α7nAChR Agonist Research Articles

Antiproliferative effect of GTS-21 in glioblastoma cells.

Glioblastoma multiforme (GBM) is the most common malignant brain tumour in adults. The poor prognosis and short median overall survival of patients with GBM is associated with resistance to therapy after surgical and adjuvant treatment. The expression of various acetylcholine receptors (AChR) in GBM has been widely reported. The present study aimed to investigate the expression of cholinergic system-related genes in primary GBM and to explore the antiproliferative effect of 3-(2,4-dimethoxybenzylidene) anabaseine (GTS-21) in GBM cell lines. Therefore, the expression of 28 genes associated with the cholinergic system was detected using a customized RT2 Profiler PCR Array in 44 GBM and 5 healthy control brain tissue samples. In addition, the activity of GTS-21, an alpha 7 subunit nicotinic AChR (α7 nAChR) agonist, and that of α-bungarotoxin (α-BTX), an α7 nAChR antagonist, was determined in primary and established GBM cells. Therefore, the A172, U87 and G28 cell lines and primary GBM cells were treated with GTS-21, ACh or nicotine. Cell viability was evaluated using MTT assay at 24, 48 and 72 h following cell treatment with the corresponding compounds. The results revealed that the expression of cholinergic system-related components was notably downregulated, except that of cholinergic receptor nicotinic alpha 7 subunit (CHRNA7), in primary GBM and U87 cells. However, the dominant-negative duplicate form of CHRNA7 was also downregulated. Furthermore, A172 and G28 cells exhibited a heterogeneous gene expression pattern. Additionally, GTS-21 inhibited the proliferation of GBM cells in a dose- and time-dependent manner. Interestingly, treatment with α-BTX restored the proliferation of U87 cells, but not that of A172 and G28 cells. Collectively, the findings of the present study suggested that GTS-21 may inhibit the proliferation of GBM cells and may therefore serve as a novel therapeutic approach to the treatment of GBM, which warrants further investigation.

Geniposide protects pulmonary arterial smooth muscle cells from lipopolysaccharide‑induced injury via α7nAchR‑mediated TLR‑4/MyD88 signaling

Geniposide is a bioactive iridoid glucoside derived from Gardenia jasminoides that has proven anti-inflammatory effects against acute lung injury. The aim of this study was to determine whether geniposide could protect pulmonary arterial smooth muscle cells (PASMCs) from lipopolysaccharide (LPS)-induced injury and to explore the participation of α7 nicotinic acetylcholine receptor (α7nAChR), which was previously reported to suppress pro-inflammatory cytokine production in LPS-stimulated macrophages. In the present study, rat PASMCs were isolated and stimulated using LPS. The effect of geniposide on LPS-induced PASMC injury was then explored. Geniposide exerted anti-apoptotic and anti-inflammatory effects on LPS-treated PASMCs, as demonstrated by the downregulation of pro-apoptotic proteins and pro-inflammatory cytokines, respectively. Furthermore, the α7nAChR agonist PNU282987 accentuated the protective effect of geniposide against LPS-induced injury in PASMCs by inhibiting toll-like receptor-4/myeloid differentiation primary response 88 (TLR-4/MyD88) signaling and downregulating nuclear factor (NF)-κB expression. Conversely, methyllycaconitine, an inhibitor of α7nAChR, attenuated the effects of geniposide. These findings collectively suggested that in conjunction with geniposide, the activation of α7nAChR may contribute to further mitigating LPS-induced PASMC apoptosis and inflammation. In addition, the underlying mechanisms critically involve the NF-κB/MyD88 signaling axis. These results may provide novel insights into the treatment and management of lung diseases via geniposide administration.

Sinomenine increases adenosine A2A receptor and inhibits NF-κB to inhibit arthritis in adjuvant-induced-arthritis rats and fibroblast-like synoviocytes through α7nAChR.

Sinomenine (SIN) is a clinical drug for treating rheumatoid arthritis (RA) in China. Our previous study found SIN inhibited inflammation via alpha7 nicotinic acetylcholine receptor (α7nAChR) in macrophages in vitro. Adenosine receptor A2A has anti-inflammatory and immunosuppressive function. However, the mechanisms of SIN acting on α7nAChR and the effect on adenosine A2A receptor (A2A R) in RA are not clear. In the present study, the effects of SIN on adjuvant-induced-arthritis (AIA) rats in vivo and on fibroblast-like synoviocytes (FLSs) in vitro were investigated. Indomethacin (Indo) and methotrexate (MTX), the clinical anti-arthritis drugs, were used as controls. Nicotine (Nic), a specific agonist of α7nAChR, was used as a control for targeting α7nAChR. Alpha-bungarotoxin (α-BTX), the antagonist of α7nAChR or small interference RNA (siRNA) was used to block or knock down α7nAChR. Results showed that SIN decreased arthritis index, hind paw volume, erythrocyte sedimentation (ESR) and serum TNF-α in AIA rats, and α-BTX attenuated the earlier-mentioned effects of SIN and Nic, but not Indo and MTX. The expressions of A2A R in synovium declined in AIA rats, but remarkably increased after the intervention of SIN. The expression of A2A R decreased by LPS or TNF-α, but increased by SIN; cAMP also increased by SIN in FLSs in vitro. SIN inhibited the expression of MCP-1, IL-6, and vascular endothelial growth factor in LPS-induced FLSs. SIN inhibited the activation of NF-κB. Meanwhile, α-BTX or α7nAChR siRNA blocked the earlier-mentioned effects of SIN in FLSs. Results suggested the expressions of A2A R in synovium and FLSs are negatively correlated with the arthritis progression of AIA rats and the activation of FLSs. SIN increases A2A R and inhibits the activation of NF-κB pathway via α7nAChR in AIA rats and FLSs.

Synaptic Components, Function and Modulation Characterized by GCaMP6f Ca2+ Imaging in Mouse Cholinergic Myenteric Ganglion Neurons.

The peristaltic contraction and relaxation of intestinal circular and longitudinal smooth muscles is controlled by synaptic circuit elements that impinge upon phenotypically diverse neurons in the myenteric plexus. While electrophysiological studies provide useful information concerning the properties of such synaptic circuits, they typically involve tissue disruption and do not correlate circuit activity with biochemically defined neuronal phenotypes. To overcome these limitations, mice were engineered to express the sensitive, fast Ca2+ indicator GCaMP6f selectively in neurons that express the acetylcholine (ACh) biosynthetic enzyme choline acetyltransfarse (ChAT) thereby allowing rapid activity-driven changes in Ca2+ fluorescence to be observed without disrupting intrinsic connections, solely in cholinergic myenteric ganglion (MG) neurons. Experiments with selective receptor agonists and antagonists reveal that most mouse colonic cholinergic (i.e., GCaMP6f+/ChAT+) MG neurons express nicotinic ACh receptors (nAChRs), particularly the ganglionic subtype containing α3 and β4 subunits, and most express ionotropic serotonin receptors (5-HT3Rs). Cholinergic MG neurons also display small, spontaneous Ca2+ transients occurring at ≈ 0.2 Hz. Experiments with inhibitors of Na+ channel dependent impulses, presynaptic Ca2+ channels and postsynaptic receptor function reveal that the Ca2+ transients arise from impulse-driven presynaptic activity and subsequent activation of postsynaptic nAChRs or 5-HT3Rs. Electrical stimulation of axonal connectives to MG evoked Ca2+ responses in the neurons that similarly depended on nAChRs or/and 5-HT3Rs. Responses to single connective shocks had peak amplitudes and rise and decay times that were indistinguishable from the spontaneous Ca2+ transients and the largest fraction had brief synaptic delays consistent with activation by monosynaptic inputs. These results indicate that the spontaneous Ca2+ transients and stimulus evoked Ca2+ responses in MG neurons originate in circuits involving fast chemical synaptic transmission mediated by nAChRs or/and 5-HT3Rs. Experiments with an α7-nAChR agonist and antagonist, and with pituitary adenylate cyclase activating polypeptide (PACAP) reveal that the same synaptic circuits display extensive capacity for presynaptic modulation. Our use of non-invasive GCaMP6f/ChAT Ca2+ imaging in colon segments with intrinsic connections preserved, reveals an abundance of direct and modulatory synaptic influences on cholinergic MG neurons.

Varenicline limits ischemia reperfusion injury following testicular torsion in mice

Torsion of the spermatic cord and the resulting testicular ischemia leads to the production of inflammatory cytokines and cell death due to impaired aerobic metabolism. Following reperfusion of the testis, a robust innate inflammatory response furthers tissue injury due to the production of reactive oxygen species and disruption of normal capillary function. Blunting the innate immune response with antioxidants, anti-inflammatory medications and targeted genetic interventions reduces long term testicular injury in animal models of torsion, however these approaches have limited clinical applicability. Mediated via α7 nACh receptors, the cholinergic anti-inflammatory pathway limits NFKB signaling and prevents renal fibrosis following warm renal ischemia. We identified varenicline as an FDA approved α7 nAChR agonist and hypothesized that varenicline administration would decrease long-term testicular atrophy and fibrosis in a murine model of testicular torsion. Using an established model, unilateral testicular torsion was induced in mature male CD1 mice by rotating the right testicle 720° for 2h. In the treatment group, 4 doses of varenicline (1mg/grm) were administered via intraperitoneal injection every 12h, with the first dose given 1h after the creation of testicular torsion. The acute inflammatory response was evaluated 48h following reperfusion of the testis. Long term outcomes were evaluated 30 days following testicular perfusion. 48h following reperfusion, the testis of animals treated with varenicline demonstrated a significant reduction in the inflammatory response as measured by the acute immune cell infiltrate, myeloperoxidase activity, concentration of reduced glutathione and expression of downstream NF-KB targets. 30 days following reperfusion, animals treated with varenicline, demonstrated decreased testicular atrophy (Summary Figure), fibrosis and expression of pro-fibrotic genes. Activation of a central immunosuppressive cascade with varenicline after the onset of testicular torsion reduces ischemia reperfusion injury and prevents long term testicular atrophy and fibrosis. Further studies are needed to define the optimum dose and varenicline administration regimen. Our results suggest that varenicline offers a novel, FDA approved, adjunct to the current management of testicular torsion.

Curcumin Potentiates α7 Nicotinic Acetylcholine Receptors and Alleviates Autistic-Like Social Deficits and Brain Oxidative Stress Status in Mice.

Autistic spectrum disorder (ASD) refers to a group of neurodevelopmental disorders characterized by impaired social interaction and cognitive deficit, restricted repetitive behaviors, altered immune responses, and imbalanced oxidative stress status. In recent years, there has been a growing interest in studying the role of nicotinic acetylcholine receptors (nAChRs), specifically α7-nAChRs, in the CNS. Influence of agonists for α7-nAChRs on the cognitive behavior, learning, and memory formation has been demonstrated in neuro-pathological condition such as ASD and attention-deficit hyperactivity disorder (ADHD). Curcumin (CUR), the active compound of the spice turmeric, has been shown to act as a positive allosteric modulator of α7-nAChRs. Here we hypothesize that CUR, acting through α7-nAChRs, influences the neuropathology of ASD. In patch clamp studies, fast inward currents activated by choline, a selective agonist of α7-nAChRs, were significantly potentiated by CUR. Moreover, choline induced enhancement of spontaneous inhibitory postsynaptic currents was markedly increased in the presence of CUR. Furthermore, CUR (25, 50, and 100 mg/kg, i.p.) ameliorated dose-dependent social deficits without affecting locomotor activity or anxiety-like behaviors of tested male Black and Tan BRachyury (BTBR) mice. In addition, CUR (50 and 100 mg/kg, i.p.) mitigated oxidative stress status by restoring the decreased levels of superoxide dismutase (SOD) and catalase (CAT) in the hippocampus and the cerebellum of treated mice. Collectively, the observed results indicate that CUR potentiates α7-nAChRs in native central nervous system neurons, mitigates disturbed oxidative stress, and alleviates ASD-like features in BTBR mice used as an idiopathic rodent model of ASD, and may represent a promising novel pharmacological strategy for ASD treatment.

Modulation of α7nAchR by Melatonin Alleviates Ischemia and Reperfusion-Compromised Integrity of Blood-Brain Barrier Through Inhibiting HMGB1-Mediated Microglia Activation and CRTC1-Mediated Neuronal Loss.

The only food and drug administration (FDA)-approved drug currently available for the treatment of acute ischemic stroke is tissue plasminogen activator (tPA), yet the therapeutic benefits of this drug are partially outweighed by the increased risk of hemorrhagic transformation (HT). Analysis of the NIH trial has shown that cigarette smoking protected tPA-treated patients from HT; however, the underlying mechanism is not clear. Nicotinic acetylcholine receptors (nAChR) has shown anti-inflammatory effect and modulation nAChR could be a strategy to reduce ischemia/reperfusion-induced blood-brain barrier (BBB) damage. Since melatonin could regulate the expression of α7nAchR and melatonin's neuroprotective effect against ischemic injury is mediated via α7nAChR modulation, here, we aim to test the hypothesis that melatonin reduces ischemia and reperfusion (I/R)-induced BBB damage through modulation of α7nACh receptor (α7nAChR). Mice were subjected to 1.5 h ischemia and 24 h reperfusion and at the onset of reperfusion, mice received intraperitoneal administration (i.p.) of either drug or saline. Mice were randomly assigned into five groups: Saline; α7nAChR agonist PNU282987; Melatonin; Melatonin+Methyllycaconitine (MLA, α7nAChR antagonist), and MLA group. BBB permeability was assessed by detecting the extravasation of Evan's blue and IgG. Our results showed that I/R significantly increased BBB permeability accompanied by occludin degradation, microglia activation, and high mobility group box 1 (HMGB1) release from the neuron. In addition, I/R significantly induced neuronal loss accompanied by the decrease of CREB-regulated transcriptional coactivator 1 (CRTC1) and p-CREB expression. Melatonin treatment significantly inhibited the above changes through modulating α7nAChR. Taken together, these results demonstrate that melatonin provides a protective effect on ischemia/reperfusion-induced BBB damage, at least in part, depending on the modulation of α7nAChR.

The selective alpha7 nicotinic acetylcholine receptor agonist AR-R17779 does not affect ischemia-reperfusion brain injury in mice.

Inflammation plays a central role in stroke-induced brain injury. The alpha7 nicotinic acetylcholine receptor (α7nAChR) can modulate immune responses in both the periphery and the brain. The aims of the present study were to investigate α7nAChR expression in different brain regions and evaluate the potential effect of the selective α7nAChR agonist AR-R17779 on ischemia–reperfusion brain injury in mice. Droplet digital PCR (ddPCR) was used to evaluate the absolute expression of the gene encoding α7nAChR (Chrna7) in hippocampus, striatum, thalamus and cortex in adult, naïve mice. Mice subjected to transient middle cerebral artery occlusion (tMCAO) or sham surgery were treated with α7nAChR agonist AR-R17779 (12 mg/kg) or saline once daily for 5 days. Infarct size and microglial activation 7 days after tMCAO were analyzed using immunohistochemistry. Chrna7 expression was found in all analyzed brain regions in naïve mice with the highest expression in cortex and hippocampus. At sacrifice, white blood cell count was significantly decreased in AR-R17779 treated mice compared with saline controls in the sham groups, although, no effect was seen in the tMCAO groups. Brain injury and microglial activation were evident 7 days after tMCAO. However, no difference was found between mice treated with saline or AR-R17779. In conclusion, α7nAChR expression varies in different brain regions and, despite a decrease in white blood cells in sham mice receiving AR-R17779, this compound does not affect stroke-induced brain injury.

Expression of the α7 Nicotinic Acetylcholine Receptor Is Critically Required for the Antifibrotic Effect of PHA-543613 on Skin Fibrosis

Introduction: Targeting the α7 nicotinic acetylcholine receptor (α7nAChR) has recently been suggested as a potential new treatment for fibrotic skin diseases. Here, we performed a genetic and pharmacologic approach to clarify the role of this receptor in the bleomycin (BLM) mouse model of skin fibrosis using α7nAChR KO mice. Methods: We analyzed the expression of extracellular matrix (ECM) components in murine skin using quantitative RT-PCR, pepsin digestion/SDS-PAGE of proteins and performed hydroxyproline assays as well as histological/immunohistochemical staining of skin sections. To identity the target cells of the α7nAChR agonist PHA-543613, we used murine dermal fibroblasts (MDF). We tested their response to the profibrotic cytokine transforming growth factor-β₁ (TGF-β₁) and utilized gene silencing to elucidate the role of the α7nAChR. Results: We confirmed our previous findings on C3H/HeJ mice and detected a suppressive effect of PHA-543613 on BLM-induced skin fibrosis in the mouse strain C57BL/6J. This antifibrotic effect of PHA-543613 was abrogated in α7nAChR-KO mice. Interestingly, α7nAChR-KO animals exhibited a basal profibrotic signature by higher RNA expression of ECM genes and hydroxyproline content than WT mice. In WT MDF, PHA-543613 suppressed ECM gene expression induced by TGF-β₁. Gene silencing of α7nAChR by small interfering RNA neutralized the effects of PHA-543613 on TGF-β₁-mediated ECM gene expression. Conclusion: In summary, we have identified the α7nAChR as the essential mediator of the antifibrotic effect of PHA-543613. MDF are directly targeted by PHA-543613 to suppress collagen synthesis. Our findings emphasize therapeutic exploitation of α7nAChR receptor agonists in fibrotic skin diseases.

Cynandione A and PHA-543613 inhibit inflammation and stimulate macrophageal IL-10 expression following α7 nAChR activation

Cynandione A, an acetophenone isolated from Cynanchum Wilfordii Radix, attenuates inflammation. The present study aimed to study the mechanisms underlying cynandione A-induced antiinflammation. Treatment with cynandione A and the specific α7 nicotinic acetylcholine receptor (α7 nAChR) agonist PHA-543613 remarkably reduced overexpression of proinflammatory cytokines, including tumor necrosis factor (TNF)-α, interleukin (IL)-6 and IL-1β in lipopolysaccharide (LPS)-treated RAW264.7 cells and primary peritoneal macrophages, and endotoxemic mice. Both cynandione A and PHA-543613 also stimulated IL-10 expression in naïve and LPS-treated macrophages and endotoxemic mice. Cynandione A- and PHA-543613-inhibited proinflammatory cytokine expression was completely blocked by the α7 nAChR antagonist methyllycaconitine and the IL-10 antibody. The stimulatory effect of cynandione A and PHA-543613 on IL-10 expression were suppressed by methyllycaconitine and knockdown of α7 nAChRs using siRNA/α7 nAChR. Cynandione A significantly stimulated STAT3 phosphorylation, which was attenuated by methyllycaconitine and the IL-10 neutralizing antibody. The STAT3 activation inhibitor NSC74859 also blocked cynandione A-inhibited proinflammatory cytokine expression. Taken together, our results, for the first time, demonstrate that cynandione A and PHA-543613 inhibit inflammation through macrophageal α7 nAChR activation and subsequent IL-10 expression.

Anti‐inflammatory cholinergic system modulates LPS‐induced acute lung injury

Lung cholinergic system suing acetylcholine (ACh) as a neurotransmitter regulates lung inflammation and is believed to play an anti-inflammatory role via activation of nicotinic (nAChR) and/or muscarinic (mAChR) acetylcholine receptors expressed in lung tissue. Therefore, studies that evaluate the effects of lung cholinergic deficiency are relevant to understand the multiple actions of ACh in acute lung injury (ALI). Aim To evaluate whether VAChT deficiency and treatment with agonist α-7 nAChR affects lung alterations observed in an experimental model of ALI. Methods Wild-Type (WT) mice and mice with decreased expression of VAChT KDHOM, which is correlated with 70% less of ACh release, received PNU-282987 (specific α7nAChR agonist,10 mg/kg) 6 hours after LPS (1mg/kg) intratracheal instillation. We evaluated lung inflammation focusing on inflammation in bronchioalveolar lavage fluid (BALF). We also compared the expression levels of α-7 and α-4 nAChRs in lung tissue using radioligand binding assays. Results LPS treatment induced higher increase in inflammatory cells in BALF in VAChT KDHOM compared to WT mice. The activation α-7 nAChR by treatment with PNU-282987 reduced the total cells, macrophages, and neutrophils recovered in BALF (p<0.001 for all comparisons) in both VAChT-KDHOM and WT that received LPS. The LPS induced upregulate α-7 nAChR levels in WT mice but not in the VAChT KDHOM mice. However, treatment with PNU-282987 induced increase in α-7 nAChR levels in LPS-treated VAChT-KDHOM mice. Unlike α-7 nAChR, α-4 nAChR expression was not changed by VAChT reduction, LPS-induce, and PNU-282987 treatment. Conclusion Our preliminary results suggest that α-7 nAChR upregulation contributes to the anti-inflammatory effect of lung cholinergic system. Unlike WT mice, VAChT-KDHOM mice did not upregulate α-7 nAChR in response to inflammatory insult (LPS treatment) resulting in higher inflammation in VAChT-KDHOM mice than WT mice. Furthermore, treatment of VAChT-KDHOM mice with PNU-282987 resulted in increase in α-7 nAChR expression in lung tissue and reduction of LPS-induced lung inflammation. Experiments to determine mAChRs expression in WT vs. VAChT-KDHOM mice are ongoing.

Repeated treatment with alpha 7 nicotinic acetylcholine receptor ligands enhances cognitive processes and stimulates Erk1/2 and Arc genes in rats

The α7 nicotinic acetylcholine receptor (α7 nAChR) is a potential target for the treatment of cognitive decline in patients with schizophrenia, Alzheimer’s disease, and attention-deficit/hyperactivity disorder.Here we examined the promnesic activity of the α7 nAChR agonist (A582941), the type I (CCMI), and the type II (PNU120596) positive allosteric modulators (PAMs) in rats following single and repeated (once daily for seven days) treatment. To determine the neuronal mechanisms underlying the procognitive activity of the tested compounds, levels of the extracellular signal-regulated kinases (Erk1/2) and the activity-regulated cytoskeleton-associated protein (Arc) mRNAs were assessed in the frontal cortical and hippocampal brain regions.Using the novel object recognition test, we demonstrate that the lower doses of A582941 (0.1 mg/kg), CCMI (1 mg/kg), and PNU120596 (0.3 mg/kg) improved recognition memory after repeated but not single administration, suggesting a cumulative effect of repeated dosing. In contrast, the higher doses of A582941 (0.3 mg/kg), CCMI (3 mg/kg) and PNU120596 (1 mg/kg) demonstrated promnesic efficacy following both single and repeated administration. Subsequent in situ hybridization revealed that repeated treatment with A582941 and CCMI, but not PNU120596 enhanced mRNA expression of the Erk1/2 and Arc in the frontal cortex and hippocampus.Present data suggest that both the α7 nAChR agonist and PAMs exhibit procognitive effects after single and repeated administration. The increased level of the Erk1/2 and Arc genes is likely to be at least partially involved in this effect.

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.

Targeted α7 Nicotinic Acetylcholine Receptor Treatment for the Modulation of Speech MMN in Schizophrenia: An Optimized CDP-Choline and Galantamine Combination Approach Personalized to Baseline-Deviance Detection Levels

α7 nicotinic acetylcholine receptors (nAChR) pathophysiology is associated with sensory and cognitive dysfunction in schizophrenia (SCZ), a multigenic neurodevelopmental disorder. The mismatch negativity event-related potential (MMN-ERP) is sensitive to early deviance detection and sensory memory processing, which are impaired in SCZ and their non-affected relatives. Improvement in MMN amplitudes is associated with clinical and functional outcomes, making this ERP an ideal endophenotype to assess novel pharmacological treatments. As genetics, smoking status, personality, and functioning level were emphasized as trait factors underlying response variability to pharmacological therapies and specific α7 nAChR agonists, the MMN use for participant baseline electrophysiological response stratification is an increasingly applied strategy demonstrating converging evidence of deviance detection improvements in SCZ. Evidence for sensory processing improvements mediated by α7 nAChR-agonists and considering this receptor’s fast desensitization rate, a novel optimized approach combining CDP-choline (a selective α7 agonist) with galantamine (a nAChR positive allosteric modulator) was shown to increase deviance detection in healthy low-baseline groups.

Suppression of neuroinflammation by an allosteric agonist and positive allosteric modulator of the \u03b17 nicotinic acetylcholine receptor GAT107

BackgroundThe α7 nicotinic acetylcholine receptor (α7 nAChR) negatively regulates the synthesis and release of pro-inflammatory cytokines by immune cells. Our previous studies showed that in encephalitogenic T cells, α7 nAChR expression is upregulated and that activation of the cholinergic system can attenuate experimental autoimmune encephalomyelitis (EAE). GAT107 is an allosteric agonist and positive allosteric modulator (ago-PAM) of α7 nAChR that can produce persistent activation of this receptor. Therefore, in the present study, we investigated the effect of GAT107 on neuroinflammation in EAE, the animal model used for the study of multiple sclerosis (MS) via α7 nAChR, and the inflammatory pathways involved.MethodsEAE was induced by administration of myelin oligodendrocyte glycoprotein (MOG35–55) in C57BL/6 mice. EAE mice were treated with the ago-PAM GAT107 or a placebo for 9 days, starting from the day of EAE induction. Clinical assessment and immunological evaluation of immune cells and cytokine production was performed.ResultsFollowing activation of the α7 nAChR by GAT107 during EAE, disease severity was significantly reduced by 70% and was correlated with a reduction in the extent of neuroinflammation in the CNS. The treatment reduced encephalitogenic T cell proliferation and the production of pro-inflammatory cytokines, as well as increased the production of the anti-inflammatory cytokine IL-10. Furthermore, the expression of immune cell markers was altered by GAT107 treatment, which induced a significant reduction in macrophages, dendritic cells, and B cells, as well as a reduction in anti-MOG35–55 antibodies. Additionally, GAT107 was found to directly activate α7 nAChR in murine macrophage RAW264.7 cells and in human PBMCs derived from MS patients and healthy donors.ConclusionsOur results show that GAT107 can be a useful molecule for harnessing the cholinergic anti-inflammatory pathway for long-lasting and wide-ranging modulation and downregulation of neuroinflammation in EAE.

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.

Central Activation of Alpha7 Nicotinic Signaling Attenuates LPS-Induced Neuroinflammation and Sickness Behavior in Adult but Not in Aged Animals.

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.

Anti-inflammatory effects of α7-nicotinic ACh receptors are exerted through interactions with adenylyl cyclase-6.

Nicotinic ACh receptors containing the α7 sub-unit (α7-nAChRs) suppress inflammation through a wide range of pathways in immune cells. These receptors are thus potentially involved in a number of inflammatory diseases. However, the detailed mechanisms underlying the anti-inflammatory effects of α7-nAChRs remain to be described. Anti-inflammatory effects of α7-nAChR agonists were assessed in both murine macrophages (RAW 264.7) and bone marrow-derived macrophages (BMDM), stimulated with LPS, using immunoblotting, RT-PCR and luciferase reporter assays. The role of adenylyl cyclase-6 in the degradation of Toll-like receptor 4 (TLR4) following endocytosis, was explored via overexpression and knockdown. A mouse model of chronic obstructive pulmonary disease (COPD) induced by porcine pancreatic elastase was used to confirm key findings. Anti-inflammatory effects of α7-nAChRs were largely dependent on adenylyl cyclase-6 activation, as knockdown of adenylyl cyclase-6 considerably reduced the effects of α7-nAChR agonists while adenylyl cyclase-6 overexpression promoted them. We found that α7-nAChRs and adenylyl cyclase-6 are co-localized in lipid rafts of macrophages and directly interact. Activation of adenylyl cyclase-6 led to increased degradation of TLR4. Administration of the α7-nAChR agonist PNU-282987 attenuated pathological and inflammatory end points in a mouse model of COPD. The α7-nAChRs inhibit inflammation through activating adenylyl cyclase-6 and promoting degradation of TLR4. The use of α7-nAChR agonists may represent a novel therapeutic approach for treating COPD and possibly other inflammatory diseases.

α7-nAChRs-mediated therapeutic angiogenesis accounts for the advantageous effect of low nicotine doses against myocardial infarction in rats

Angiogenesis accelerates tissue regeneration in a variety of ischemic conditions including myocardial infarction (MI). Here we tested the hypothesis that angiogenesis induced by α7-nicotinic acetylcholine receptors (α7-nAChRs) mitigates histopathological, electrocardiographic, and molecular consequences of MI in rats. These profiles were evaluated in the isoprenaline (85 mg/kg/day i. p. For 2 days) MI rat model treated with or without nicotine or PHA-543613 (PHA, selective α7-nAChR agonist). Isoprenaline-insulted rats showed (i) ECG signs of MI such as significant ST-segment elevations and prolonged QT-intervals, (ii) deteriorated left ventricular histopathological scoring and elevated inflammatory cell infiltration, (iii) reduced immunohistochemical expression of cardiac CD34, a surrogate marker of capillary density, (iv) decreased cardiac expression of iNOS and α7-nAChRs, and (v) adaptive increases in cardiac HO-1 expression and plasma angiogenic markers such as vascular endothelial growth factor (VEGF) and nitric oxide (NO). These effects of isoprenaline, except cardiac iNOS and α7-nAChRs downregulation, were ameliorated in rats treated with a low dose (20 μg/kg/day s. c. For 16 days) of nicotine or PHA. We also show that concurrent α7-nAChR blockade by methyllycaconitine (MLA, 40 μg/kg/day, for 16 days) reversed the ECG, histopathological, and capillary density effects of nicotine, thereby reinforcing the advantageous cardioprotective and anti-ischemic roles of α7-nAChRs in this setting. The observed results showed promising effects on isoprenaline induced myocardial damage. In conclusion, the activation of α7-nAChRs by doses of nicotine or PHA in the microgram scale promotes neovascularization and offers a promising therapeutic strategy for MI. CategoryCardiovascular Pharmacology.

Therapeutic efficacy of α7 ligands after acute ischaemic stroke is linked to conductive states of α7 nicotinic ACh receptors

Targeting α7 nicotinic ACh receptors (nAChRs) in neuroinflammatory disorders including acute ischaemic stroke holds significant therapeutic promise. However, therapeutically relevant signalling mechanisms remain unidentified. Activation of neuronal α7 nAChRs triggers ionotropic signalling, but there is limited evidence for it in immunoglial tissues. The α7 ligands which are effective in reducing acute ischaemic stroke damage promote α7 ionotropic activity, suggesting a link between their therapeutic effects for treating acute ischaemic stroke and activation of α7 conductive states. This hypothesis was tested using a transient middle cerebral artery occlusion (MCAO) model of acute ischaemic stroke, NS6740, a known selective non-ionotropic agonist of α7 nAChRs and 4OH-GTS-21, a partial α7 agonist. NS6740-like ligands exhibiting low efficacy/potency for ionotropic activity will be referred to as non-ionotropic agonists or "metagonists". 4OH-GTS-21, used as a positive control, significantly reduced neurological deficits and brain injury after MCAO as compared to vehicle and NS6740. By contrast, NS6740 was ineffective in identical assays and reversed the effects of 4OH-GTS-21 when these compounds were co-applied. Electrophysiological recordings from acute hippocampal slices obtained from NS6740-injected animals demonstrated its remarkable brain availability and protracted effects on α7 nAChRs as evidenced by sustained (>8 h) alterations in α7 ionotropic responsiveness. These results suggest that α7 ionotropic activity may be obligatory for therapeutic efficacy of α7 ligands after acute ischaemic stroke yet, highlight the potential for selective application of α7 ligands to disease states based on their mode of receptor activation.