A, which lacks a major part of the extracellular N-terminal ligand-binding domain, co-assembles with α7, the gene product of CHRNA7. We assessed the receptor function in hiPSC-derived cortical and SCG neurons with Fura-2 calcium imaging and three different α7-specific ligands: PNU282987, choline, and 4BP-TQS. Given the short-lived open state of α7 receptors, we combined the two orthosteric agonists PNU282987 and choline with the type-2 positive allosteric modulator (PAM II) PNU120596. In line with different cellular models used previously, we demonstrate that CHRFAM7A has a major impact on nicotinic α7 nAChRs by reducing calcium transients in response to all three agonists., (© 2024 The Author(s). European Journal of Neuroscience published by Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

Alzheimer's disease (AD) is an age-dependent incurable neurodegenerative disorder accompanied by neuroinflammation, amyloid accumulation, and memory impairment. It begins decades before the first clinical symptoms appear, and identifying early biomarkers is key for developing disease-modifying therapies. We show now in a mouse model of AD that before any amyloid deposition the brains of 1.5-month-old mice contain increased levels of pro-inflammatory cytokines IL-1β and IL-6, decreased levels of nicotinic acetylcholine receptors (nAChRs) in the brain and brain mitochondria and increased amounts of α7 nAChR-bound Aβ 1-42 , along with impaired episodic memory and increased risk of apoptosis. Both acute (1-week-long) and chronic (4-month-long) treatments with α7-selective agonist PNU282987, starting at 1.5 months of age, were well tolerated. The acute treatment did not affect the levels of soluble Aβ 1-42 but consistently upregulated the α7 nAChR expression, decreased the level of α7-Aβ 1-42 complexes, and improved episodic memory of 1.5-month-old mice. The chronic treatment, covering the disease development phase, strongly upregulated the expression of all abundant brain nAChRs, reduced both free and α7-coupled Aβ 1-42 within the brain, had anti-inflammatory and antiapoptotic effects, and potently upregulated cognition, thus identifying α7 nAChRs as both early biomarker and potent therapeutic target for fighting this devastating disease., Competing Interests: Declaration of Competing Interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: Olga Garaschuk, Maryna Skok reports financial support was provided by Alexander von Humboldt Foundation. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Chronic diabetes mellitus compromises the vascular system, which causes organ injury, including in the lung. Due to the strong compensatory ability of the lung, patients always exhibit subclinical symptoms. Once sepsis occurs, the degree of lung injury is more severe under hyperglycemic conditions. The α7 nicotinic acetylcholine receptor (α7nAChR) plays an important role in regulating inflammation and metabolism and can improve endothelial progenitor cell (EPC) functions. In the present study, lung injury caused by sepsis was compared between diabetic rats and normal rats. We also examined whether α7nAChR activation combined with EPC transplantation could ameliorate lung injury in diabetic sepsis rats. A type 2 diabetic model was induced in rats via a high-fat diet and streptozotocin. Then, a rat model of septic lung injury was established by intraperitoneal injection combined with endotracheal instillation of LPS. The oxygenation indices, wet-to-dry ratios, and histopathological scores of the lungs were tested after PNU282987 treatment and EPC transplantation. IL-6, IL-8, TNF-α, and IL-10 levels were measured. Caspase-3, Bax, Bcl-2, and phosphorylated NF-κB (p-NF-κB) levels were determined by blotting. Sepsis causes obvious lung injury, which is exacerbated by diabetic conditions. α7nAChR activation and endothelial progenitor cell transplantation reduced lung injury in diabetic sepsis rats, alleviating inflammation and decreasing apoptosis. This treatment was more effective when PNU282987 and endothelial progenitor cells were administered together. p-NF-κB levels decreased following treatment with PNU282987 and EPCs. In conclusion, α7nAChR activation combined with EPC transplantation can alleviate lung injury in diabetic sepsis rats through the NF-κB signaling pathway., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Herpes simplex virus type 1 (HSV-1), a neurotropic DNA virus, establishes latency in neural tissues, with reactivation causing severe consequences like encephalitis. Emerging evidence links HSV-1 infection to chronic neuroinflammation and neurodegenerative diseases. Microglia, the central nervous system's (CNS) immune sentinels, express diverse receptors, including α7 nicotinic acetylcholine receptors (α7 nAChRs), critical for immune regulation. Recent studies suggest α7 nAChR activation protects against viral infections. Here, we show that α7 nAChR agonists, choline and PNU-282987, significantly inhibit HSV-1 replication in microglial BV2 cells. Notably, this inhibition is independent of the traditional ionotropic nAChR signaling pathway. mRNA profiling revealed that choline stimulates the expression of antiviral factors, IL-1β and Nos2, and down-regulates the apoptosis genes and type A Lamins in BV2 cells. These findings suggest a novel mechanism by which microglial α7 nAChRs restrict viral infections by regulating innate immune responses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

Pulmonary emphysema is a primary component of chronic obstructive pulmonary disease (COPD), a life-threatening disorder characterized by lung inflammation and restricted airflow, primarily resulting from the destruction of small airways and alveolar walls. Cumulative evidence suggests that nicotinic receptors, especially the α7 subtype (α7nAChR), is required for anti-inflammatory cholinergic responses. We postulated that the stimulation of α7nAChR could offer therapeutic benefits in the context of pulmonary emphysema. To investigate this, we assessed the potential protective effects of PNU-282987, a selective α7nAChR agonist, using an experimental emphysema model. Male mice (C57BL/6) were submitted to a nasal instillation of porcine pancreatic elastase (PPE) (50 µl, 0.667 IU) to induce emphysema. Treatment with PNU-282987 (2.0 mg/kg, ip) was performed pre and post-emphysema induction by measuring anti-inflammatory effects (inflammatory cells, cytokines) as well as anti-remodeling and anti-oxidant effects. Elastase-induced emphysema led to an increase in the number of α7nAChR-positive cells in the lungs. Notably, both groups treated with PNU-282987 (prior to and following emphysema induction) exhibited a significant decrease in the number of α7nAChR-positive cells. Furthermore, both groups treated with PNU-282987 demonstrated decreased levels of macrophages, IL-6, IL-1β, collagen, and elastic fiber deposition. Additionally, both groups exhibited reduced STAT3 phosphorylation and lower levels of SOCS3. Of particular note, in the post-treated group, PNU-282987 successfully attenuated alveolar enlargement, decreased IL-17 and TNF-α levels, and reduced the recruitment of polymorphonuclear cells to the lung parenchyma. Significantly, it is worth noting that MLA, an antagonist of α7nAChR, counteracted the protective effects of PNU-282987 in relation to certain crucial inflammatory parameters. In summary, these findings unequivocally demonstrate the protective abilities of α7nAChR against elastase-induced emphysema, strongly supporting α7nAChR as a pivotal therapeutic target for ameliorating pulmonary emphysema., (© 2024. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Both autonomic nervous system dysfunction and immune system activation are characteristic of chronic pain after limb injuries. Cholinergic agonists reduce immune system activation in many settings. We hypothesized, therefore, that alpha-7 nicotinic acetylcholine receptor (α7nAChR) agonist administration would reduce nociceptive and immune changes after tibia fracture and cast immobilization in mice. Fracture mice were treated with either vehicle, a low (.2 mg/kg) dose, or a high (1 mg/kg) dose of the selective α7nAChR agonist PNU-282987 for 4 weeks. We assessed hindpaw allodynia and weight bearing as behavioral outcomes. The assessment of adaptive immune responses included regional lymph node hypertrophy, germinal center formation, α7nAChR expression, and IgM deposition. Assessment of innate immune system activation focused on IL-1β and IL-6 generation in fractured hindlimb skin. We observed that mechanical allodynia and unweighting were alleviated by PNU-282987 treatment. Drug treatment also reduced popliteal lymph node hypertrophy and germinal center formation. Immunohistochemical studies localized α7nAChR to germinal center B lymphocytes, and this expression increased after fracture. Analysis of fracture limb hindpaw skin demonstrated increased inflammatory mediator (IL-1β and IL-6) levels and IgM deposition, which were abrogated by PNU-282987. Serum analyses demonstrated fracture-induced IgM reactivity against keratin 16, histone 3.2, GFAP, and NMDAR-2B. Administration of PNU-282987 reduced the enhancement of IgM reactivity. Collectively, these data suggest that the α7nAChR is involved in regulating posttraumatic innate and adaptive immune responses and the associated nociceptive sensitization. PERSPECTIVE: These studies evaluate the effects of a selective α7nAChR agonist in a tibial fracture/cast immobilization model of limb pain. Administration of the drug reduced nociceptive and functional changes 4 weeks after injury. These novel findings suggest that well-tolerated α7nAChR agonists may be viable analgesics for chronic pain after limb injuries., (Published by Elsevier Inc.)

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., (Copyright © 2022 IBRO. Published by Elsevier Ltd. All rights reserved.)

Acetylcholine (ACh), which activates muscarinic ACh receptors (mAChRs) and nicotinic ACh receptors (nAChRs), enhances airway ciliary beating by increasing the intracellular Ca 2+ concentration ([Ca 2+ ] i ). The mechanisms enhancing airway ciliary beating by nAChRs have remained largely unknown, although those by mAChRs are well understood. In this study, we focused on the effects of α7-nAChRs and voltage-gated Ca 2+ channels (Ca V s) on the airway ciliary beating. The activities of ciliary beating were assessed by frequency (CBF, ciliary beat frequency) and amplitude (CBD, ciliary bend distance) measured by high-speed video microscopy. ACh enhanced CBF and CBD by 25% mediated by an [Ca 2+ ] i increase stimulated by mAChRs and α7-nAChRs (a subunit of nAChR) in airway ciliary cells of mice. Experiments using PNU282987 (an agonist of α7-nAChR) and MLA (an inhibitor of α7-nAChR) revealed that CBF and CBD enhanced by α7-nAChR are approximately 50% of those enhanced by ACh. CBF, CBD, and [Ca 2+ ] i enhanced by α7-nAChRs were inhibited by nifedipine, suggesting activation of Ca V s by α7-nAChRs. Experiments using a high K + solution with/without nifedipine (155.5 mM K + ) showed that the activation of Ca V s enhances CBF and CBD via an [Ca 2+ ] i increase. Immunofluorescence and immunoblotting studies demonstrated that Cav1.2 and α7-nAChR are expressed in airway cilia. Moreover, IL-13 stimulated MLA-sensitive increases in CBF and CBD in airway ciliary cells, suggesting an autocrine regulation of ciliary beating by Ca V 1.2/α7-nAChR/ACh. In conclusion, a novel Ca 2+ signalling pathway in airway cilia, Ca V 1.2/α7-nAChR, enhances CBF and CBD and activates mucociliary clearance maintaining healthy airways., (© 2022. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

The α7 nicotinic acetylcholine receptor (nAChR) is widely distributed in the central and peripheral nervous systems and is closely related to a variety of nervous system diseases and inflammatory responses. The α7 nAChR subtype plays a vital role in the cholinergic anti-inflammatory pathway. In vivo, ACh released from nerve endings stimulates α7 nAChR on macrophages to regulate the NF-κB and JAK2/STAT3 signaling pathways, thereby inhibiting the production and release of downstream proinflammatory cytokines and chemokines. Despite a considerable level of recent research on α7 nAChR-mediated immune responses, much is still unknown. In this study, we used an agonist (PNU282987) and antagonists (MLA and α-conotoxin [A10L]PnIA) of α7 nAChR as pharmacological tools to identify the molecular mechanism of the α7 nAChR-mediated cholinergic anti-inflammatory pathway in RAW264.7 mouse macrophages. The results of quantitative PCR, ELISAs, and transcriptome analysis were combined to clarify the function of α7 nAChR regulation in the inflammatory response. Our findings indicate that the agonist PNU282987 significantly reduced the expression of the IL-6 gene and protein in inflammatory macrophages to attenuate the inflammatory response, but the antagonists MLA and α-conotoxin [A10L]PnIA had the opposite effects. Neither the agonist nor antagonists of α7 nAChR changed the expression level of the α7 nAChR subunit gene; they only regulated receptor function. This study provides a reference and scientific basis for the discovery of novel α7 nAChR agonists and their anti-inflammatory applications in the future.

BACKGROUND To reveal the mechanism underlying the effect of alpha7 nicotinic acetylcholine receptor (nAChR) on neurodegeneration in Alzheimer disease (AD), the influence of the receptor on recognition in APP/PS1 mice was evaluated by using its selective agonist (PNU-282987). MATERIAL AND METHODS APP/PS1 and wild-type (WT) mice were treated with PNU or saline, respectively, for 7 days at the ages of 6 and 10 months. RESULTS Morris water maze analysis showed that both at 6 and 10 months of age, PNU treatment enhanced the learning and memory of APP/PS1 mice. However, PNU treatment did not alter the number of senile plaques. Furthermore, a higher protein expression of Nrf2/HO-1, ADAM10, SYP, and SNAP-25, and a lower level of oxidative stress, were observed in the hippocampus of APP/PS1 mice treated with PNU compared with the control group. CONCLUSIONS The results indicated that the activation of alpha7 nAChR by PNU improved the learning and memory of mice carrying the APP/PS1 mutation, regulated the levels of enzymes that mediate APP metabolization to reduce ß-amyloid peptide damage, and decreased the level of oxidative stress and maintained synaptic plasticity, in which the mechanism might be enhancement of the Nrf2/HO-1 pathway.

Osteoarthritis (OA) is a chronic degenerative joint disease characterized by cartilage degradation. Alpha 7 nicotinic acetylcholine receptor (α7nAChR) is associated with inflammatory and metabolic responses in OA. However, the mechanisms underlying the pathological process of OA remain unclear. The aim of the present study was to examine the role and mechanisms of α7nAChR-mediated autophagy and anti-inflammatory response in chondroprotection. Monosodium iodoacetate (MIA)-induced Wistar rat OA model was used to assess the in vivo effects of the ɑ7nAChR agonist (PNU-282987). The histopathological characteristics of OA were evaluated by immunohistochemistry (IHC), and the levels of autophagy markers were determined by western blotting and transmission electron microscopy. The anti-inflammatory effect of the ɑ7nAChR agonist was assessed by IHC, quantitative real-time polymerase chain reaction, and western blotting. Parallel experiments to determine the molecular mechanisms through which the ɑ7nAChR agonist prevents OA were performed using interleukin-1β (IL-1β)-treated chondrocytes. Our results showed that PNU-282987 reduced cartilage degeneration and matrix metalloproteinase (MMP)-1 and MMP-13 expressions. Activating α7nAChR with PNU-282987 significantly promoted MIA/IL-1β-induced chondrocyte autophagy, as demonstrated by the increase in LC3-II/LC3-I ratio, Beclin-1 levels, and autophagosome number. Furthermore, treating chondrocyte with ULK1 siRNA attenuated the PNU282987-induced enhancement of LC3-II/LC3-I ratio and Beclin-1 level. Additionally, PNU282987 suppressed NF-κB/NLRP3 inflammasome activation by inhibiting the ROS/TXNIP pathway and suppressed tumor necrosis factor-ɑ and IL-1β secretion in MIA/IL-1β-treated chondrocytes. Our results demonstrate that the activation of α7nAChR promotes chondrocyte autophagy and attenuates inflammation to mitigate OA progression, providing a novel target for the treatment of OA., Competing Interests: The authors have declared that no competing interests exist.

Background: Cholinergic anti-inflammatory pathway (CAP) is implicated in cardioprotection in chronic heart failure (CHF) by downregulating inflammation response. Mitochondrial injuries play an important role in ventricular remodeling of the CHF process. Herein, we aim to investigate whether CAP elicitation prevents ventricular remodeling in CHF by protecting myocardial mitochondrial injuries and its underlying mechanisms., Methods and Results: CHF models were established by ligation of anterior descending artery for 5 weeks. Postoperative survival rats were assigned into 5 groups: the sham group (sham, n = 10), CHF group (CHF, n = 11), Vag group (CHF+vagotomy, n = 10), PNU group (CHF+PNU-282987 for 4 weeks, n = 11), and Vag+PNU group (CHF+vagotomy+PNU-282987 for 4 weeks, n = 10). The antiventricular remodeling effect of cholinergic elicitation was evaluated in vivo, and H9C2 cells were selected for the TNF- α gradient stimulation experiment in vitro. In vivo, CAP agitated by PNU-282987 alleviated the left ventricular dysfunction and inhibited the energy metabolism remodeling. Further, cholinergic elicitation increased myocardium ATP levels and reduced systemic inflammation. CAP induction alleviates macrophage infiltration and cardiac fibrosis, of which the effect is counteracted by vagotomy. Myocardial mitochondrial injuries were ameliorated by CAP activation, including the reserved ultrastructural integrity, declining ROS overload, reduced myocardial apoptosis, and enhanced mitochondrial fusion. In vitro, TNF- α intervention significantly exacerbated the mitochondrial damage in H9C2 cells., Conclusion: CAP elicitation effectively improves ischemic ventricular remodeling by suppressing systemic and cardiac inflammatory response, attenuating cardiac fibrosis and potentially alleviating the mitochondrial dysfunction linked to hyperinflammation reaction., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2021 Yang Zhao et al.)

The adult mammal lacks the ability to regenerate neurons lost to retinal damage or disease in a meaningful capacity. However, previous studies from this laboratory have demonstrated that PNU-282987, an α7 nicotinic acetylcholine receptor agonist, elicits a robust neurogenic response in the adult murine retina. With eye drop application of PNU-282987, Müller glia cells re-enter the cell cycle and produce progenitor-like cells that can differentiate into various types of retinal neurons. In this study, we analyzed the regenerative capability of PNU-282987 in two retinal disease models and identified the source of newly regenerated neurons. Wild-type mice and mice with a transgenic Müller-glia lineage tracer were manipulated to mimic loss of retinal cells associated with glaucoma or photoreceptor degeneration. Following treatment with PNU-282987, the regenerative response of retinal neurons was quantified and characterized. After onset of photoreceptor degeneration, PNU-282987 was able to successfully regenerate both rod and cone photoreceptors. Quantification of this response demonstrated significant regeneration, restoring photoreceptors to near wild-type density. In mice that had glaucoma-like conditions induced, PNU-282987 treatment led to a significant increase in retinal ganglion cells. Retrograde labeling of optic nerve axon fibers demonstrated that newly regenerated axons projected into the optic nerve. Lineage tracing analysis demonstrated that these new neurons were derived from Müller glia. These results demonstrate that PNU-282987 can induce retinal regeneration in adult mice following onset of retinal damage. The ability of PNU-282987 to regenerate retinal neurons in a robust manner offers a new direction for developing novel and potentially transformative treatments to combat neurodegenerative disease., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

(1) Background: The lung cholinergic pathway is important for controlling pulmonary inflammation in acute lung injury, a condition that is characterized by a sudden onset and intense inflammation. This study investigated changes in the expression levels of nicotinic and muscarinic acetylcholine receptors (nAChR and mAChR) in the lung during acute lung injury. (2) Methods: acute lung injury (ALI) was induced in wild-type and cholinergic-deficient (VAChT-KD HOM ) mice using intratracheal lipopolysaccharide (LPS) instillation with or without concurrent treatment with nicotinic ligands. Bronchoalveolar lavage fluid was collected to evaluate markers of inflammation, and then the lung was removed and processed for isolation of membrane fraction and determination of acetylcholine receptors level using radioligand binding assays. (3) Results: LPS-induced increase in lung inflammatory markers (e.g., neutrophils and IL-1β) was significantly higher in VAChT-KD HOM than wild-type mice. In contrast, LPS treatment resulted in a significant increase in lung's α7 nicotinic receptor level in wild-type, but not in VAChT-KD HOM mice. However, treatment with PNU 282987, a selective α7 nicotinic receptor agonist, restored VAChT-KD HOM mice's ability to increase α7 nicotinic receptor levels in response to LPS-induced acute lung injury and reduced lung inflammation. LPS also increased muscarinic receptors level in VAChT-KD HOM mice, and PNU 282987 treatment reduced this response. (4) Conclusions: Our data indicate that the anti-inflammatory effects of the lung cholinergic system involve an increase in the level of α7 nicotinic receptors. Pharmacological agents that increase the expression or the function of lung α7 nicotinic receptors have potential clinical uses for treating acute lung injury.

We previously reported that lipopolysaccharide (LPS) challenge caused microglial-mediated neuroinflammation and sickness behavior that was amplified in aged mice. As α7 nAChRs are implicated in the "Cholinergic anti-inflammatory pathway", we aimed to determine how α7 nAChR stimulation modulates microglial phenotype in an LPS-induced neuroinflammation model in adult and aged mice. For this, BALB/c mice were injected intraperitoneally with LPS (0.33 mg/kg) and treated with the α7 nAChR agonist PNU282987, using different administration protocols. LPS challenge reduced body weight and induced lethargy and social withdrawal in adult mice. Peripheral (intraperitoneal) co-administration of the α7 nAChR agonist PNU282987 with LPS, attenuated body weight loss and sickness behavior associated with LPS challenge in adult mice, and reduced microglial activation with suppression of IL-1β and TNFα mRNA levels. Furthermore, central (intracerebroventricular) administration of the α7 nAChR agonist, even 2 h after LPS injection, attenuated the decrease in social exploratory behavior and microglial activation induced by peripheral administration of LPS, although this recovery was not achieved if activation of α7 nAChRs was performed peripherally. Finally, we observed that the positive results of central activation of α7 nAChRs were lost in aged mice. In conclusion, we provide evidence that stimulation of α7 nAChR signaling reduces microglial activation in an in vivo LPS-based model, but this cholinergic-dependent regulation seems to be dysfunctional in microglia of aged mice.

Background: The anti-inflammatory effect of an α7nAChR agonist, PNU-282987, has previously been explored in the context of inflammatory disease. However, the effects of PNU-282987 on type 2 innate lymphoid cells (ILC2s)-mediated allergic airway inflammation has not yet been established., Aims: To determine the effects of PNU-282987 on the function of ILC2s in the context of IL-33- or Alternaria Alternata (AA)- induced airway inflammation., Methods: PNU-282987 was administered to mice that received recombinant IL-33 or AA intranasal challenges. Lung histological analysis and flow cytometry were performed to determine airway inflammation and the infiltration and activation of ILC2s. The previously published α7nAChR agonist GTS-21 was employed as a comparable reagent. ILC2s were isolated from murine lung tissue and cultured in vitro in the presence of IL-33, IL-2, and IL-7 with/without either PNU-282987 or GTS-21. The expression of the transcription factors GATA3, IKK, and NF-κB were also determined., Results: PNU-282987 and GTS-21 significantly reduced goblet cell hyperplasia in the airway, eosinophil infiltration, and ILC2s numbers in BALF, following IL-33 or AA challenge. In vitro IL-33 stimulation of isolated lung ILC2s showed a reduction of GATA3 and Ki67 in response to PNU-282987 or GTS-21 treatments. There was a significant reduction in IKK and NF-κB phosphorylation in the PNU-282987-treated group when compared to the GTS-21-treated ILC2s., Conclusion: PNU-282987 inhibits ILC2-associated airway inflammation, where its effects were comparable to that of GTS-21., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Yuan, Jiang, Li, Sokulsky, Wanyan, Wang, Liu, Zhou, Tay, Zhang, Yang and Li.)

Acute pancreatitis (AP) is associated with impaired acinar cell autophagic flux, intracellular zymogen activation, cell necrosis and inflammation. Activation of the cholinergic system of vagus nerve has been shown to attenuate AP, but the effect of organ-intrinsic cholinergic system on pancreatitis remains unknown. In this study, we aim to examine the effect of α7 nicotinic acetylcholine receptor (α7nAChR) stimulation within the pancreas during AP. In vivo, AP was induced by caerulein plus LPS or ethanol plus palmitoleic acid in mice. In vitro, pancreatic acini were isolated and subjected to cholecystokinin (CCK) stimulation. Mice or acini were pre-treated with PNU-282987 (selective α7nAChR agonist) or methyllycaconitine citrate salt (selective α7nAChR antagonist). Pancreatitis severity, acinar cell injury, autophagic flux, and transcription factor EB (TFEB) pathway were analyzed. Both caerulein plus LPS in vivo and CCK in vitro led to an up-regulation of α7nAChR, indicating activation of pancreas-intrinsic α7nAChR signaling during AP. PNU-282987 decreased acinar cell injury, trypsinogen activation and pancreatitis severity. Conversely, methyllycaconitine citrate salt increased acinar cell injury and aggravated AP. Moreover, activation of α7nAChR by PNU-282987 promoted autophagic flux as indicated by reduced p62, increased LysoTracker staining and decreased number of autolysosomes with undegraded contents. Furthermore, PNU-282987 treatment significantly increased TFEB activity in pancreatic acinar cells. α7nAChR activation also attenuated pancreatic inflammation and NF-κB activation. Our results showed that activation of α7nAChR protected against experimental pancreatitis through enhancing TFEB-mediated acinar cell autophagy, suggesting that activation of pancreas-intrinsic α7nAChR may serve as an endogenous protective mechanism during AP., (Copyright © 2020. Published by Elsevier B.V.)

The nicotinic acetylcholine receptors (nAChRs) are essential for acetylcholine-mediated signaling. Two major functional subtypes of nAChR in the brain, α7-type and α4β2-type, have a high affinity for nicotine. Here, we demonstrated that chronic exposure to nicotine at 0.03-0.3 mg/kg for 14 days rescued depressive-like behavior in calcium/calmodulin-dependent protein kinase IV (CaMKIV) null mice. Chronic exposure to nicotine together with methyllycaconitine, an α7-type nAChR antagonist, but not with dihydro-β-erythroidine, an α4β2-type nAChR antagonist, failed to rescue the depressive-like behavior and restore the reduced number of BrdU-positive cells in the dentate gyrus (DG) of CaMKIV null mice. Furthermore, chronic exposure to nicotine enhanced the PI3K/Akt and ERK/CREB pathways and increased BDNF expression in the DG of CaMKIV null mice. Similar to chronic exposure to nicotine, both PNU-282987 and GTS-21, α7-type nAChR agonists, significantly rescued depressive-like behavior, with a reduction in the number of BrdU-positive cells in the DG of CaMKIV null mice. Both PNU-282987 and GTS-21 also enhanced the PI3K/Akt and ERK/CREB pathways and increased brain-derived neurotrophic factor (BDNF) expression in the DG of CaMKIV null mice. Taken together, we demonstrated that chronic exposure to nicotine rescues depressive-like behavior via α7-type nAChR through the activation of both PI3K/Akt and ERK/CREB pathways in CaMKIV null mice.

The cholinergic anti-inflammatory pathway has been shown to regulate lung inflammation and cytokine release in acute models of inflammation, mainly via α7 nicotinic receptor (α7nAChR). We aimed to evaluate the role of endogenous acetylcholine in chronic allergic airway inflammation in mice and the effects of therapeutic nAChR stimulation in this model. We first evaluated lung inflammation and remodeling on knock-down mice with 65% of vesicular acetylcholine transport (VAChT) gene reduction (KDVAChT) and wild-type(WT) controls that were subcutaneously sensitized and then inhaled with ovalbumin(OVA). We then evaluated the effects of PNU-282987(0.5-to-2mg/kg),(α7nAChR agonist) treatment in BALB/c male mice intraperitoneal sensitized and then inhaled with OVA. Another OVA-sensitized-group was treated with PNU-282987 plus Methyllycaconitine (MLA,1 mg/kg, α7nAChR antagonist) to confirm that the effects observed by PNU were due to α7nAChR. We showed that KDVAChT-OVA mice exhibit exacerbated airway inflammation when compared to WT-OVA mice. In BALB/c, PNU-282987 treatment reduced the number of eosinophils in the blood, BAL fluid, and around airways, and also decreased pulmonary levels of IL-4,IL-13,IL-17, and IgE in the serum of OVA-exposed mice. MLA pre-treatment abolished all the effects of PNU-282987. Additionally, we showed that PNU-282987 inhibited STAT3-phosphorylation and reduced SOCS3 expression in the lung. These data indicate that endogenous cholinergic tone is important to control allergic airway inflammation in a murine model. Moreover, α7nAChR is involved in the control of eosinophilic inflammation and airway remodeling, possibly via inhibition of STAT3/SOCS3 pathways. Together these data suggest that cholinergic anti-inflammatory system mainly α7nAChR should be further considered as a therapeutic target in asthma., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Patients with Alzheimer's disease often undergo anxiety and depression. Our previous studies have shown that α7nAChR protects against Aβ-induced neurotoxicity via downregulation of p38 and JNK MAPKs, but the role of α7nAChR on Aβ-induced anxiety and depressive-like behaviors and the effect of α7nAChR on the regulation of MAPKs pathways remain unknown. To examine the effects of α7nAChR and MAPKs pathways on Aβ-induced anxiety and depression-like behaviors and to explore their relationships between them, elevated plus maze, open field and forced swim tests were performed. Protein levels of 5-HT 1A receptor, 5-HT 2C receptor, α7nAChR, t-ERK1/2 and p-ERK1/2 in the amygdala were analyzed by western blotting and immunostaining. Our study found out that Aβ oligomers induced anxiety and depression-like behaviors in C56BL/6 mice with open field, elevated plus maze and forced swim tests. However, activation of α7nAChR or inhibition of ERK pathways showed significant antidepressant and anxiolytic-like effects on Aβ-injected mice. Moreover, Aβ significantly decreased the level of 5-HT 1A receptor but increased the level of 5-HT 2C receptor in the basolateral amygdala. Treatment with α7nAChR agonist PNU282987 or ERK inhibitor U0126 reversed Aβ-induced 5-HT 1A and 5-HT 2C receptor changes. Moreover, activation of α7nAChR inhibited ERK pathway in the amygdala of Aβ 1-42 -injected mice. Our study provides a new insight into the mechanism of α7nAChR in Aβ-induced depression and anxiety-related symptoms through the regulation of ERK1/2 pathway and the potential association with serotonin receptors. Together, our data suggests that α7nAChR is protective against Aβ-induced anxiety and depression-like behaviors in mice., Competing Interests: Declaration of Competing Interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Microvascular injury is a common pathological process in ischemia-reperfusion injury. Endothelial progenitor cells (EPCs) are vital cells for angiogenesis and endothelial repair. These cells can home to injury sites and secrete angiogenic growth factors. α7nAChRs are pivotal in cholinergic angiogenesis, which is associated with endothelial cells and EPCs. Our study was designed to determine whether activating α7nAChRs enhances the function of EPCs and to explore the underlying mechanism. EPCs were derived from the bone marrow of male Sprague-Dawley rats and treated with an α7nAChR agonist (PNU282987), an α7nAChR antagonist (MLA) and a JAK2 antagonist (AG490). We then assayed the angiogenic abilities of the EPCs, including proliferation ability, adhesion ability, migration ability and in vitro tube formation ability. The levels of total JAK2 (t-JAK2), phosphorylated JAK2 (p-JAK2), total STAT3 (t-STAT3) and phosphorylated STAT3 (p-STAT3) were estimated by western blot analysis. PNU282987 treatment facilitated the angiogenic abilities of EPCs compared with the control regimen. The western blot data suggested that PNU282987 increased the levels of p-JAK2 and p-STAT3. However, the differences in t-JAK2 levels and t-STAT3 levels between the agonist-treated group and the control group were not significant. Moreover, treating EPCs with AG490 reduced STAT3 phosphorylation and attenuated the PNU282987-induced enhancement of EPCs. We demonstrated that activating α7nAChRs can enhance EPC functions partially through the JAK2/STAT3 signaling pathway. This study reveals that α7nAChRs are potential therapeutic targets for angiogenesis and that the JAK2/STAT3 pathway plays a vital role in the associated therapeutic mechanism., Competing Interests: Declaration of competing interest The authors declare no conflicts of interest., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Immune response during sepsis is characterized by hyper-inflammation followed by immunosuppression. The crucial role of macrophages is well-known for both septic stages, since they are involved in immune homeostasis and inflammation, their dysfunction being implicated in immunosuppression. The cholinergic anti-inflammatory pathway mediated by macrophage α7 nicotinic acetylcholine receptor (nAChR) represents possible drug target. Although α7 nAChR activation on macrophages reduces the production of proinflammatory cytokines, the role of these receptors in immunological changes at the cellular level is not fully understood. Using α7 nAChR selective agonist PNU 282,987, we investigated the influence of α7 nAChR activation on the expression of cytokines and, for the first time, of the macrophage membrane markers: cluster of differentiation 14 (CD14), human leukocyte antigen-DR (HLA-DR), CD11b, and CD54. Application of PNU 282,987 to THP-1Mϕ (THP-1 derived macrophages) cells led to inward ion currents and Ca 2+ increase in cytoplasm showing the presence of functionally active α7 nAChR. Production of cytokines tumor necrosis factor-α (TNF-α), interleukin (IL)-6, and IL-10 was estimated in classically activated macrophages (M1) and treatment with PNU 282,987 diminished IL-10 expression. α7 nAChR activation on THP-1Mϕ, THP-1M1, and monocyte-derived macrophages (MDMs) increased the expression of HLA-DR, CD54, and CD11b molecules, but decreased CD14 receptor expression, these effects being blocked by alpha (α)-bungarotoxin. Thus, PNU 282,987 enhances the macrophage-mediated immunity via α7 nAChR by regulating expression of their membrane receptors and of cytokines, both playing an important role in preventing immunosuppressive states.

Ligands of nicotinic acetylcholine receptors (nAChRs) are widely considered as potential therapeutic agents. The present study used primary hippocampus cells and APPswe/PSEN1dE9 double-transgenic mice models to study the possible therapeutic effect and underlying mechanism of the specific activation of α7 nAChR by PNU-282987 in the pathogenesis of Alzheimer's disease. The results indicated that activation of α7 nAChR attenuated the Aβ-induced cell apoptosis, decreased the deposition of Aβ, increased the expression of synaptic-associated proteins, and maintained synaptic morphology. Furthermore, in the APP/PS1_DT mice model, activation of α7 nAChR attenuated Aβ-induced synaptic loss, reduced the deposition of Aβ in the hippocampus, maintained the integral structure of hippocampus-derived synapse, and activated the calmodulin (CaM)-calmodulin-dependent protein kinase II (CaMKII)-cAMP response element-binding protein signaling pathway by upregulation of its key signaling proteins. In addition, activation of α7 nAChR improved the learning and memory abilities of the APP/PS1_DT mice. Collectively, the activation of α7 nAChR by PNU-282987 attenuated the toxic effect of Aβ in vivo and in vitro , which including reduced deposition of Aβ in the hippocampus, maintained synaptic morphology by partially reversing the expression levels of synaptic-associated proteins, activation of the Ca 2+ signaling pathway, and improvement of the cognitive abilities of APP/PS1_DT mice.

The release of inflammatory cytokines and chemokines and autophagy has been reported to be involved in the pathogenic mechanism of acute lung injury (ALI). Reportedly, alpha-7 nicotinic acetylcholine receptors (α7nAchR) might play a protective role in LPS-induced ALI. In the current research, we established LPS-induced ALI model in mice and α7nAchR agonist PNU-282987 improved LPS-induced injury. In MH-S cells, LPS stimulation inhibited, whereas α7nAchR agonist PNU-282987 enhanced the autophagy. α7nAchR agonist PNU-282987 protected MH-S cells from LPS-induced inflammation by reducing the concentrations of IL-6, TNF-α, and IL-1β. Finally, LPS stimulation dramatically inhibited MH-S cell viability but enhanced cell apoptosis, whereas PNU-282987 treatment exerted opposite effects; α7nAchR might regulate the cellular homeostasis via affecting the crosstalk between the autophagy and apoptosis in MH-S cells; in other words, α7nAChR agonist enhances MH-S cell autophagy and inhibits MH-S cell apoptosis. In conclusion, α7nAchR promote the protective autophagy in LPS-induced ALI model in mice and MH-S cells. The application of α7nAchR agonist is considered a potent target for LPS-induced ALI, which needs further clinical investigation.

Neuroimaging and preclinical studies showing that nicotinic receptors (nAChR) may play a role in mood control has increased interest in targeting the cholinergic system for treatment of major depressive disorder. Indeed, modulation of nAChRs in the basolateral amygdala (BLA) are sufficient to produce an anti-immobility effect in the mouse tail suspension test. However, how α7 nAChR modulation impacts BLA neuronal activity in vivo as well as the downstream mechanisms involved in its mood-related effects are not understood. In this work, we used the unpredictable chronic mild stress (CMS) model to investigate the mechanisms underlying the antidepressant-like effect of an α7 nAChR full agonist on BLA-induced changes in dopaminergic neurotransmission. Male adult Sprague-Dawley rats were exposed to four weeks of CMS. Behavioral and electrophysiological experiments were performed within one week following stress. CMS exposure increased rats' immobility time in the forced swimming test, decreased the number of spontaneously active dopamine neurons in the ventral tegmental area and increased the firing rate of putative projection neurons in the BLA. Stress-induced behavioral and electrophysiological changes were reversed by a single systemic administration of PNU282987. In summary, our findings corroborate previous descriptions of a potential rapid antidepressant effect for the α7 nAChR full agonist. This effect appears to involve a mechanism distinct from those of classic antidepressants: normalization of BLA hyperactivity and, consequently, of DA hypofunction. These observations corroborate the role of α7 nAChR as a potential target for novel antidepressant drug development., (Copyright © 2019. Published by Elsevier B.V.)

Background and Purpose: PDGF-BB is an angiogenic factor involved in cardiovascular diseases. Here, we investigated the possible effects of activation of the nicotinic ACh receptor α7 subtype (α7nAChR) on PDGF-BB-induced proliferation and migration in vascular smooth muscle cells (VSMCs)., Experimental Approach: PNU-282987, a selective α7nAChR pharmacological agonist, was used to activate α7nAChR. The influences of α7nAChR activation on PDGF-BB-induced proliferation and migration, as well as the phosphorylation of focal adhesion kinase (FAK)/Src, a pro-migration signalling pathway, were determined in VSMCs. A variety of biochemical assays were applied to explore the underlying molecular mechanisms., Key Results: PDGF-BB induced pronounced migration and proliferation of VSMCs. Activation of α7nAChRs by PNU-282987 blocked PDGF-BB-induced migration but not proliferation in wild-type (WT) VSMCs, whereas this effect was absent in α7nAChR-knockout VSMCs. Accordingly, PNU-282987 attenuated PDGF-BB-induced phosphorylation of FAK Tyr397 and Src Tyr416 in WT VSMCs. Mechanistically, PNU-282987 suppressed PDGF-BB-induced oxidative stress, as demonstrated by the alterations in ROS, H 2 O 2 content, superoxide anion and total antioxidant activity. A sirtuin 3 (SIRT3) inhibitor 3-(1H-1,2,3-triazol-4-yl) pyridine or shRNA-mediated SIRT3 knockdown abolished the inhibitory effect of PNU-282987. PNU-282987 did not modulate SIRT3 protein expression but enhanced mitochondrial SIRT3 deacetylase activity. In line with this action, PNU-282987 enhanced the deacetylation of mitochondrial FoxO3. Lastly, PNU-282987 corrected the PDGF-BB-induced mitochondrial dysfunction by increasing mitochondrial citrate synthase activity, ATP content and nicotinamide adenine dinucleotide pool., Conclusions: Pharmacological activation of α7nAChRs inhibits PDGF-BB-induced VSMC migration by activating the mitochondrial deacetylase SIRT3, implying an important role for α7nAChRs in mitochondria biology and PDGF-related diseases., Linked Articles: This article is part of a themed section on Mitochondrial Pharmacology: Featured Mechanisms and Approaches for Therapy Translation. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.22/issuetoc., (© 2018 The British Pharmacological Society.)

Diffuse axonal injury (DAI) is the predominant effect of severe traumatic brain injury and significantly contributes to cognitive deficits. The mechanisms that underlie these cognitive deficits are often associated with complex molecular alterations. α7nAChR, one of the abundant and widespread nicotinic acetylcholine receptors (nAChRs) in the brain, plays important physiological functions in the central nervous system. However, the relationship between temporospatial alterations in the α7nAChR and DAI-related learning and memory dysfunction are not completely understood. Our study detected temporospatial alterations of α7nAChR in vulnerable areas (hippocampus, internal capsule, corpus callosum and brain stem) of DAI rats and evaluated the development and progression of learning and memory dysfunction via the Morris water maze (MWM). We determined that α7nAChR expression in vulnerable areas was mainly reduced at the recovery of DAI in rats. Moreover, the escape latency of the injured group increased significantly and the percentages of the distance travelled and time spent in the target quadrant were significantly decreased after DAI. Furthermore, α7nAChR expression in the vulnerable area was significantly positively correlated with MWM performance after DAI according to regression analysis. In addition, we determined that a selective α7nAChR agonist significantly improved learning and memory dysfunction. Rats in the α7nAChR agonist group showed better learning and memory performance than those in the antagonist group. These results demonstrate that microstructural injury-induced alterations of α7nAChR in the vulnerable area are significantly correlated with learning and memory dysfunctions after DAI and that augmentation of the α7nAChR level by its agonist contributes to the improvement of learning and memory function., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background and Purpose: Progressive dysfunction of cholinergic transmission is a well-known characteristic of Alzheimer's disease (AD). Amyloid β (Aβ) peptide oligomers are known to play a central role in AD and are suggested to impair the function of the cholinergic nicotinic ACh receptor α7 (α7nAChR). However, the mechanism underlying the effect of Aβ on α7nAChR function is not fully understood, limiting the therapeutic exploration of this observation in AD. Here, we aimed to detect and characterize Aβ binding to α7nAChR, including the possibility of interfering with this interaction for therapeutic purposes., Experimental Approach: We developed a specific and quantitative time-resolved FRET (TR-FRET)-based binding assay for Aβ to α7nAChR and pharmacologically characterized this interaction., Key Results: We demonstrated specific and high-affinity (low nanomolar) binding of Aβ to the orthosteric binding site of α7nAChR. Aβ binding was prevented and reversed by the well-characterized orthosteric ligands of α7nAChR (epibatidine, α-bungarotoxin, methylylcaconitine, PNU-282987, S24795, and EVP6124) and by the type II positive allosteric modulator (PAM) PNU-120596 but not by the type I PAM NS1738., Conclusions and Implications: Our TR-FRET Aβ binding assay demonstrates for the first time the specific binding of Aβ to α7nAChR, which will be a crucial tool for the development, testing, and selection of a novel generation of AD drug candidates targeting Aβ/α7nAChR complexes with high specificity and fewer side effects compared to currently approved α7nAChR drugs., Linked Articles: This article is part of a themed section on Therapeutics for Dementia and Alzheimer's Disease: New Directions for Precision Medicine. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v176.18/issuetoc., (© 2019 The British Pharmacological Society.)

Epidemiologic studies have shown a reduced risk of developing Parkinson's disease (PD) among cigarette smokers. Nicotine, as a key component in tobacco products, is thought as a possible candidate for action of smoking in neuroprotection. α7 nicotinic acetylcholine receptors (α7-nAChRs) is one of the most abundant nAChRs in the mammalian brain. Although nicotine is thought to exert this protective action by acting on nicotinic receptors, including the α7-nAChRs; the mechanisms underlying how α7-nAChRs protect against dopaminergic neuron loss are highly complex. Using nicotine and a selective α7-nAChR agonist PNU-282987, we first confirmed that their addition to SH-SY5Y cells challenged with 1-methyl-4-phenylpyridinium (MPP + ) could afford neuroprotection and result in a reduction in apoptotic cell death. Then, we found that the pretreatment with nicotine and PNU-282987 showed the neuroprotective antiapoptotic effects via activating the α7-nAChRs/MAPK/p53 axis. Furthermore, we used RNA interference to silence the expression of α7-nAChRs in SH-SY5Y cells and found that suppressing α7-nAChR expression diminished the antiapoptotic effects of nicotine and PNU-282987, not the toxic effects of MPP + . Moreover, α7-nAChR knockdown could only decrease the inhibitory effects of nicotine and PNU-282987 on the phosphorylated extracellular signal-regulated kinase (ERK), not c-Jun amino-terminal kinase and p38. Therefore, our findings indicate the important roles of ERK/MAPK signaling in the neuroprotective effects of α7-nAChRs and suggest that α7-nAChR agonists may be validated as novel treatments for PD., (© 2019 Wiley Periodicals, Inc.)

Background: Several lines of investigations support the idea that nicotinic acetylcholine receptors modulate neuronal pathways involved in anxiety and depression., Aims: The purpose of this study was to determine whether 3-furan-2-yl-N-p-tolyl-acrylamide, a highly selective positive allosteric modulator of α7 nicotinic acetylcholine receptors, influences anxiety-like behaviour in mice, and to determine the modulatory activity of 3-furan-2-yl-N-p-tolyl-acrylamide on mice pretreated with either nicotine or selective α7-agonists (i.e. PNU-282987 or (2.4)-dimethoxybenzylidene anabaseine dihydrochloride)., Methods: The elevated plus maze and novelty suppressed feeding tests were selected to evaluate 3-furan-2-yl-N-p-tolyl-acrylamide and other nicotinic ligands on anxiety-like behaviour in mice., Results: The results indicated that: (a) 3-furan-2-yl-N-p-tolyl-acrylamide induces anxiolytic-like activity at 0.5 (elevated plus maze) and 1.0 (novelty suppressed feeding) mg/kg, respectively, after acute treatment, whereas its efficacy is increased after chronic treatments (i.e. active at 0.1 mg/kg; elevated plus maze). This is the first time showing anxiolytic-like activity elicited by 3-furan-2-yl-N-p-tolyl-acrylamide, contrary to the lack of activity for PNU-120596 (0.1 mg/kg); (b) the anxiolytic-like activity of 0.5 mg/kg 3-furan-2-yl-N-p-tolyl-acrylamide is inhibited by methyllycaconitine, a selective α7-antagonist, suggesting that α7 nicotinic acetylcholine receptors are involved in this process; (c) 0.5 mg/kg 3-furan-2-yl-N-p-tolyl-acrylamide reverses the anxiogenic effects induced by 0.1 mg/kg nicotine but not by 10.0 mg/kg PNU-282987; and (d) inactive doses of both 3-furan-2-yl-N-p-tolyl-acrylamide (0.1 mg/kg) and (2.4)-dimethoxybenzylidene anabaseine dihydrochloride (1.0 mg/kg) produce anxiolytic-like effects, suggesting drug interactions, probably synergistic., Conclusions: Our findings indicated that anxiolytic-like activity is mediated by α7 nicotinic acetylcholine receptors, supporting the concept that these receptors modulate anxiety processes. The results indicating that the chronic treatment with 3-furan-2-yl-N-p-tolyl-acrylamide is more efficient than the acute treatment in eliciting anxiolytic-like activity, and that 3-furan-2-yl-N-p-tolyl-acrylamide reverses the anxiogenic effects induced by nicotine, might be of therapeutic importance during smoking cessation.

Purpose: Motion detection is performed by a unique neural network in the mouse retina. Starburst amacrine cells (SACs), which release acetylcholine and gamma-aminobutyric acid (GABA) into the network, are key neurons in the motion detection pathway. Although GABA contributions to the network have been extensively studied, the role of acetylcholine is minimally understood. Acetylcholine receptors are present in a subset of bipolar, amacrine, and ganglion cells. We focused on α7-nicotinic acetylcholine receptor (α7-nAChR) expression in bipolar cells, and investigated which types of bipolar cells possess α7-nAChRs., Methods: Retinal slice sections were prepared from C57BL/6J and Gus8.4-GFP mice. Specific expression of α7-nAChRs in bipolar cells was examined using α-bungarotoxin (αBgTx)-conjugated Alexa dyes co-labeled with specific bipolar cell markers. Whole-cell recordings were conducted from bipolar cells in retinal slice sections. A selective α7-nAChR agonist, PNU282987, was applied by a puff and responses were recorded., Results: αBgTx fluorescence was observed primarily in bipolar cell somas. We found that α7-nAChRs were expressed by the majority of type 1, 2, 4, and 7 bipolar cells. Whole-cell recordings revealed that type 2 and 7 bipolar cells depolarized by PNU application. In contrast, α7-nAChRs were not detected in most of type 3, 5, 6, and rod bipolar cells., Conclusions: We found that α7-nAChRs are present in bipolar cells in a type-specific manner. Because these bipolar cells provide synaptic inputs to SACs and direction selective ganglion cells, α7-nAChRs may play a role in direction selectivity by modulating these bipolar cells' outputs.

The cholinergic anti-inflammatory pathway modulates cytokine release by activating alpha-7 nicotinic acetylcholine receptors (α7nAChR) in monocytes/macrophages. We aimed to determine the role of α7nAChR in lupus nephritis (LN). We enrolled 36 inactive and 35 active LN patients, 34 primary glomerulonephritis patients, and 35 healthy controls. Peripheral blood monocytes were isolated, and mRNA expression of α7nAChR, interleukin (IL)-1β, IL-6, IL-10, and tumor necrosis factor-alpha (TNF-α) in monocytes was measured. α7nAChR and IL-10 mRNA levels were significantly decreased, but IL-6 was increased, in LN patients compared with healthy controls or glomerulonephritis patients (all P < 0.01). Interestingly, α7nAChR mRNA levels were negatively correlated to SLEDAI (r = -0.68, P < 0.01), anti-dsDNA (r = -0.38, P < 0.05), and proteinuria (r = -0.49, P < 0.01) levels, and positively correlated to serum complement C3 levels (r = 0.38, P < 0.05) in patients with active LN. Furthermore, α7nAChR mRNA levels were negatively correlated to TNF-α (r = -0.50, P < 0.01), IL-1β (r = -0.42, P < 0.05), IL-6 (r = -0.69, P < 0.01) mRNA levels, and positively correlated to IL-10 (r = 0.45, P < 0.01). TNF-α, IL-1β, and IL-6 protein levels in the supernatant of cultured monocytes from active LN patients were significantly higher, while IL-10 was lower, than that of healthy controls. PNU-282987, an α7nAChR agonist, significantly decreased TNF-α, IL-1β, and IL-6 but increased IL-10 in the monocyte culture supernatant of active LN patients, which were abolished by an α7nAChR antagonist methyllycaconitine. The effects of PNU-282987 were confirmed in lipopolysaccharides-stimulated monocytes. Taken together, these findings suggest that decrease in α7nAChR mRNA levels may play a role in LN and that activation of α7nAChR may inhibit inflammation in LN., (Copyright © 2018 Elsevier Masson SAS. All rights reserved.)

Purpose: The adult mammalian retina is typically incapable of regeneration when damaged by disease or trauma. Restoration of function would require generation of new adult neurons, something that until recently, mammals were thought to be incapable of doing. However, previous studies from this laboratory have shown that the α7 nicotinic acetylcholine receptor (α7 nAChR) agonist, PNU-282987, induces cell cycle reentry of Müller glia and generation of mature retinal neurons in adult rats, in the absence of detectible injury. This study analyzes how PNU-282987 treatment in RPE leads to robust BrdU incorporation in Müller glia in adult mice and leads to generation of Müller-derived retinal progenitors and neuronal differentiation., Methods: Retinal BrdU incorporation was examined after eye drop application of PNU-282987 in adult wild-type and transgenic mice that contain tamoxifen-inducible tdTomato Müller glia, or after intraocular injection of conditioned medium from PNU-282987-treated cultured RPE cells., Results: PNU-282987 induced robust incorporation of BrdU in all layers of the adult mouse retina. The α7 nAChR agonist was found to stimulate cell cycle reentry of Müller glia and their generation of new retinal progenitors indirectly, via the RPE, in an α7 nAChR-dependent fashion., Conclusions: The results from this study point to RPE as a contributor to Müller glial neurogenic responses. The manipulation of the RPE to stimulate retinal neurogenesis offers a new direction for developing novel and potentially transformative treatments to reverse the loss of neurons associated with neurodegenerative disease, traumatic injury, or aging.

Background/aims: Cholinergic signalling mediated by the activation of muscarinic and nicotinic receptors has been described in the literature as a classic and important signalling pathway in the regulation of the inflammatory response. Recent research has investigated the role of acetylcholine, the physiological agonist of these receptors, in the control of energy homeostasis at the central level. Studies have shown that mice that do not express acetylcholine in brain regions regulating energy homeostasis present with excessive weight gain and hyperphagia. However, it has not yet been well-described in the literature which cholinergic receptor subunits are involved in this response; moreover, the signalling pathways responsible for the observed effects are not fully delineated. The hypothalamus is the regulating centre of energy homeostasis, and the α7 subunit of the nicotinic acetylcholine receptor (α7nAChR) is highly expressed in this region. When active, α7nAChR recruits proteins such as JAK2/STAT3 to mediate its signalling; the same intracellular components are required by leptin, an anorexigenic hormone. The aim of the present study was to evaluate the role of the hypothalamic α7nAChR in the control of energy homeostasis., Methods: The work was performed on Swiss male mice. Initially, using immunofluorescent staining on brain sections, the presence of α7nAChR in hypothalamic cells regulating energy homeostasis was evaluated. Animals were submitted to stereotaxis in the lateral ventricle and intracerebroventricular stimulation (ICV) was used for the administration of an agonist (PNU) or antagonist (α-bungarotoxin) of α7nAChR. Metabolic parameters were evaluated and the expression of neuropeptides was evaluated in the hypothalamus by real-time PCR and western blot. The expression of hypothalamic neuropeptides was evaluated in mice treated with siRNA or inhibitors of JAK2/STAT3 (AG490 and STATTIC) proteins. We also evaluated food intake in α7nAChR knockout animals (α7KO). Additionally, in mouse hypothalamic cell culture (the mypHoA-POMC/GFP lineage), we evaluated the expression of neuropeptides and pSTAT3 after stimulation with PNU., Results: Our results indicate co-localisation of α7nAChR with α-MSH, AgRP and NPY in hypothalamic cells. Pharmacological activation of α7nAChR reduced food intake and increased hypothalamic POMC expression and decreased NPY and AgRP mRNA levels and the protein content of pAMPK. Inhibition of α7nAChR with an antagonist increased the mRNA content of NPY and AgRP. Inhibition of α7nAChR with siRNA led to the suppression of POMC expression and an increase in AgRP mRNA levels. α7KO mice showed no changes in food intake. Inhibition of proteins involved in the JAK2/STAT3 signalling pathway reversed the effects observed after PNU stimulation. POMC-GFP cells, when treated with PNU, showed increased POMC expression and nuclear translocation of pSTAT3., Conclusion: Thus, selective activation of α7nAChR is able to modulate important markers of the response to food intake, suggesting that α7nAChR activation can suppress the expression of orexigenic markers and favour the expression of anorexics using the intracellular JAK2/STAT3 machinery., Competing Interests: The authors declare no conflicts of interest., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)

Effects of curcumin, a major ingredient of turmeric, were tested on the function of the α 7 -subunit of the human nicotinic acetylcholine ( α 7 -nACh) receptor expressed in Xenopus oocytes and on nociception in mouse models of tonic and visceral pain. Curcumin caused a significant potentiation of currents induced by acetylcholine (ACh; 100 μ M) with an EC 50 value of 0.2 µ M. The effect of curcumin was not dependent on the activation of G-proteins and protein kinases and did not involve Ca 2+ -dependent Cl - channels expressed endogenously in oocytes. Importantly, the extent of curcumin potentiation was enhanced significantly by decreasing ACh concentrations. Curcumin did not alter specific binding of [ 125 I] α -bungarotoxin. In addition, curcumin attenuated nociceptive behavior in both tonic and visceral pain models without affecting motor and locomotor activity and without producing tolerance. Pharmacological and genetic approaches revealed that the antinociceptive effect of curcumin was mediated by α 7 -nACh receptors. Curcumin potentiated the antinociceptive effects of the α 7 -nACh receptor agonist N -(3R)-1-azabicyclo[2.2.2]oct-3-yl-4-chlorobenzamide (PNU282987). Collectively, our results indicate that curcumin is a positive allosteric modulator of α 7 -nACh receptor and reverses nociception in mouse models of tonic and visceral pain., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Alzheimer's disease (AD) is one of the most devastating neurodegenerative disorders. Intracellular β-amyloid protein (Aβ) is an early event in AD. It induces the formation of amyloid plaques and neuron damage. The α7 nicotinic acetylcholine receptor (α7nAChR) has been suggested to play an important role in Aβ caused cognition. It has high affinity with Aβ and could mediate Aβ internalization in vitro. However, whether in mouse brain the p38 MAPK signaling pathway is involved in the regulation of the α7nAChR mediated Aβ internalization and their role in mitochondria remains little known. Therefore, in this study, we revealed that Aβ is internalized by cholinergic and GABAergic neurons. The internalized Aβ were found deposits in lysosomes/endosomes and mitochondria. Aβ could form Aβ-α7nAChR complex with α7nAChR, activates the p38 mitogen activated protein kinase (MAPK). And the increasing of α7nAChR could in return mediate Aβ internalization in the cortex and hippocampus. In addition, by using the α7nAChR agonist PNU282987, the p38 phosphorylation level decreases, rescues the biochemical changes which are tightly associated with Aβ-induced apoptosis, such as Bcl2/Bax level, cytochrome c (Cyt c) release. Collectively, the p38 MAPK signaling pathway could regulate the α7nAChR-mediated internalization of Aβ. The activation of α7nAChR or the inhibition of p38 MAPK signaling pathway may be a beneficial therapy to AD., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The α7 nicotinic acetylcholine receptor (α7-nAChR) is associated with inflammation, re-epithelialization, and angiogenesis in wound healing process. A recent study demonstrated that PNU-282987, a selective agonist of α7-nAChR, accelerates the repair of diabetic excisional wounds. Whether α7-nAChR activation promotes non-diabetic wounds healing is unknown. The aim of this study was to evaluate the effects of α7-nAChR activation on non-diabetic wound healing. The effects were evaluated in two wound models. In the first model, the wound was covered with a semi-permeable transparent dressing. In the second model, the wound was left uncovered. In both models, the mice were randomly assigned to two treatment groups: saline or PNU282987 (25 mice in each group). In covered wounds, we found that α7-nAChR activation inhibited re-epithelialization, angiogenesis, and epithelial cells proliferation, promoted neo-epithelial detachment, and suppressed neutrophil infiltration and the expression of interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF). However, in uncovered wounds, we observed that α7-nAChR activation promoted re-epithelialization and angiogenesis, inhibited neutrophil infiltration and the expression of high mobility group box (HMGB)-1, epidermal growth factor (EGF), and VEGF. In conclusion, this data demonstrated that α7-nAChR activation inhibited wound healing in covered wounds but played an opposite role in uncovered wounds. The opposite effect might be primarily due to inhibition of inflammation.

Objective: Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide; yet, the pathogenesis of the disorder is not completely understood. The nicotinic acetylcholine receptor α7 subunit (α7nAChR) plays an indispensable role in the vagus nerve-regulated cholinergic anti-inflammatory pathway., Methods: In the present study, we investigated the key role of α7nAChR in NAFLD development. Male wild-type (WT) and α7nAChR knockout (α7nAChR -/- ) mice were fed a normal chow or a high-fat diet (HFD) for 16weeks to induce NAFLD., Results: We found that both the mRNA and protein levels of α7nAChR in the liver tissue of NAFLD mice were significantly higher than those in mice fed normal chow. There were no differences in food intake, body weight, hepatic cholesterol and triglyceride contents, and insulin sensitivity between WT and α7nAChR -/- mice under normal condition. When the WT and α7nAChR -/- mice were challenged with HFD, the body weight of α7nAChR -/- mice became higher than that of WT mice. The oxygen consumption and energy expenditure in HFD-fed α7nAChR -/- mice were significantly lower than that in HFD-fed WT mice. The HFD-fed α7nAChR -/- mice also showed more aggravated hepatic lipid accumulation, steatosis and oxidative stress than HFD-fed WT mice. Macrophage infiltration; mRNA levels of tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and IL-1β; and liver fibrosis were significantly accelerated in HFD-fed α7nAChR -/- mice compared to that in HFD-fed WT mice. In addition, the bolus insulin injection-activated insulin signaling pathway, which was reflected by the phosphorylation of insulin receptor at Tyr1162/Tyr1163 site (p-IR Tyr1162/Tyr1163 ), insulin receptor substrate-1 at Tyr612 site (p-IRS-1 Tyr612 ) and Akt at Ser473 (p-Akt Ser473 ), was significantly compromised in liver tissues of HFD-fed α7nAChR -/- mice relative to HFD-fed WT mice. Finally, pharmacologically activation of α7nAChR in HFD-fed mice, with a selective agonist PNU-282987, remarkably ameliorated the hepatic steatosis, inflammatory cell infiltration and fibrosis., Conclusion: In conclusion, our results demonstrate that activation of α7nAChR improves energy homeostasis and inhibits inflammation in nonalcoholic fatty liver disease., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Aims: To evaluate whether activating α7 nicotinic acetylcholine receptor (α7nAChR) could inhibit the NOD-like receptor family, pyrin domain containing 3 (NLRP3) inflammasome through regulation of β-arrestin-1 in monocyte/macrophage system, thus contributing to the control of neuroinflammation., Methods: The protein levels of NLRP3, caspase-1 (Casp-1) p20 and proCasp-1, interleukin-1β (IL-1β) p17 and proIL-1β, IL-18 and proIL-18 were measured using Western blotting. The mRNA levels of Casp-1 and IL-1β were detected by real-time PCR (RT-PCR). The colocalization and interaction of NLRP3 protein and β-arrestin-1 were measured by immunofluorescence staining and immunoprecipitation., Results: The expression of β-arrestin-1 was significantly increased and colocalized with CD45-positive cells in spinal cord of experimental auto-immune encephalomyelitis (EAE) mice when compared with the sham mice, which was attenuated by pretreatment with PNU282987, a specific α7nAChR agonist. PNU282987 also significantly inhibited the activation of NLRP3 inflammasome and thus decreased the production of IL-1β and IL-18 both in lipopolysaccharide (LPS)/ATP-stimulated BV2 microglia in vitro and spinal cord from EAE mice in vivo, while inverse effects were observed in α7nAChR knockout mice. Furthermore, overexpression of β-arrestin-1 attenuated the inhibitory effect of PNU282987 on NLRP3 inflammasome activation in LPS/ATP-stimulated BV2 microglia. PNU282987 inhibited the interaction between β-arrestin-1 and NLRP3 protein in vitro., Conclusions: The present study demonstrates that activating α7nAChR can lead to NLRP3 inflammasome inhibition via regulation of β-arrestin-1 in monocyte/microglia system., (© 2017 John Wiley & Sons Ltd.)

Recently, α7 nicotinic acetylcholine receptors (nAChRs), primarily activated by binding of orthosteric agonists, represent a target for anti-inflammatory and analgesic drug development. These receptors may also be modulated by positive allosteric modulators (PAMs), ago-allosteric ligands (ago-PAMs), and α7-silent agonists. Activation of α7 nAChRs has been reported to increase the brain levels of endogenous ligands for nuclear peroxisome proliferator-activated receptors type-α (PPAR-α), palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), in a Ca 2+ -dependent manner. Here, we investigated potential crosstalk between α7 nAChR and PPAR-α, using the formalin test, a mouse model of tonic pain. Using pharmacological and genetic approaches, we found that PNU282987, a full α7 agonist, attenuated formalin-induced nociceptive behavior in α7-dependent manner. Interestingly, the selective PPAR-α antagonist GW6471 blocked the antinociceptive effects of PNU282987, but did not alter the antinociceptive responses evoked by the α7 nAChR PAM PNU120596, ago-PAM GAT107, and silent agonist NS6740. Moreover, GW6471 administered systemically or spinally, but not via the intraplantar surface of the formalin-injected paw blocked PNU282987-induced antinociception. Conversely, exogenous administration of the naturally occurring PPAR-α agonist PEA potentiated the antinociceptive effects of PNU282987. In contrast, the cannabinoid CB 1 antagonist rimonabant and the CB 2 antagonist SR144528 failed to reverse the antinociceptive effects of PNU282987. These findings suggest that PPAR-α plays a key role in a putative antinociceptive α7 nicotinic signaling pathway., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Chronic tobacco use dramatically increases health burdens and financial costs. Limitations of current smoking cessation therapies indicate the need for improved molecular targets. The main addictive component of tobacco, nicotine, exerts its dependency effects via nicotinic acetylcholine receptors (nAChRs). Activation of the homomeric α7 nAChR reduces nicotine's rewarding properties in conditioned place preference (CPP) test and i.v. self-administration models, but the mechanism underlying these effects is unknown. Recently, the nuclear receptor peroxisome proliferator-activated receptor type-α (PPARα) has been implicated as a downstream signaling target of the α7 nAChR in ventral tegmental area dopamine cells. The present study investigated PPARα as a possible mediator of the effect of α7 nAChR activation in nicotine dependence. Our results demonstrate the PPARα antagonist GW6471 blocks actions of the α7 nAChR agonist PNU282987 on nicotine reward in an unbiased CPP test in male ICR adult mice. These findings suggests that α7 nAChR activation attenuates nicotine CPP in a PPARα-dependent manner. To evaluate PPARα activation in nicotine dependence we used the selective and potent PPARα agonist, WY-14643 and the clinically used PPARα activator, fenofibrate, in nicotine CPP and we observed attenuation of nicotine preference, but fenofibrate was less potent. We also studied PPARα in nicotine dependence by evaluating its activation in nicotine withdrawal. WY-14643 reversed nicotine withdrawal signs whereas fenofibrate had modest efficacy. This suggests that PPARα plays a role in nicotine reward and withdrawal and that further studies are warranted to elucidate its function in mediating the effects of α7 nAChRs in nicotine dependence., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

Alpha-7 nicotinic acetylcholine receptor (α7-nAChR) agonists can prevent glutamate-induced excitotoxicity in cultured retinal ganglion cells (RGCs). However, the neuroprotective effects and the mechanism of action of PNU-282987, an α7-nAChR agonist, in a chronic in vivo rat glaucoma model are poorly understood. We found that elevated intraocular pressure (IOP) downregulated retinal α7-nAChR expression. Electroretinography revealed that the amplitude of the photopic negative response (PhNR) decreased in parallel with the loss of RGCs caused by elevated IOP. PNU-282987 enhanced RGC viability and function and decreased terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive signals in RGCs. Patch-clamp recordings revealed differences in the baseline frequencies and decay times of the miniature GABAergic inhibitory postsynaptic currents (mIPSCs) of RGCs between control and glaucomatous retinal slices. The results of western blotting and immunostaining showed that glutamic acid decarboxylase 65/67 and GABA deficits persisted in glaucomatous retinas and that these deficits were reversed by PNU-282987. Patch-clamp recordings also showed that PNU-282987 significantly increased the frequency and amplitude of the GABAergic mIPSCs of RGCs. The protective effects of PNU-292987 were blocked by intravitreal administration of selective GABA A receptor antagonists. The modulation of GABAergic synaptic transmission by PNU-282987 causes de-excitation of ganglion cell circuits and suppresses excitotoxic processes.

Background: Cholinergic deficits play an important role in both cognitive and behavioural alterations in Alzheimer's disease. This study was aimed at evaluating the possible therapeutic role of PNU-282987 (PNU), an α7 nicotinic cholinergic receptor agonist, and the possible effects of stress in precipitating the onset of behavioural deficits in animals with susceptibility to Alzheimer's disease., Methods: B6C3-Tg mice with susceptibility to Alzheimer's disease and wild-type mice either with or without restraint stress received 0- or 1-mg/kg PNU. At 12 months old, mice were evaluated for activity levels, anxiety-like levels, and spatial learning and memory., Results: Data did not show the effects of PNU on activity and anxiety-like behaviour. No effect of PNU on acquisition of a spatial learning task was detected, but a reversal of stress effects on retention in the Morris water maze was observed in transgenic mice., Conclusions: Further studies are needed in order to better understand the role of α7 nicotinic cholinergic receptor agonists in motor activity, anxiety, and spatial learning and memory and to develop more accurate pharmacological treatment of psychopathological diseases., (© 2016 The Authors. Psychogeriatrics © 2016 Japanese Psychogeriatric Society.)