α7nAChR Knockout Mice Research Articles

Huperzine a ameliorates sepsis-induced acute lung injury by suppressing inflammation and oxidative stress via α7 nicotinic acetylcholine receptor

Sepsis, characterized by high mortality rates, causes over 50 % of acute lung injury (ALI) cases, primarily due to the heightened susceptibility of the lungs during this condition. Suppression of the excessive inflammatory response is critical for improving the survival of patients with sepsis; nevertheless, no specific anti-sepsis drugs exist. Huperzine A (HupA) exhibits neuroprotective and anti-inflammatory properties; however, its underlying mechanisms and effects on sepsis-induced ALI have yet to be elucidated. In this study, we demonstrated the potential of HupA for treating sepsis and explored its mechanism of action. To investigate the in vivo impacts of HupA, a murine model of sepsis was induced through cecal ligation and puncture (CLP) in both wild-type (WT) and α7 nicotinic acetylcholine receptor (α7nAChR) knockout mice. Our results showed that HupA ameliorates sepsis-induced acute lung injury by activating the α7nAChR. We used the CLP sepsis model in wild-type and α7nAChR −/− mice and found that HupA significantly increased the survival rate through α7nAChR, reduced the pro-inflammatory cytokine levels and oxidative stress, ameliorated histopathological lung injury, altered the circulating immune cell composition, regulated gut microbiota, and promoted short-chain fatty acid production through α7nAChR in vivo. Additionally, HupA inhibited Toll-like receptor NF-κB signaling by upregulating the α7nAChR/protein kinase B/glycogen synthase kinase-3 pathways. Our data elucidate HupA’s mechanism of action and support a “new use for an old drug” in treating sepsis. Our findings serve as a basis for further in vivo studies of this drug, followed by application to humans. Therefore, the findings have the potential to benefit patients with sepsis.

Electroacupuncture alleviates intestinal inflammation via a distinct neuro-immune signal pathway in the treatment of postoperative ileus

BackgroundThe induction of intestinal inflammation as a result of abdominal surgery is an essential factor in postoperative ileus (POI) development. Electroacupuncture (EA) at ST36 has been demonstrated to relieve intestinal inflammation and restore gastrointestinal dysmotility in POI. This study aims to elucidate the neuroimmune pathway involved in the anti-inflammatory properties of EA in POI. MethodsAfter intestinal manipulation (IM) was performed to induce POI, intestinal inflammation and motility were assessed 24 h post-IM, by evaluating gastrointestinal transit (GIT), cytokines expression, and leukocyte infiltration. Experimental surgery, pharmacological intervention, and genetic knockout mice were used to elucidate the neuroimmune mechanisms of EA. ResultsEA at ST36 significantly improved GIT and reduced the expression of pro-inflammatory cytokines and leukocyte infiltration in the intestinal muscularis following IM in mice. The anti-inflammatory effectiveness of EA treatment was abolished by sub-diaphragmatic vagotomy, whereas splenectomy did not hinder the anti-inflammatory benefits of EA treatment. The hexamethonium chloride (HEX) administration contributes to a notable reduction in the EA capacity to suppress inflammation and enhance motility dysfunction, and EA is ineffective in α7 nicotinic acetylcholine receptor (α7nAChR) knockout mice. ConclusionsEA at ST36 prevents intestinal inflammation and dysmotility through a neural circuit that requires vagal innervation but is independent of the spleen. Further findings revealed that the process involves enteric neurons mediating the vagal signal and requires the presence of α7nAChR. These findings suggest that utilizing EA at ST36 may represent a possible therapeutic approach for POI and other immune-related gastrointestinal diseases.

The Alpha 7 Nicotinic Acetylcholine Receptor Does Not Affect Neonatal Brain Injury.

Inflammation plays a central role in the development of neonatal brain injury. The alpha 7 nicotinic acetylcholine receptor (α7nAChR) can modulate inflammation and has shown promising results as a treatment target in rodent models of adult brain injury. However, little is known about the role of the α7nAChR in neonatal brain injury. Hypoxic-ischemic (HI) brain injury was induced in male and female C57BL/6 mice, α7nAChR knock-out (KO) mice and their littermate controls on postnatal day (PND) 9–10. C57BL/6 pups received i.p. injections of α7nAChR agonist PHA 568487 (8 mg/kg) or saline once daily, with the first dose given directly after HI. Caspase-3 activity and cytokine mRNA expression in the brain was analyzed 24 h after HI. Motor function was assessed 24 and 48 h after HI, and immunohistochemistry was used to assess tissue loss at 24 h and 7 days after HI and microglial activation 7 days after HI. Activation of α7nAChR with the agonist PHA 568487 significantly decreased CCL2/MCP-1, CCL5/RANTES and IL-6 gene expression in the injured brain hemisphere 24 h after HI compared with saline controls in male, but not female, pups. However, α7nAChR activation did not alter caspase-3 activity and TNFα, IL-1β and CD68 mRNA expression. Furthermore, agonist treatment did not affect motor function (24 or 48 h), neuronal tissue loss (24 h or 7 days) or microglia activation (7 days) after HI in either sex. Knock-out of α7nAChR did not influence neuronal tissue loss 7 days after HI. In conclusion, targeting the α7nAChR in neonatal brain injury shows some effect on dampening acute inflammatory responses in male pups. However, this does not lead to an effect on overall injury outcome.

Alpha7 nicotinic acetylcholine receptor mediates chronic nicotine inhalation-induced cardiopulmonary dysfunction.

Cigarette smoking remains the leading modifiable risk factor for cardiopulmonary diseases; however, the effects of nicotine alone on cardiopulmonary function remain largely unknown. Previously, we have shown that chronic nicotine vapor inhalation in mice leads to the development of pulmonary hypertension (PH) with right ventricular (RV) remodeling. The present study aims to further examine the cardiopulmonary effects of nicotine and the role of the α7 nicotinic acetylcholine receptor (α7-nAChR), which is widely expressed in the cardiovascular system. Wild-type (WT) and α7-nAChR knockout (α7-nAChR-/-) mice were exposed to room air (control) or nicotine vapor daily for 12 weeks. Consistent with our previous study, echocardiography and RV catheterization reveal that male WT mice developed increased RV systolic pressure with RV hypertrophy and dilatation following 12-week nicotine vapor exposure; in contrast, these changes were not observed in male α7-nAChR-/- mice. In addition, chronic nicotine inhalation failed to induce PH and RV remodeling in female mice regardless of genotype. The effects of nicotine on the vasculature were further examined in male mice. Our results show that chronic nicotine inhalation led to impaired acetylcholine-mediated vasodilatory response in both thoracic aortas and pulmonary arteries, and these effects were accompanied by altered endothelial nitric oxide synthase phosphorylation (enhanced inhibitory phosphorylation at threonine 495) and reduced plasma nitrite levels in WT but not α7-nAChR-/- mice. Finally, RNA sequencing revealed up-regulation of multiple inflammatory pathways in thoracic aortas from WT but not α7-nAChR-/- mice. We conclude that the α7-nAChR mediates chronic nicotine inhalation-induced PH, RV remodeling and vascular dysfunction.

Alpha-7 Nicotinic Receptor Dampens Murine Osteoblastic Response to Inflammation and Age-Related Osteoarthritis

IntroductionOsteoarthritis (OA) is a whole-joint disease characterized by a low-grade inflammation that is involved in both cartilage degradation and subchondral bone remodeling. Since subchondral bone has a cholinergic innervation and that acetylcholine (Ach) might have an anti-inflammatory effect through the α7 nicotinic Ach receptor (α7nAchR), we aimed (i) to determine the expression of non-neuronal cholinergic system and nicotinic receptor subunits by murine and human osteoblasts, (ii) to address the role of α7nAchR in osteoblastic response to inflammation, and (iii) to study the role of α7nAchR in a spontaneous aging OA model.MethodsPrimary cultures of WT and α7nAchR knock-out mice (Chrna7-/-) murine osteoblasts and of subchondral bone human OA osteoblasts were performed. The expressions of the non-neuronal cholinergic system and of the nAchR subunits were assessed by PCR. In vitro, IL1β-stimulated WT, Chrna7-/-, and human osteoblasts were pretreated with nicotine. At 24 h, expressions of interleukin-6 (IL6) and metalloproteinase-3 and -13 (MMP), RANK-ligand (RANKL), and osteoprotegerin (OPG) were quantified by qPCR and ELISA. Spontaneous aging OA was evaluated and compared between male WT and Chrna7-/- mice of 9 and 12 months.ResultsMurine WT osteoblasts express the main components of the cholinergic system and α7 subunit composing α7nAchR. Nicotine partially prevented the IL1β-induced expression and production of IL6, MMP3, and RANKL in WT osteoblasts. The effect for IL6 and MMP was mediated by α7nAchR since nicotine had no effect on Chrna7-/- osteoblasts while the RANKL decrease persisted. Chrna7-/- mice displayed significantly higher cartilage lesions than their WT counterparts at 9 and 12 months, without difference in subchondral bone remodeling. Human OA osteoblasts also expressed the non-neuronal cholinergic system and α7 subunit as well as CHRFAM7A, the dominant negative duplicate of Chrna7. Nicotine pretreatment did not significantly reduce IL6 and MMP3 production in IL-1β-stimulated human osteoarthritic osteoblasts (n = 4), possibly due to CHRFAM7A.ConclusionCholinergic system counteracts murine osteoblastic response to IL-1β through α7nAchR. Since α7nAchR deletion may limit cartilage degradation during murine age-related OA, enhancing cholinergic system could be a new therapeutic target in OA but may depend on CHRFAM7A expression.

The alpha-7 nicotinic acetylcholine receptor agonist GTS-21 engages the glucagon-like peptide-1 incretin hormone axis to lower levels of blood glucose in db/db mice.

To establish if alpha-7 nicotinic acetylcholine receptor (α7nAChR) agonist GTS-21 exerts a blood glucose-lowering action in db/db mice, and to test if this action requires coordinate α7nAChR and GLP-1 receptor (GLP-1R) stimulation by GTS-21 and endogenous GLP-1, respectively. Blood glucose levels were measured during an oral glucose tolerance test (OGTT) using db/db mice administered intraperitoneal GTS-21. Plasma GLP-1, peptide tyrosine tyrosine 1-36(PYY1-36), glucose-dependent insulinotropic peptide (GIP), glucagon, and insulin levels were measured by ELISA. A GLP-1R-mediated action of GTS-21 that is secondary to α7nAChR stimulation was evaluated using α7nAChR and GLP-1R knockout (KO) mice, or by co-administration of GTS-21 with the dipeptidyl peptidase-4 inhibitor, sitagliptin, or the GLP-1R antagonist, exendin (9-39). Insulin sensitivity was assessed in an insulin tolerance test. Single or multiple dose GTS-21 (0.5-8.0mg/kg) acted in a dose-dependent manner to lower levels of blood glucose in the OGTT using 10-14 week-old male and female db/db mice. This action of GTS-21 was reproduced by the α7nAChR agonist, PNU-282987, was enhanced by sitagliptin, was counteracted by exendin (9-39), and was absent in α7nAChR and GLP-1R KO mice. Plasma GLP-1, PYY1-36, GIP, glucagon, and insulin levels increased in response to GTS-21, but insulin sensitivity, body weight, and food intake were unchanged. α7nAChR agonists improve oral glucose tolerance in db/db mice. This action is contingent to coordinate α7nAChR and GLP-1R stimulation. Thus α7nAChR agonists administered in combination with sitagliptin might serve as a new treatment for type 2 diabetes.

Endogenous α7 nAChR Agonist SLURP1 Facilitates Escherichia coli K1 Crossing the Blood-Brain Barrier.

Alpha 7 nicotinic acetylcholine receptor (α7 nAChR) is critical for the pathogenesis of Escherichia coli (E. coli) K1 meningitis, a severe central nervous system infection of the neonates. However, little is known about how E. coli K1 manipulates α7 nAChR signaling. Here, through employing immortalized cell lines, animal models, and human transcriptional analysis, we showed that E. coli K1 infection triggers releasing of secreted Ly6/Plaur domain containing 1 (SLURP1), an endogenous α7 nAChR ligand. Exogenous supplement of SLURP1, combined with SLURP1 knockdown or overexpression cell lines, showed that SLURP1 is required for E. coli K1 invasion and neutrophils migrating across the blood-brain barrier (BBB). Furthermore, we found that SLURP1 is required for E. coli K1-induced α7 nAChR activation. Finally, the promoting effects of SLURP1 on the pathogenesis of E. coli K1 meningitis was significantly abolished in the α7 nAChR knockout mice. These results reveal that E. coli K1 exploits SLURP1 to activate α7 nAChR and facilitate its pathogenesis, and blocking SLURP1-α7 nAChR interaction might represent a novel therapeutic strategy for E. coli K1 meningitis.

Activation of α7 nicotinic acetylcholine receptor ameliorates HIV-associated neurology and neuropathology.

HIV-associated neurocognitive disorders (HAND) in the era of combination antiretroviral therapy are primarily manifested as impaired behaviours, glial activation/neuroinflammation and compromised neuronal integrity, for which there are no effective treatments currently available.In the current study, we used doxycycline-inducible astrocyte-specific HIV Tat transgenic mice (iTat), a surrogate HAND model, and determined effects of PNU-125096, a positive allosteric modulator of α7 nicotinic acetylcholine receptor (α7 nAChR) on Tat-induced behavioural impairments and neuropathologies.We showed that PNU-125096 treatment significantly improved locomotor, learning and memory deficits of iTat mice while inhibited glial activation and increased PSD-95 expression in the cortex and hippocampus of iTat mice. Using α7 nAChR knockout mice, we showed that α7 nAChR knockout eliminated the protective effects of PNU-125096 on iTat mice. In addition, we showed that inhibition of p38 phosphorylation by SB239063, a p38 MAPK-specific inhibitor exacerbated Tat neurotoxicity in iTat mice. Last, we used primary mouse cortical individual cultures and neuron-astrocytes co-cultures and in vivo staining of iTat mouse brain tissues and showed that glial activation was directly involved in the interplay among Tat neurotoxicity, α7 nAChR activation and the p38 MAPK signalling pathway.Taken together, these findings demonstrated for the first time that α7 nAChR activation led to protection against HAND and suggested that α7 nAChR modulator PNU-125096 holds significant promise for development of therapeutics for HAND.

Chronic Nicotine Inhalation‐Induced Vascular Dysfunction is Mediated by the Alpha 7 Nicotinic Acetylcholine Receptor

Cigarette smoking remains a leading risk factor for cardiopulmonary pathologies. However, the effects of nicotine, the addictive component of cigarettes, on cardiopulmonary function is not fully understood. Our laboratory has shown previously that chronic nicotine inhalation in mice leads to the development of pulmonary hypertension with right ventricular (RV) remodeling. This study aims to further examine the molecular mechanisms of nicotine-induced vascular dysfunction. We hypothesize that chronic nicotine inhalation-induced cardiopulmonary dysfunction is mediated by the α7-nicotinic acetylcholine receptor (nAChR). To investigate our hypothesis, male C57BL6/J wildtype (WT) and α7-nAChR global knockout (KO) mice were exposed to air (WT, n=10; α7-nAChR KO, n=7) or nicotine vapor (WT, n=10; α7-nAChR KO, n=10) daily, 12 hour on/12 hour off, for 3 months. After the 3-month exposure, mice were subjected to right heart catheterization to measure right ventricular systolic pressure (RVSP) as an index of pulmonary artery pressure. In contrast to WT mice, which developed increased RVSP (31.4 ± 1.5 mmHg in nicotine-exposed compared to 24.0 ± 1.3 mmHg in air controls, p = 0.002), α7-nAChR KO mice were protected from these nicotine effects (25.7 ± 0.8 mmHg in nicotine-exposed compared to 21.6 ± 2.0 mmHg in air controls, p = 0.218). In addition, thoracic aortas were isolated from WT and α7-nAChR KO mice following air or nicotine exposure and subjected to RNA-sequencing analysis. Aortas isolated from WT mice showed 123 differentially expressed genes (112 up and 11 down) in nicotine-exposed compared to controls, with 18 upregulated and 3 downregulated pathways as identified by the Ingenuity Pathway Analysis (IPA). Many of the top upregulated pathways are involved in inflammatory response, including acute phase response signaling (p = 4.91E-06), complement system (p = 5.71E-06), and interleukin (IL)-6 signaling (p = 1.88E-03). Furthermore, the Angiotensin II receptor type 1, an important effector controlling blood pressure and volume, was upregulated 4.48-fold (Adjusted p = 0.038). In contrast, only 7 none-overlapping genes were differentially regulated in α7-nAChR KO mice by nicotine compared with the air controls. Finally, vasoreactivity assays were performed on aortas isolated from air and nicotine-exposed WT mice, and aortas isolated from nicotine-exposed mice exhibited impaired endothelium-dependent vasodilation. In conclusion, we found that chronic nicotine inhalation-induced cardiopulmonary dysfunction is mediated by the α7-nAChR.

Autophagy is Involved in Neuroprotective Effect of Alpha7 Nicotinic Acetylcholine Receptor on Ischemic Stroke.

The α7 nicotinic acetylcholine receptor (α7nAChR) belongs to the superfamily of cys loop cationic ligand-gated channels, which consists of homogeneous α7 subunits. Although our lab found that activation of α7nAChR could alleviate ischemic stroke, the mechanism is still unknown. Herein, we explored whether autophagy is involved in the neuroprotective effect mediated by α7nAChR in ischemic stroke. Transient middle cerebral artery occlusion (tMCAO) and oxygen and glucose deprivation (OGD/R) exposure were applied to in vivo and in vitro models of ischemic stroke, respectively. Neurological deficit score and infarct volume were used to evaluate outcomes of tMCAO in the in vivo study. Autophagy-related proteins were detected by Western blot, and autophagy flux was detected by using tandem fluorescent mRFP-GFP-LC3 lentivirus. At 24 h after tMCAO, α7nAChR knockout mice showed worse neurological function and larger infarct volume than wild-type mice. PNU282987, an α7nAChR agonist, protected against OGD/R-induced neuronal injury, enhanced autophagy, and promoted autophagy flux. However, the beneficial effects of PNU282987 were eliminated by 3-methyladenine (3-MA), an autophagy inhibitor. Moreover, we found that PNU282987 treatment could activate the AMPK-mTOR-p70S6K signaling pathway in the in vitro study, while the effect was attenuated by compound C, an AMPK inhibitor. Our results demonstrated that the beneficial effect on neuronal survival via activation of α7nAChR was associated with enhanced autophagy, and the AMPK-mTOR-p70S6K signaling pathway was involved in α7nAChR activation–mediated neuroprotection.

α7 Nicotinic Acetylcholine Receptor Agonists Regulate Inflammation and Growth Hormone Resistance in Sepsis.

During sepsis the normal induction of circulating insulin-like growth factor-I (IGF-I) by growth hormone (GH) action on liver is attenuated, a phenomenon called hepatic GH resistance. Hepatic GH resistance can be caused by cytokine-mediated activation of the NF-κB pathway which interferes with normal GH-signaling. The afferent and efferent fibers of the vagus nerve are integral to the cholinergic anti-inflammatory pathway (CAP) which attenuates hepatic TNFα production by activating the α7 nicotinic acetylcholine receptor (α7nAChR). We examined the effects of selective afferent vagotomy (SAV) and α7nAChR activation on sepsis-induced mortality, hepatic and systemic inflammation, the GH/IGF system and hepatic GH resistance using Sprague Dawley (SD) rats, C57BL/6 wild type (WT) mice, and α7nAChR knockout (KO) mice. Capsaicin was used to perform SAV and GTS-21 (α7nAChR agonist) was used to activate the α7nAChR. Sepsis-induced mortality, hepatic NF-κB activation, and plasma cytokine levels were increased in SAV rats and reduced in GTS-21-treated mice. The effects of sepsis on the GH/IGF-I system plasma IGF-I, IGF binding protein-1 (IGFBP-1), hepatic IGF-I, IGFBP-1, and GH receptor (GHR) mRNA and rhGH-responsiveness in mice were improved by GTS-21. Collectively these results confirm the protective effects of the anti-inflammatory CAP and α7nAChR activation in sepsis. They also provide evidence the CAP and α7nAChR activation could be used to attenuate hepatic GH resistance and anabolic failure in sepsis.

Melatonin Reduces NLRP3 Inflammasome Activation by Increasing α7 nAChR-Mediated Autophagic Flux.

Microglia controls the immune system response in the brain. Specifically, the activation and dysregulation of the NLRP3 inflammasome is responsible for the initiation of the inflammatory process through IL-1β and IL-18 release. In this work, we have focused on studying the effect of melatonin on the regulation of the NLRP3 inflammasome through α7 nicotinic receptor (nAChR) and its relationship with autophagy. For this purpose, we have used pharmacological and genetic approaches in lipopolysaccharide (LPS)-induced inflammation models in both in vitro and in vivo models. In the BV2 cell line, LPS inhibited autophagy, which increased NLRP3 protein levels. However, melatonin promoted an increase in the autophagic flux. Treatment of glial cultures from wild-type (WT) mice with LPS followed by extracellular adenosine triphosphate (ATP) produced the release of IL-1β, which was reversed by melatonin pretreatment. In cultures from α7 nAChR knock-out (KO) mice, melatonin did not reduce IL-1β release. Furthermore, melatonin decreased the expression of inflammasome components and reactive oxygen species (ROS) induced by LPS; co-incubation of melatonin with α-bungarotoxin (α-bgt) or luzindole abolished the anti-inflammatory and antioxidant effects. In vivo, melatonin reverted LPS-induced cognitive decline, reduced NLRP3 levels and promoted autophagic flux in the hippocampi of WT mice, whereas in α7 nAChR KO mice melatonin effect was not observed. These results suggest that melatonin may modulate the complex interplay between α7 nAChR and autophagy signaling.

The α7 nicotinic acetylcholine receptor is an important determinant of monocyte-derived macrophage migration

Abstract Inflammation is regulated by the nervous system. The α7 nicotinic acetylcholine receptor (α7nAChR) on macrophages plays a key role in this regulation by meditating cholinergic suppression of pro-inflammatory cytokine release within the vagus nerve-based inflammatory reflex during endotoxemia and other conditions. However, the involvement of of α7nAChR in other macrophage functions remains poorly understood. The objective of this study was to provide insight into the role of α7nAChR in macrophage migration and accumulation in organs during murine endotoxemia. Briefly, monocyte progenitors isolated from bone marrow of wild type (WT) and α7nAChR knockout (KO) mice were labeled with red PKH26 (WT) or green PKH67 (α7nAChR−/−) fluorescent dyes, mixed in equal amounts and injected in tail vein of WT mice 20 min before LPS administration. 48 hours later, lungs, livers and spleens were isolated, digested and analyzed using multicolor flow cytometry (Fortessa-X20) and imaging flow cytometry (ImageStream X Mark II). α7nAChR deficiency resulted in significantly decreased accumulation of macrophages in liver (~10 folds), lung (~4 folds) and spleen (~3 folds) (P<0.01). The morphology of migrated macrophage was verified by imaging flow cytometry. These results identify α7nAChR as an important determinant of the recruitment of monocytes to the site of acute inflammation. They also provide a rationale for further studies on the molecular mechanisms, including the involvement of adhesive receptors in α7nAChR-dependent monocyte/macrophage migration.

Preoperative administration of the 5-HT4 receptor agonist prucalopride reduces intestinal inflammation and shortens postoperative ileus via cholinergic enteric neurons

ObjectivesVagus nerve stimulation (VNS), most likely via enteric neurons, prevents postoperative ileus (POI) by reducing activation of alpha7 nicotinic receptor (α7nAChR) positive muscularis macrophages (mMφ) and dampening surgery-induced intestinal inflammation....

Nicotinic acetylcholine receptor subunit α7-knockout mice exhibit degraded auditory temporal processing.

The CHRNA7 gene that encodes the α7-subunit of the nicotinic acetylcholine receptor (α7-nAChR) has been associated with some autism spectrum disorders and other neurodevelopmental conditions characterized, in part, by auditory and language impairment. These conditions may include auditory processing disorders that represent impaired timing of neural activity, often accompanied by problems understanding speech. Here, we measure timing properties of sound-evoked activity via the auditory brainstem response (ABR) of α7-nAChR knockout mice of both sexes and wild-type colony controls. We find a significant timing delay in evoked ABR signals that represents midbrain activity in knockouts. We also examine spike-timing properties of neurons in the inferior colliculus, a midbrain nucleus that exhibits high levels of α7-nAChR during development. We find delays of evoked responses along with degraded spiking precision in knockout animals. We find similar timing deficits in responses of neurons in the superior paraolivary nucleus and ventral nucleus of the lateral lemniscus, which are brainstem nuclei thought to shape temporal precision in the midbrain. In addition, we find that other measures of temporal acuity including forward masking and gap detection are impaired for knockout animals. We conclude that altered temporal processing at the level of the brainstem in α7-nAChR-deficient mice may contribute to degraded spike timing in the midbrain, which may underlie the observed timing delay in the ABR signals. Our findings are consistent with a role for the α7-nAChR in types of neurodevelopmental and auditory processing disorders and we identify potential neural targets for intervention.NEW & NOTEWORTHY Disrupted signaling via the α7-nicotinic acetylcholine receptor (α7-nAChR) is associated with neurodevelopmental disorders that include impaired auditory processing. The underlying causes of dysfunction are not known but a common feature is abnormal timing of neural activity. We examined temporal processing of α7-nAChR knockout mice and wild-type controls. We found degraded spike timing of neurons in knockout animals, which manifests at the level of the auditory brainstem and midbrain.

Nicotinic alpha-7 acetylcholine receptor deficiency exacerbates hepatic inflammation and fibrosis in a mouse model of non-alcoholic steatohepatitis.

Aims/IntroductionNon‐alcoholic steatohepatitis (NASH), which occurs in association with insulin resistance and hepatic fat accumulation, is characterized by chronic liver injury and fibrosis. NASH onset and progression is closely related to hepatic inflammation, which is partly regulated by the vagus nerve through the α7 nicotinic acetylcholine receptor (α7nAchR). Hepatic α7nAchR action is impeded in obesity and insulin resistance. In the present study, using α7nAchR knockout (α7KO) mice, we elucidated the effect of α7nAchR deficiency on NASH‐related inflammation and fibrosis.Materials and Methodsα7KO mice were fed an atherogenic high‐fat diet (AD) for 32 weeks or methionine/choline‐deficient diet (MCD) for 6 weeks, both of which induce NASH. Mice were then examined for the degree of NASH‐related inflammation and fibrosis by hepatic gene expression analysis and Sirius red histological staining.ResultsHepatic triglyceride accumulation and elevated plasma transaminase levels were observed in both AD and MCD mice, but the plasma transaminase level increase was higher in α7KO mice than in control mice. α7KO mice fed an AD showed significant upregulation of the Col1a1 gene encoding alpha‐1 type I collagen, which is involved in liver fibrosis, and the Ccl2 gene encoding C‐C motif chemokine ligand 2, a pro‐inflammatory chemokine; α7KO mice fed an MCD had significant upregulation of the Col1a1 gene and the Tnf gene, an inflammatory cytokine. Histological analysis showed that AD and MCD exacerbated liver fibrosis in α7KO mice.ConclusionsThe results of this study suggest that α7nAchR deficiency exacerbates hepatic inflammation and fibrosis in a diet‐induced mouse model of NASH.

Neuronal nicotinic acetylcholine receptors mediate ∆9 -THC dependence: Mouse and human studies.

Cessation from prolonged use of ∆9 -tetrahydrocannabinol (THC), the primary active compound responsible for the cannabimimetic effects of cannabis, results in a mild to moderate withdrawal syndrome in humans and laboratory animals. Whereas manipulations of the endogenous cannabinoid system (eg, cannabinoid receptors and endocannabinoid regulating enzymes) alter nicotine withdrawal, in this study we asked the reciprocal question. Do nicotinic acetylcholine receptors (nAChRs) modulate THC withdrawal? To assess the role of different nAChR subtypes in THC withdrawal, we used transgenic mouse, preclinical pharmacological, and human genetic correlation approaches. Our findings show that selective α3β4* nAChR antagonist, AuIB, and α3β4* nAChR partial agonist, AT-1001, dose-dependently attenuated somatic withdrawal signs in THC-dependent mice that were challenged with the cannabinoid-1 receptor antagonist rimonabant. Additionally, THC-dependent α5 and α6 nAChR knockout (KO) mice displayed decreased rimonabant precipitated somatic withdrawal signs compared with their wild-type counterparts. In contrast, β2 and α7 nAChR KO mice showed no alterations in THC withdrawal signs. Moreover, deletion of β2 nAChR did not alter the reduced expression of somatic signs by the preferred α6β4* antagonist, BulA [T5A;P60]. Finally, the human genetic association studies indicated that variations in the genes that code for the α5, α3, β4, and α6 nAChRs were associated with cannabis disorder phenotypes. Overall, these findings suggest that α3β4* and α6β4* nAChR subtypes represent viable targets for the development of medications to counteract THC dependence.

Possible anti-inflammatory role of Zingiberis processum rhizoma, one component of the Kampo formula daikenchuto, against neutrophil infiltration through muscarinic acetylcholine receptor activation

Zingiberis processum rhizoma (ZPR) is a major active component of daikenchuto (DKT), which induces anti-inflammatory action by inhibiting macrophage infiltration. However, it is unclear whether ZPR is related to DKT-induced anti-inflammatory action via a reduction of neutrophil infiltration against postoperative ileus (POI). In this study, we orally administered individual herbal components of DKT to mice four times before and after intestinal manipulation (IM). The anti-inflammatory action of each crude drug was evaluated by histochemical analysis of relevant molecules. The results showed that treatment with all herbal components of DKT significantly inhibits neutrophil infiltration. This inhibition of neutrophil infiltration by ZPR was significantly reduced in 5-hydroxytryptamine receptor 4 (5-HT4R) knockout (KO) mice but not in alpha-7 nicotinic acetylcholine receptor (α7nAChR) KO mice. Also, transient receptor potential ankyrin 1 (TRPA1) and muscarinic acetylcholine receptor (mAChR) antagonists partly and significantly inhibited the amelioration of neutrophil infiltration by ZPR. Therefore, DKT-induced anti-inflammatory action, mediated by inhibition of neutrophil infiltration in POI, depends, in part, on the effects of ZPR. ZPR activates TRPA1 channels, possibly in enterochromaffin (EC) cells, to release 5-HT. This 5-HT stimulates 5-HT4R in the myenteric plexus neurons to release acetylcholine, which, in turn, activates mAChR to inhibit inflammation in POI.

Abstract 602: The α7 nicotinic acetylcholine receptor silent agonist R-47 prevents and reverses paclitaxel-induced peripheral neuropathy without enhancing the proliferation of lung cancer cells or interfering with paclitaxel-induced antitumor activity

Abstract While cancer chemotherapy continues to significantly contribute to the number of cancer survivors, exposure to these drugs can often result in chemotherapy-induced peripheral neuropathy (CIPN), a consequence of peripheral nerve fiber dysfunction or degeneration. CIPN is characterized by sensory symptoms such as numbness, burning, and allodynia, resulting in an overall decrease in quality of life. Paclitaxel (Taxol), a microtubule poison that is commonly used to treat breast, lung, and ovarian cancers, has been found to cause CIPN in 59-78% of cancer patients. There is currently no effective preventative or therapeutic treatment for this side effect, which can be a dose-limiting factor for chemotherapy or delay treatment. Our laboratories have previously shown that nicotine, the prototypical nicotinic acetylcholine receptor (nAChR) agonist, can prevent and reverse paclitaxel-induced peripheral neuropathy without increasing lung tumor growth in mice. Our current studies have demonstrated that the α7 nAChR silent agonist R-47 may be a promising preventative and therapeutic treatment for CIPN as well. R-47 (10 mg/kg, p.o.) was administered to male C57BL/6J mice treated with paclitaxel (8 mg/kg, i.p., every other day for a total of four injections). Von Frey filament testing revealed that R-47 can both prevent and reverse paclitaxel-induced mechanical allodynia; the latter effect can be inhibited by the α7 nAChR antagonist methyllycaconitine. In support of these findings, R-47 also prevents the reduction of intra-epidermal nerve fibers in the hind paws of paclitaxel-treated mice. In addition, α7 nAChR knockout mice exhibit a greater and sustained increase in mechanical allodynia after paclitaxel administration when compared to wild-type mice, suggesting the involvement of this receptor subtype in hindering CIPN development and/or maintenance. With regard to cancer, R-47 fails to increase A549 and H460 non-small cell lung cancer cell viability, colony formation, or proliferation alone, and does not interfere with paclitaxel-induced growth arrest, apoptosis, or DNA fragmentation. Most importantly, R-47 does not increase the growth of A549 tumors or interfere with the antitumor activity of paclitaxel in tumor-bearing mice. These data suggest that the use of an α7 nAChR silent agonist may be a safe and efficacious approach for the prevention and treatment of CIPN. Citation Format: S Lauren Kyte, Wisam Toma, Ganeshsingh Thakur, M Imad Damaj, David A. Gewirtz. The α7 nicotinic acetylcholine receptor silent agonist R-47 prevents and reverses paclitaxel-induced peripheral neuropathy without enhancing the proliferation of lung cancer cells or interfering with paclitaxel-induced antitumor activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 602.

Cigarette toxin 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) induces experimental pancreatitis through α7 nicotinic acetylcholine receptors (nAChRs) in mice.

Clinical studies have shown that cigarette smoking is a dose-dependent and independent risk factor for acute pancreatitis. Cigarette smoke contains nicotine which can be converted to the potent receptor ligand and toxin, NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone]. Previously, we have shown that NNK induces premature activation of pancreatic zymogens in rats, an initiating event in pancreatitis, and this activation is prevented by pharmacologic inhibition of nicotinic acetylcholine receptors (nAChR). In this study, we determined whether NNK mediates pancreatitis through the α7 isoform of nAChR using α7nAChR knockout mice. PCR analysis confirmed expression of non-neuronal α7nAChR in C57BL/6 (WT) mouse and human acinar cells. NNK treatment stimulated trypsinogen activation in acini from WT but not α7nAChR-/- mice. NNK also stimulated trypsinogen activation in human acini. To further confirm these findings, WT and α7nAChR-/- mice were treated with NNK in vivo and markers of pancreatitis were measured. As observed in acini NNK treatment induced trypsinogen activation in WT but not α7nAChR-/- mice. NNK also induced other markers of pancreatitis including pancreatic edema, vacuolization and pyknotic nuclei in WT but not α7nAChR-/- animals. NNK treatment led to increased neutrophil infiltration, a marker of inflammation, in WT mice and to a significantly lesser extent in α7nAChR-/- mice. We also examined downstream targets of α7nAChR activation and found that calcium and PKC activation are involved down stream of NNK stimulation of α7nAChR. In this study we used genetic deletion of the α7nAChR to confirm our previous inhibitor studies that demonstrated NNK stimulates pancreatitis by activating this receptor. Lastly, we demonstrate that NNK can also stimulate zymogen activation in human acinar cells and thus may play a role in human disease.