Α7 nACh Receptors Research Articles

Protective effect of menthol against thioacetamide-induced hepatic encephalopathy by suppressing oxidative stress and inflammation, augmenting expression of BDNF and α7-nACh receptor, and improving spatial memory

Hepatic encephalopathy (HE) is a neuropsychiatric syndrome that can occur in people with acute or chronic liver disease. Here, we investigated the effects of menthol, a natural monoterpene, on HE induced by thioacetamide (TA) in male Wistar rats. The rats received 200 mg/kg of TA twice a week for four weeks and were administered 10 mg/kg of menthol intraperitoneally daily for the same period. The results showed that menthol treatment reduced oxidative stress and inflammation in the livers and hippocampi of the rats that received TA. It also lowered the levels of ammonium and liver enzymes AST, ALT, ALP, and GGT in the serum of these animals and prevented liver histopathological damage. In addition, the expression and activity of acetylcholinesterase in the hippocampus of HE model rats were decreased by menthol. Likewise, this monoterpene reduced the expression of TLR4, MyD88, and NF-κB in the hippocampus while increasing the expression of BDNF and α7-nACh receptor. Menthol also reduced neuronal death in the hippocampal cornu ammonis-1 and dentate gyrus regions and reduced astrocyte swelling, which led to improved learning and spatial memory in rats with HE. In conclusion, the study suggests that menthol may have strong protective effects on the liver and brain, making it a potential treatment for HE and neurodegenerative diseases.

The role of nicotinic acetylcholine receptors in the pathophysiology and pharmacotherapy of autism spectrum disorder: Focus on α7 nicotinic receptors

Postmortem studies have revealed that brains of individuals with autism spectrum disorder (ASD) exhibit abnormalities in various components of the cholinergic system including cholinergic receptors, projections, and nuclei. Deletions in the 15q13.3 region which encompasses CHRNA7, the gene that encodes the α7-nACh receptor, have been linked to various neurodevelopmental disorders, including ASD. In addition, the involvement of α7-nACh receptors in biological phenomena known to play a role in the pathophysiology of ASD such as cognitive functions, learning, memory, neuroinflammation, and oxidative stress, as well as the excitation-inhibition balance in neuronal circuits and maternal immune activation have been reported in previous studies. Furthermore, evolving preclinical and clinical literature supports the potential therapeutic benefits of using selectively acting cholinergic compounds, particularly those targeting the α7-nACh receptor subtype, in the treatment of ASD. This study reviews the previous literature on the involvement of nACh receptors in the pathophysiology of ASD and focuses on the α7-nACh receptor as a potential therapeutic target.

Vitamin D and rosuvastatin alleviate type-II diabetes-induced cognitive dysfunction by modulating neuroinflammation and canonical/noncanonical Wnt/β-catenin signaling.

Type-II diabetes mellitus (T2DM) is a major risk factor for cognitive impairment. Protecting the brain environment against inflammation, and neurodegeneration, as well as preservation of the BBB veracity through modulating the crosstalk between insulin/AKT/GSK-3β and Wnt/β-catenin signaling, might introduce novel therapeutic targets. This study aimed at exploring the possible neuroprotective potential of vitamin D3 (VitD) and/or rosuvastatin (RSV) in T2DM-induced cognitive deficits. T2DM was induced by a high-fat sucrose diet and a single streptozotocin (STZ) dose. Diabetic rats were allocated into a diabetic control and three groups treated with RSV (15 mg/kg/day, PO), VitD (500 IU/kg/day, PO), or their combination. Administration of VitD and/or RSV mitigated T2DM-induced metabolic abnormalities and restored the balance between the anti-inflammatory, IL 27 and the proinflammatory, IL 23 levels in the hippocampus. In addition, they markedly activated both the canonical and noncanonical Wnt/β-catenin cassettes with stimulation of their downstream molecular targets. VitD and/or RSV upregulated insulin and α7 nicotinic acetylcholine (α7nACh) receptors gene expression, as well as blood-brain barrier integrity markers including Annexin A1, claudin 3, and VE-cadherin. Also, they obliterated hippocampal ApoE-4 content, Tau hyperphosphorylation, and Aβ deposition. These biochemical changes were reflected as improved behavioral performance in Morris water maze and novel object recognition tests and restored hippocampal histological profile. The current findings have accentuated the neuroprotective potential of VitD and RSV and provide new incentives to expand their use in T2DM-induced cognitive and memory decline. This study also suggests a superior benefit of combining both treatments over either drug alone.

Modulation of the mammalian GABAA receptor by type I and type II positive allosteric modulators of the α7 nicotinic acetylcholine receptor.

Positive allosteric modulators of the α7 nicotinic acetylcholine (nACh) receptor (α7-PAMs) possess promnesic and procognitive properties and have potential in the treatment of cognitive and psychiatric disorders including Alzheimer's disease and schizophrenia. Behavioural studies in rodents have indicated that α7-PAMs can also produce antinociceptive and anxiolytic effects that may be associated with positive modulation of the GABAA receptor. The overall goal of this study was to investigate the modulatory actions of selected α7-PAMs on the GABAA receptor. We employed a combination of cell fluorescence imaging, electrophysiology, functional competition and site-directed mutagenesis to investigate the functional and structural mechanisms of modulation of the GABAA receptor by three representative α7-PAMs. We show that the α7-PAMs at micromolar concentrations enhance the apparent affinity of the GABAA receptor for the transmitter and potentiate current responses from the receptor. The compounds were equi-effective at binary αβ and ternary αβγ GABAA receptors. Functional competition and site-directed mutagenesis indicate that the α7-PAMs bind to the classic anaesthetic binding sites in the transmembrane region in the intersubunit interfaces, which results in stabilization of the active state of the receptor. We conclude that the tested α7-PAMs are micromolar-affinity, intermediate- to low-efficacy allosteric potentiators of the mammalian αβγ GABAA receptor. Given the similarities in the in vitro sensitivities of the α7 nACh and α1β2γ2L GABAA receptors to α7-PAMs, we propose that doses used to produce nACh receptor-mediated behavioural effects in vivo are likely to modulate GABAA receptor function.

Recurring Cholinergic Inputs Induce Local Hippocampal Plasticity through Feedforward Disinhibition.

The CA1 pyramidal neurons are embedded in an intricate local circuitry that contains a variety of interneurons. The roles these interneurons play in the regulation of the excitatory synaptic plasticity remains largely understudied. Recent experiments showed that recurring cholinergic activation of α7 nACh receptors expressed in oriens-lacunosum-moleculare (OLMα2) interneurons can directly induce LTP in Schaffer collateral (SC)–CA1 synapses. Here, we pair in vitro studies with biophysically based modeling to uncover the underlying mechanisms. According to our model, α7 nAChR activation increases OLM GABAergic activity. This results in the inhibition of the fast-spiking interneurons that provide feedforward inhibition onto CA1 pyramidal neurons. This disinhibition, paired with tightly timed SC stimulation, can induce potentiation at the excitatory synapses of CA1 pyramidal neurons. Our work details the role of cholinergic modulation in disinhibition-induced hippocampal plasticity. It relates the timing of cholinergic pairing found experimentally in previous studies with the timing between disinhibition and hippocampal stimulation necessary to induce potentiation and suggests the dynamics of the involved interneurons plays a crucial role in determining this timing.

Evaluation of galantamine and deconstructed analogs as α7 nAChR and AChE ligands

Galantamine (1), one of only five agents currently approved for the treatment of Alzheimer’s disease, is thought to act primarily as an inhibitor of acetylcholinesterase (AChE) and possibly as a positive allosteric modulator (PAM) of neuronal α7 nicotinic acetylcholine (nACh) receptors. Here, the structure of 1 was deconstructed to ten “abbreviated” analogs and examined electrophysiologically at α7 nACh receptors and as inhibitors of AChE. Neither 1, nor its reduced analog lycoramine (3), displayed PAM action, but at high concentrations both inhibited the agonist action of 100 μM ACh. The other deconstructed analogs lacked a PAM effect and also the inhibitory action of 1/3 indicating that the cyclohexa(e)nyl ring and/or hydroxyl group of 1/3 are important for the latter. All of the deconstructed analogs examined behaved as inhibitors of AChE but were >400-fold less potent than 1, again indicating that the cyclohexanol portion of 1 is a major determinant for optimal activity.

HTS-based discovery and optimization of novel positive allosteric modulators of the α7 nicotinic acetylcholine receptor

HTS campaign of the corporate compound collection resulted in a novel, oxalic acid diamide scaffold of α7 nACh receptor positive allosteric modulators. During the hit expansion, several derivatives, such as 4, 11, 17 demonstrated not only high invitro potency, but also invivo efficacy in the mouse place recognition test. The advanced hit molecule 11 was further optimized by the elimination of the putatively mutagenic aromatic-amine building block that resulted in a novel, aminomethylindole compound family. The most balanced physico-chemical and pharmacological profile was found in case of compound 55. Docking study revealed an intersubunit binding site to be the most probable for our compounds. 55 demonstrated favorable cognitive enhancing profile not only in scopolamine-induced amnesia (place recognition test in mice) but also in natural forgetting (novel object recognition test in rats). Compound 55 was, furthermore, active in a cognitive paradigm of high translational value, namely in the rat touch screen visual discrimination test. Therefore, 55 was selected as a lead compound for further optimization. Based on the obtained favorable results, the invented aminomethylindole cluster may provide a viable approach for cognitive enhancement through positive allosteric modulation of α7 nAChRs.

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.

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 protective effect of α7-nACh receptor and its interaction with 5-HT1B/1D receptors in acute intestinal ischemia-reperfusion injury in rats.

Over the past decades, great attention has been given to the nervous system modulating effects on the immune response in inflammation-associated injuries, such as acute intestinal ischemia-reperfusion (IR). Recently, we proved the anti-inflammatory and antioxidant effects of 5-hydroxytryptamine (5-HT)1B/1D receptors in intestinal IR injury in rats. Also, the alpha7 nicotinic acetylcholine (α7-nACh) receptor has anti-inflammatory effects in different inflammation-associated injuries. Starting from these premises, we aimed to examine the function of the α7-nACh receptors and the functional interactions between the anti-inflammatory and antioxidant effects of α7-nACh and 5-HT1B/1D receptors in acute intestinal IR injury. To confirm the expression and localization of α7-nACh receptors on the ileum nerves, an immunofluorescence-based method was applied. Then, intestinal IR injury was induced by 30-min occlusion of superior mesenteric artery and reperfusion for 2h in rats. Acute systemic administration of α7-nACh receptor agonist PNU-282987 and antagonist methyllycaconitine, and 5-HT1B/1D receptors agonist (sumatriptan) and antagonist (GR127, 935) were used in the model of intestinal IR injury. Finally, biochemical and histological parameters were assessed. Α7-nACh receptors were expressed by 9% on the ileum nerves. Likewise, activation of the α7-nACh receptor showed anti-inflammatory and antioxidant effects in intestinal IR injury but not as well as 5-HT1B/1D receptors. Interestingly, 5-HT1B/1D receptors via attenuation of glutamate (Glu) release indirectly activated the α7-nACh receptor and its protective effects against inflammation and oxidative stress. The protective effect of the α7-nACh receptor on intestinal IR injury was activated indirectly through the 5-HT1B/1D receptors' modulatory impact on Glu release.

Apigenin and Structurally Related Flavonoids Allosterically Potentiate the Function of Human α7-Nicotinic Acetylcholine Receptors Expressed in SH-EP1 Cells

Phytochemicals, such as monoterpenes, polyphenols, curcuminoids, and flavonoids, are known to have anti-inflammatory, antioxidant, neuroprotective, and procognitive effects. In this study, the effects of several polyhydroxy flavonoids, as derivatives of differently substituted 5,7-dihydroxy-4H-chromen-4-one including apigenin, genistein, luteolin, kaempferol, quercetin, gossypetin, and phloretin with different lipophilicities (cLogP), as well as topological polar surface area (TPSA), were tested for induction of Ca2+ transients by α7 human nicotinic acetylcholine (α7 nACh) receptors expressed in SH-EP1 cells. Apigenin (10 μM) caused a significant potentiation of ACh (30 μM)-induced Ca2+ transients, but did not affect Ca2+ transients induced by high K+ (60 mM) containing solutions. Co-application of apigenin with ACh was equally effective as apigenin preincubation. However, the effect of apigenin significantly diminished by increasing ACh concentrations. The flavonoids tested also potentiated α7 nACh mediated Ca2+ transients with descending potency (highest to lowest) by genistein, gossypetin, kaempferol, luteolin, phloretin, quercetin, and apigenin. The specific binding of α7 nACh receptor antagonist [125I]-bungarotoxin remained unchanged in the presence of any of the tested polyhydroxy flavonoids, suggesting that these compounds act as positive allosteric modulators of the α7-nACh receptor in SH-EP1 cells. These findings suggest a clinical potential for these phytochemicals in the treatment of various human diseases from pain to inflammation and neural disease.

Activation of alpha7 nicotinic acetylcholine receptor protects bovine endometrial tissue against LPS-induced inflammatory injury via JAK2/STAT3 pathway and COX-2 derived prostaglandin E2

Bovine endometritis is one of the major postpartum diseases associated with infertility and subfertility, decreasing the benefit of dairy industry. It is important to develop alternate therapies for endometritis in the context of drug residues in the milk and hormone disorder in the estrous cycle. α7 nicotine acetylcholine receptor has been identified as the core of ‘cholinergic anti-inflammatory pathway (CAP)’, which is a potential drug target to inflammatory diseases. However, there has been still no study on its anti-inflammatory effects and mechanism on lipopolysaccharide (LPS)-induced bovine endometritis. This study aimed to demonstrate the underlying anti-inflammatory effects and mechanism of α7-nACh receptor on LPS-induced inflammation in bovine endometrial tissues cultured in vitro. The results suggested that activation of α7-nACh receptor significantly suppressed the mRNA expression levels of interleukin 1β (IL-1β), IL-6, IL-8, and tumor necrosis factor alpha (TNF-α) in bovine endometrial tissues. Western blot and enzyme-linked immunosorbent assay (ELISA) detection results showed that activation of α7-nACh receptor inhibited LPS-induced phosphorylation of Janus kinase 2 (JAK2) and signal transducer and activator of transcription 3 (STAT3). Moreover, α7-nACh receptor agonist decreased the expression of cyclooxygenase 2 (COX-2) and microsomal prostaglandin E synthase-1(mPGES-1), as well as prostaglandin E2 (PGE2) secretion. Interestingly, in COX-2 inhibition experiment, activation of α7-nACh receptor increased COX-2 expression and PGE2 production, compared with COX-2 inhibitor treatment. In conclusion, activation of the cholinergic system through α7-nACh receptor agonist has suppressed inflammation of bovine endometrial tissues via JAK2/STAT3 pathway and potential COX-2-derived PGE2.

Discovery of novel positive allosteric modulators of the α7 nicotinic acetylcholine receptor: Scaffold hopping approach.

The paper focuses on the scaffold hopping-based discovery and characterization of novel nicotinic alpha 7 receptor positive modulator (α7 nAChR PAM) ligands around the reference molecule (A-867744). First, substantial efforts were carried out to assess the importance of the various pharmacophoric elements on the invitro potency (SAR evaluation) by chemical modifications. Subsequently, several new derivatives with versatile, heteroaromatic central cores were synthesized and characterized. A promising, pyrazole-containing new chemotype with good physicochemical and invitro parameters was identified. Retrospective analysis based on homology modeling was also carried out. Besides its favorable invitro characteristics, the most advanced derivative 69 also showed invivo efficacy in a rodent model of cognition (scopolamine-induced amnesia in the mouse place recognition test) and acceptable pharmacokinetic properties. Based on the invivo data, the resulting molecule with advanced drug-like characteristics has the possibility to improve cognitive performance in a biologically relevant dose range, further strengthening the view of the supportive role of α7 nACh receptors in the cognitive processes.

Cholinergic Modulation of Glial Function During Aging and Chronic Neuroinflammation.

Aging is a complex biological process that increases the risk of age-related cognitive degenerative diseases such as dementia, including Alzheimer’s disease (AD), Lewy Body Dementia (LBD), and mild cognitive impairment (MCI). Even non-pathological aging of the brain can involve chronic oxidative and inflammatory stress, which disrupts the communication and balance between the brain and the immune system. There has been an increasingly strong connection found between chronic neuroinflammation and impaired memory, especially in AD. While microglia and astrocytes, the resident immune cells of the central nervous system (CNS), exerting beneficial effects during the acute inflammatory phase, during chronic neuroinflammation they can become more detrimental. Central cholinergic circuits are involved in maintaining normal cognitive function and regulating signaling within the entire cerebral cortex. While neuronal-glial cholinergic signaling is anti-inflammatory and anti-oxidative, central cholinergic neuronal degeneration is implicated in impaired learning, memory sleep regulation, and attention. Although there is evidence of cholinergic involvement in memory, fewer studies have linked the cholinergic anti-inflammatory and anti-oxidant pathways to memory processes during development, normal aging, and disease states. This review will summarize the current knowledge of cholinergic effects on microglia and astroglia, and their role in both anti-inflammatory and anti-oxidant mechanisms, concerning normal aging and chronic neuroinflammation. We provided details on how stimulation of α7 nicotinic acetylcholine (α7nACh) receptors can be neuroprotective by increasing amyloid-β phagocytosis, decreasing inflammation and reducing oxidative stress by promoting the nuclear factor erythroid 2-related factor 2 (Nrf2) pathways and decreasing the release of pro-inflammatory cytokines. There is also evidence for astroglial α7nACh receptor stimulation mediating anti-inflammatory and antioxidant effects by inhibiting the nuclear factor-κB (NF-κB) pathway and activating the Nrf2 pathway respectively. We conclude that targeting cholinergic glial interactions between neurons and glial cells via α7nACh receptors could regulate neuroinflammation and oxidative stress, relevant to the treatment of several neurodegenerative diseases.

Activation of α7nACh receptor protects against acute pancreatitis through enhancing TFEB-regulated autophagy

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.

Meta-analysis of randomized controlled trials of galantamine in schizophrenia: significant cognitive enhancement

Cognitive impairments are core features of schizophrenia and the best predictor of functional outcome. Cholinergic system and alpha-7 nicotinic acetylcholine (α7nACh) receptors are strongly implicated in the pathophysiologic mechanisms associated with cognitive impairments in schizophrenia. Galantamine is not only a reversible, competitive inhibitor of acetylcholinesterase but also a type I positive allosteric modulator of α7nACh receptors. The objective of this meta-analysis was to examine the efficacy of galantamine for cognitive symptoms of schizophrenia. In the meta-analysis that included six randomized controlled trials (RCTs, N=226), cognitive impairments significantly improved with galantamine compared to placebo, with a small Hedges’ g effect size of 0.233. This finding is consistent with other RCTs in schizophrenia with medications with a similar mechanism of action. On the basis of the results from all the failed (although some efficacy has been shown) RCTs to date in schizophrenia, targeting only one pathophysiologic mechanism may be insufficient to detect a clinically meaningful signal. Nicotinergic medications, like any other add-on medications, are unlikely to be effective as stand-alone medications. Hence, these medications may have to be combined with other medications with complementary mechanisms such as glutamatergic/N-methyl-D-aspartate systems to detect a meaningful effect size for the three domains of psychopathology.

The M4 Helix Is Involved in α7 nACh Receptor Function.

Nicotinic acetylcholine receptors (nAChR) are the archetypal members of the pentameric ligand-gated ion channel (pLGIC) family, an important class of cell signaling proteins. In all members of this family, each of the five subunits has four transmembrane α-helices (M1-M4) with M2 lining the pore and then M1 and M3, with M4 outermost and adjacent to the membrane lipids. M4 has a variety of roles: its interaction with neighboring M1 and M3 helices is important for receptor assembly, it can a transmit information on the lipid content of the membrane to the gating mechanism, and it may form a vital link to the extracellular domain via the Cys-loop. This study examines the role of M4 receptor residues in the α7 nAChR using site-directed mutagenesis and subsequent expression in Xenopus oocytes. The data indicate that many of the residues in M4 play a role in receptor function, as substitution with Ala can modify functional parameters; 11 of 24 mutants showed a small gain of function (<10-fold decrease in EC50), and 1 (D446A) did not respond to the agonist; it was also not expressed at the cell surface. Removal or addition of aromatic residues had small or no effects. These results demonstrate the α7 nAChR M4 has a role in receptor function, and a structural model suggests possible interactions of some of these residues with their neighbors.

Memantine treatment prevents okadaic acid induced neurotoxicity at the systemic and molecular levels

The present study was designed to investigate the effects of okadaic acid intracerebroventricular (ICV) injection on memory function and expression level of α7 subunit of nicotinic acetylcholine receptor (nAChR) and NR2B subunit of NMDA glutamate receptors in the hippocampus, as well as effect of the antidementic drug memantine on okadaic acid induced changes at systemic and molecular levels in rats. Okadaic acid was dissolved in artificial cerebrospinal fluid (aCSF) and injected ICV 200 ng/10 μl. Vehicle control received 10 μl of aCSF ICV bilaterally. Control and okadaic acid injected rats were divided into two subgroups: treated i.p. with saline or memantine (5 mg/kg daily for 13 days starting from the day of okadaic acid injection). Rats were trained in the dual-solution plus-maze task that can be solved by using place or response strategies. The Western immunoblotting was used to determine relative amount of hippocampal receptors protein levels. Obtained data revealed that okadaic acid ICV injected rats were severely impaired at acquiring the place version of the maze accompanied by significant decline in hippocampal α7 subunit of nACh receptors protein levels. Memantine treatment can prevent okadaic acid induced impairment of hippocampal-dependent spatial memory and accompanied by modulation of the expression level of α7 subunit of nACh and NR2B subunit of NMDA receptors in the hippocampus. Thus, our results support the presumption that α7 nACh receptors may play an important role in the cognitive enhancer effects of memantine and emphasize the role of cholinergic-glutamatergic interactions in memory.

Dysfunction of inflammation-resolving pathways is associated with postoperative cognitive decline in elderly mice

BackgroundPostoperative cognitive dysfunction (POCD) refers to a reversible, perioperative mental disorder. POCD increases the likelihood of postoperative complications and the risk for postoperative mortality, typically among elderly patients (age 65 or older). The importance of the cholinergic anti-inflammatory pathway (CAP) in resolving neuro-inflammatory and cognitive decline caused by sterile trauma has been recognized. We speculate that the POCD in elderly mice is associated with dysfunction of CAP. MethodsMice were assigned to several groups (n = 5 in each group): AM (adult mice) Sham, AM (adult mice) Surgery, EM (elderly mice) Sham, EM (elderly mice) Surgery, and EMP (elderly mice with PNU) Surgery. At 24 h after surgery, assessed the cognitive levels. Pro-inflammatory cytokines in peripheral blood and splenic monocytes (TNF-α, IL-6 and IL-10) were assessed by ELISA and qPCR. Levels of M2 macrophages in hippocampus were visualized by immunofluorescence. Detecting CD11b/c+α7 nAChR+ cells in the spleens with flow cytometry. ResultsAt postoperative 24 h, elderly mice exhibited significantly increased POCD compared with adult mice. The proinflammatory factor TNF-α and IL-6 were higher among elderly surgery mice (EM) compared with adult surgery (AM) and elderly-P surgery mice (EM-P); the anti-inflammatory factor IL-10 and M2 macrophages were lower among EM surgery mice compared with AM surgery and EM-P surgery mice. The CD11b/c+α7 nAChR+ population of splenocytes was reduced in the EM surgery mice. ConclusionsThe exaggerated and persistent cognitive decline and inflammatory response among elderly mice were associated with dysfunction of CAP, and these phenomena were reversed by α7nAch receptor agonists.

AE Succinimide, an Analogue of Methyllycaconitine, When Bound Generates a Nonconducting Conformation of the α4β2 Nicotinic Acetylcholine Receptor

Nicotinic acetylcholine (nACh) receptors are pentameric ligand-gated ion channels that mediate fast synaptic transmission. The α4β2 nACh receptor is highly expressed in the brain and exists in two functional stoichiometries: the (α4)2(β2)3 and (α4)3(β2)2 that differ by an ACh-binding site at the α4-α4 interface of (α4)3(β2)2 receptors. Methyllycaconitine (MLA) is an nACh receptor antagonist, and while potent at both α7 and α4β2 nACh receptors, it has a higher selectivity for the α7 nACh receptor. The anthranilate-succinimide ester side-chain is important for its activity and selectivity. Here we identify a simplified MLA analogue that contains only the A and E ring skeleton of MLA, AE succinimide, that binds close to the channel lumen to display insurmountable inhibition at α4β2 nACh receptors. Although inhibition by AE succinimide was found to be voltage-dependent indicating a possible pore channel blocker, substituted-cysteine accessibility experiments indicated it did not bind between 2'-16' region of the channel pore. Instead, we found that upon binding and in the presence of ACh, there is a conformational change to the channel membrane that was identified when the compound was assessed against (α4 V13'C)β2 nACh receptors. It was found that in the 3:2 stoichiometry the two adjacent α4 subunits containing 13' cysteine mutations formed a disulfide bond and occluded ion conductance. This was reversed by treatment with the reducing agent, dithiothreitol. Thus, AE succinimide has a different mechanism of inhibition to both MLA and other AE analogues, such as AE bicyclic alcohol, in that upon binding to an as yet unidentified site, AE succinimide in the presence of ACh induces a conformational change to the channel that generates a ligand-bound closed state.