Nicotinic Binding Research Articles

Molecular blueprint of allosteric binding sites in a homologue of the agonist-binding domain of the α7 nicotinic acetylcholine receptor

The α7 nicotinic acetylcholine receptor (nAChR) belongs to the family of pentameric ligand-gated ion channels and is involved in fast synaptic signaling. In this study, we take advantage of a recently identified chimera of the extracellular domain of the native α7 nicotinic acetylcholine receptor and acetylcholine binding protein, termed α7-AChBP. This chimeric receptor was used to conduct an innovative fragment-library screening in combination with X-ray crystallography to identify allosteric binding sites. One allosteric site is surface-exposed and is located near the N-terminal α-helix of the extracellular domain. Ligand binding at this site causes a conformational change of the α-helix as the fragment wedges between the α-helix and a loop homologous to the main immunogenic region of the muscle α1 subunit. A second site is located in the vestibule of the receptor, in a preexisting intrasubunit pocket opposite the agonist binding site and corresponds to a previously identified site involved in positive allosteric modulation of the bacterial homolog ELIC. A third site is located at a pocket right below the agonist binding site. Using electrophysiological recordings on the human α7 nAChR we demonstrate that the identified fragments, which bind at these sites, can modulate receptor activation. This work presents a structural framework for different allosteric binding sites in the α7 nAChR and paves the way for future development of novel allosteric modulators with therapeutic potential.

Pharmacological and neuroprotective profile of an essential oil derived from leaves of Aloysia citrodora Palau.

The Jordanian 'Melissa', (Aloysia citrodora) has been poorly studied both pharmacologically and in the clinic. Essential oils (EO) derived from leaves of A. citrodora were obtained by hydrodistillation, analysed by gas chromatography-mass spectrometry (GC-MS) and were investigated for a range of neurobiological and pharmacological properties, as a basis for potential future use in drug discovery. A selection of central nervous system (CNS) receptor-binding profiles was carried out. Antioxidant activity and ferrous iron-chelating assays were adopted, and the neuroprotective properties of A. citrodora EO assessed using hydrogen peroxide-induced and β-amyloid-induced neurotoxicity with the CAD (Cath.-a-differentiated) neuroblastoma cell line. The major chemical components detected in the A. citrodora EOs, derived from dried and fresh leaves, included limonene, geranial, neral, 1, 8-cineole, curcumene, spathulenol and caryophyllene oxide, respectively. A. citrodora leaf EO inhibited [(3) H] nicotine binding to well washed rat forebrain membranes, and increased iron-chelation in vitro. A. citrodora EO displays effective antioxidant, radical-scavenging activities and significant protective properties vs both hydrogen peroxide- and β-amyloid-induced neurotoxicity. A. citrodora EO displays a range of pharmacological properties worthy of further investigation to isolate the compounds responsible for the observed neuroactivities, to further analyse their mode of action and determine their clinical potential in neurodegenerative diseases.

Sex differences in conditioned nicotine reward are age-specific

Women constitute half of all smokers and many studies suggest that adult males and females differ in factors that maintain tobacco smoking, yet there is limited information about sex differences in nicotine reward during adolescence. Limited studies suggest that adolescent male rats self-administer more nicotine than adults, suggesting that drug administration during adolescence leads to different behavioral effects than during adulthood. In the present study, male rats developed a significant conditioned place preference (CPP) to lower doses of nicotine than females, regardless of age. In addition, adolescents were more sensitive than adults. In female rats, adolescents exhibited a CPP of greater magnitude than adult females. In males, the magnitude of the CPP did not differ as a function of age, but adolescents exhibited CPP to lower doses than adults. There also were differences in nicotinic acetylcholinergic receptor binding in nucleus accumbens and caudate putamen in response to nicotine across age and sex. These findings suggest that it is necessary to consider sex- and age-specific effects of drugs such as nicotine when developing strategies for improving smoking cessation treatments.

Changes in CSF cholinergic biomarkers in response to cell therapy with NGF in patients with Alzheimer's disease

IntroductionThe extensive loss of central cholinergic functions in Alzheimer's disease (AD) brain is linked to impaired nerve growth factor (NGF) signaling. The cardinal cholinergic biomarker is the acetylcholine synthesizing enzyme, choline acetyltransferase (ChAT), which has recently been found in cerebrospinal fluid (CSF). The purpose of this study was to see if EC-NGF therapy will alter CSF levels of cholinergic biomarkers, ChAT, and acetylcholinesterase. MethodEncapsulated cell implants releasing NGF (EC-NGF) were surgically implanted bilaterally in the basal forebrain of six AD patients for 12 months and cholinergic markers in CSF were analyzed. ResultsActivities of both enzymes were altered after 12 months. In particular, the activity of soluble ChAT showed high correlation with cognition, CSF tau and amyloid-β, in vivo cerebral glucose utilization and nicotinic binding sites, and morphometric and volumetric magnetic resonance imaging measures. DiscussionA clear pattern of association is demonstrated showing a proof-of-principle effect on CSF cholinergic markers, suggestive of a beneficial EC-NGF implant therapy.

Differential expression of the beta4 neuronal nicotinic receptor subunit affects tolerance development and nicotinic binding sites following chronic nicotine treatment

The role of neuronal nicotinic acetylcholine receptors (nAChR) containing the β4 subunit in tolerance development and nicotinic binding site levels following chronic nicotine treatment was investigated. Mice differing in expression of the β4-nAChR subunit [wild-type (β4++), heterozygote (β4+−) and null mutant (β4−−)] were chronically treated for 10days with nicotine (0, 0.5, 1.0, 2.0 or 4.0mg/kg/h) by constant intravenous infusion. Chronic nicotine treatment elicited dose-dependent tolerance development. β4−− mice developed significantly more tolerance than either β4++ or β4+− mice which was most evident following treatment with 4.0mg/kg/h nicotine. Subsets of [125I]-epibatidine binding were measured in several brain regions. Deletion of the β4 subunit had little effect on initial levels of cytisine-sensitive [125I]-epibatidine binding (primarily α4β2-nAChR sites) or their response (generally increased binding) to chronic nicotine treatment. In contrast, β4 gene-dose-dependent decreases in expression 5IA-85380 resistant [125I]-epibatidine binding sites (primarily β4*-nAChR) were observed. While these β4*-nAChR sites were generally resistant to regulation by chronic nicotine treatment, significant increases in binding were noted for habenula and hindbrain. Comparison of previously published tolerance development in β2−− mice (less tolerance) to that of β4−− mice (more tolerance) supports a differential role for these receptor subtypes in regulating tolerance following chronic nicotine treatment.

The effect of nicotine on the expressions of the α7 nicotinic receptor gene and Bax and Bcl-2 proteins in the mammary gland epithelial-7 breast cancer cell line and its relationship to drug resistance.

The binding of nicotine with nicotinic acetylcholine receptors (nAChRs) stimulates cell division and increases drug resistance in cancer. Experiments with specific inhibitors such as RNAi, hexamethonium, and α-bungarotoxin showed that α7 nicotinic receptor plays a key role in the pro-proliferation activity of nicotine. However, the mechanism of nicotine in the progress of breast cancer, the commonest malignancy in women, remains unknown. This study focuses on the effect of nicotine on the expressions of the α7 nicotinic receptor gene and Bax and Bcl-2 proteins in mammary gland epithelial-7 (MCF-7) breast cancer cells and its relationship to drug resistance. To evaluate the effect on drug resistance, human mammary gland epithelial adenocarcinomas from the MCF-7 line were exposed to 100 μl of nicotine at a concentration of 9.2 mg/ml for varying periods of time. Then, the cells were treated with 1, 2, 3 or 5 μl/ml of doxorubicin, either with or without the continued presence of nicotine. Cell viability was determined using the MTT assay. The biochemical parameters of apoptosis, including the expressions of Bax, Bcl-2 and α7 nicotinic receptor proteins were determined via western blotting, and the α7 nicotinic receptor gene expression level was assessed via real-time qPCR using the 2(-ΔΔCt) method. Differences in the target gene expression levels were evaluated with ANOVA with p ≤ 0.05 considered significant. We found a novel and effective signaling pathway of nicotine in the MCF-7 breast cancer cell line. The levels of α7 nicotinic receptor and Bcl-2 protein increased but the Bax protein levels decreased, while the α7 nicotinic receptor gene expression level was not significantly different compared with the control.

Predicting the intermediate syndrome in organophosphorus poisoning.

Intentional organophosphorus (OP) poisoning is estimated to kill between 230,000 and 350,000 people a year worldwide, with a further 20,000 dying following accidental exposure.[1] The occurrence of a “type II paralysis,” usually proximal muscle paralysis, in survivors after the resolution of cholinergic features was recognized as complicating up to 18% of OP related admissions during an early case-series.[2] Senanayake and Karalliedde[3] classified this paralysis as the “intermediate syndrome” and described it as a distinct clinical entity on the basis of electromyographic (EMG) evidence of postsynaptic neuromuscular junction (NMJ) failure that occurred between 24 and 96 h after the resolution of the cholinergic crisis and prior to the onset expected for delayed neuropathy. The respiratory muscles are often affected and a prolonged period of ventilator support is required with all the associated risks of clinical complications and healthcare costs that such treatment entails. The pathophysiological mechanism of the syndrome remains obscure and empirical data are limited, although oxidative stress and changes in receptor-ligand binding kinetics have been reported. Muscle biopsies taken during the Bleecker et al. case series reported a limited degree of fiber necrosis that was considered to be insufficient to account for the clinical features observed during the study.[4] Biomarkers of oxidative stress, at least in erythrocytes, have been identified in patients with the intermediate syndrome.[5] A rat model of dimethoate poisoning described changes in skeletal muscle and lymphocyte, but not the central nervous system, nicotinic receptor binding function 48 h postexposure.[6] Several risk factors have been proposed for the development of intermediate syndrome. The type of OP and the severity of poisoning may be important. In a small prospective case series (n = 19, eight of which developed intermediate syndrome), Bleecker et al.,[4] noted that dimethyl OP agents were more commonly implicated in comparison with diethyl OP compounds. An observation later confirmed by Indira et al.[7] Bleecker et al.,[4] also observed that the clinical features and recovery correlated closely with EMG features and hypothesized that the NMJ dysfunction related to both pre- and post-synaptic changes. The degree of acetylcholinesterase inhibition has also found to be greater in individuals who went on to develop the syndrome.[4] The 1st day serum acetylcholinesterase activity does not, however, appear to predict the onset of the intermediate syndrome but may predict survival.[8] A finding not inconsistent with the observations reporting by Indira et al.,[7] of an increased relative risk of the intermediate syndrome in patients with a higher poisons severity score or reduced Glasgow Coma Scale. In this edition of the Journal, a prospective observational study by Kumar et al.[9] reported a significant rise in serum creatine phosphokinase (CPK) on admission and again, 48 h postadmission in three individuals with OP poisoning who subsequently developed intermediate syndrome compared to 72 h with OP poisoning who did not. Serum CPK concentrations were also noted to correlate with markers of poisoning severity such as the Peradeniya organophosphorus poisoning score, and the total dose of atropine administered, and inversely correlated with the degree of butyrylcholinesterase activity. These findings are consistent with those of Bhattacharyya et al.[10] who also described a correlation between admission CPK; the severity of poisoning and the risk of developing the intermediate syndrome. The study by Kumar et al.[9] has several strengths: The diagnosis of OP exposure was confirmed by cholinesterase activity measurement in each case; a standard treatment protocol was adhered to and the investigators excluded for the potential confounders that may cause an elevated serum CPK concentration, like seizures. The sample size of 75 makes this one of the larger studies focusing on the intermediate syndrome. The actual number of individuals who developed the syndrome was small. The authors acknowledge this limitation and note that this was due to the exclusion of those with severe poisoning. It remains unclear, therefore, if CPK is truly an independent risk factor for the intermediate syndrome or, instead, reflects disease severity; which in turn may be the principle risk factor for the onset of the syndrome. Determining the relative contributions of these and other potential risk factors for intermediate syndrome will require more research. A predictive test to risk-stratify those most likely to develop the syndrome is, however, a goal worth pursuing. If a successful test can be validated, using a resource that is readily available in the majority of healthcare settings then it should considerably aid the early identification of patients who are likely to require transfer to a center that can provide ventilator support over several days duration and also may facilitate further research into preventative therapies. The authors are to be commended for undertaking research into this problem, which is highly prevalent and disproportionately affects the more socioeconomically disadvantaged in our community. Financial support and sponsorship Nil. Conflicts of interest There are no conflicts of interest.

Prenatal nicotine alters the developmental neurotoxicity of postnatal chlorpyrifos directed toward cholinergic systems: better, worse, or just "different?".

This study examines whether prenatal nicotine exposure sensitizes the developing brain to subsequent developmental neurotoxicity evoked by chlorpyrifos, a commonly-used insecticide. We gave nicotine to pregnant rats throughout gestation at a dose (3mg/kg/day) producing plasma levels typical of smokers; offspring were then given chlorpyrifos on postnatal days 1-4, at a dose (1mg/kg) that produces minimally-detectable inhibition of brain cholinesterase activity. We evaluated indices for acetylcholine (ACh) synaptic function throughout adolescence, young adulthood and later adulthood, in brain regions possessing the majority of ACh projections and cell bodies; we measured nicotinic ACh receptor binding, hemicholinium-3 binding to the presynaptic choline transporter and choline acetyltransferase activity, all known targets for the adverse developmental effects of nicotine and chlorpyrifos given individually. By itself nicotine elicited overall upregulation of the ACh markers, albeit with selective differences by sex, region and age. Likewise, chlorpyrifos alone had highly sex-selective effects. Importantly, all the effects showed temporal progression between adolescence and adulthood, pointing to ongoing synaptic changes rather than just persistence after an initial injury. Prenatal nicotine administration altered the responses to chlorpyrifos in a consistent pattern for all three markers, lowering values relative to those of the individual treatments or to those expected from simple additive effects of nicotine and chlorpyrifos. The combination produced global interference with emergence of the ACh phenotype, an effect not seen with nicotine or chlorpyrifos alone. Given that human exposures to nicotine and chlorpyrifos are widespread, our results point to the creation of a subpopulation with heightened vulnerability.

Novel Anti-Nicotine Vaccine Using a Trimeric Coiled-Coil Hapten Carrier.

Tobacco addiction represents one of the largest public health problems in the world and is the leading cause of cancer and heart disease, resulting in millions of deaths a year. Vaccines for smoking cessation have shown considerable promise in preclinical models, although functional antibody responses induced in humans are only modestly effective in preventing nicotine entry into the brain. The challenge in generating serum antibodies with a large nicotine binding capacity is made difficult by the fact that this drug is non-immunogenic and must be conjugated as a hapten to a protein carrier. To circumvent the limitations of traditional carriers like keyhole limpet hemocyanin (KLH), we have synthesized a short trimeric coiled-coil peptide (TCC) that creates a series of B and T cell epitopes with uniform stoichiometry and high density. Here we compared the relative activities of a TCC-nic vaccine and two control KLH-nic vaccines using Alum as an adjuvant or GLA-SE, which contains a synthetic TLR4 agonist formulated in a stable oil-in-water emulsion. The results showed that the TCC's high hapten density correlated with a better immune response in mice as measured by anti-nicotine Ab titer, affinity, and specificity, and was responsible for a reduction in anti-carrier immunogenicity. The Ab responses achieved with this synthetic vaccine resulted in a nicotine binding capacity in serum that could prevent >90% of a nicotine dose equivalent to three smoked cigarettes (0.05 mg/kg) from reaching the brain.

In vitro labelling of muscle type nicotinic receptors using a fluorophore-conjugated pinnatoxin F derivative

Fluorescent molecules are regularly utilised to study ligand–receptor interactions. Many ligands for nicotinic receptors have been conjugated with fluorophores to study receptor kinetics, recycling and ligand binding characteristics. These include small agonist molecules, as well as large peptidic antagonists. However, no small molecule antagonists have been investigated using this method. Pinnatoxin F is a newly discovered non-peptidic muscle type nicotinic receptor antagonist produced by the marine dinoflagellate species Vulcanodinium rugosum. This molecule has the potential for conjugation to a fluorophore, allowing subsequent visualisation of interactions with nicotinic receptors. Pinnatoxin F was modified by addition of diaminopolyether spacers, to which a fluorophore (VivoTag® 645) was conjugated. The fluorescent pinnatoxin was then applied to muscle sections from thy1-YFP-H transgenic mice, which express YFP in motor nerves, to allow direct visualization of fluorescent binding at the neuromuscular junction. The addition of both the diaminopolyether spacer and the VivoTag® 645 reduced the potency of pinnatoxin F, as evidenced by a reduction in in vitro neuromuscular blocking activity and in vivo toxicity. Despite this reduced potency, the fluorescent molecule selectively labelled endplate regions in thy1-YFP mouse muscle sections and this labelling was inhibited by pre-exposure of muscle sections to native pinnatoxin F or the nicotinic antagonist α-bungarotoxin. This study proves nicotinic receptor binding activity of pinnatoxin F and is the first example of a fluorophore-conjugated small-molecule antagonist for nicotinic receptors. These results indicate the potential for other small-molecule nicotinic receptor antagonists to be fluorescently labelled and used as probes for specific nicotinic receptor subtypes.

Pyrilamine inhibits nicotine-induced catecholamine secretion

Function of nicotine, which induces activation of all parts of the body including our brain, has been receiving much attention for a long period of time and also been actively studied by researchers for its pharmacological actions in the central nervous system. The modulation of nicotine concentration and the inhibition of nicotine binding on target receptors in the brain are the key factors for smoking addiction therapy. In previous studies showed that influx of nicotine at the blood–brain barrier was through the pyrilamine-sensitive organic cation transporters. But the direct interacting mechanism of pyrilamine on the nicotine binding target receptors has not yet been clarified. The aim of the present study is to investigate the direct binding mechanisms of a pyrilamine on the nicotinic acetylcholine receptors (nAChRs). We found that pyrilamine shares the same ligand binding pocket of nicotine (NCT) on nAChRs but interacts with more amino acid residues than NCT does. The extended part of pyrilamine interacts with additional residues in the ligand binding pocket of nAChRs which are located nearby the entrance of the binding pocket. The catecholamine (CA) secretion induced by nAChR agonist (NCT′) was significantly inhibited by the pyrilamine pretreatment. Real time carbon-fiber amperometry confirmed the inhibition of the NCT′-induced exocytosis by pyrilamine in a single cell level. We also found that pyrilamine inhibited the NCT′-induced [Ca2+]i. In contrast, pyrilamine did not affect the increase in calcium induced by high K+. Overall, these data suggest that pyrilamine directly docks into the ligand binding site of nAChRs and specifically inhibits the nAChR-mediated effects thereby causing inhibition of CA secretion. Therefore, pyrilamine may play an important role to explore new treatments to aid smoking cessation.

The Pro-Proliferative Effects of Nicotine and Its Underlying Mechanism on Rat Airway Smooth Muscle Cells

Recent studies have shown that nicotine, a major component of cigarette smoke, can stimulate the proliferation of non-neuronal cells. Cigarette smoking can promote a variety of pulmonary and cardiovascular diseases, such as chronic obstructive pulmonary disease (COPD), atherosclerosis, and cancer. A predominant feature of COPD is airway remodeling, which includes increased airway smooth muscle (ASM) mass. The mechanisms underlying ASM remodeling in COPD have not yet been fully elucidated. Here, we show that nicotine induces a profound and time-dependent increase in DNA synthesis in rat airway smooth muscle cells (RASMCs) in vitro. Nicotine also significantly increased the number of RASMCs, which was associated with the increased expression of Cyclin D1, phosphorylation of the retinoblastoma protein (RB) and was dependent on the activation of Akt. The activation of Akt by nicotine occurred within minutes and depended upon the nicotinic acetylcholine receptors (nAchRs). Activated Akt increased the phosphorylation of downstream substrates such as GSK3β. Our data suggest that the binding of nicotine to the nAchRs on RASMCs can regulate cellular proliferation by activating the Akt pathway.

(304) The decreased antinociceptive efficacy of epibatidine in the rostral ventromedial medulla after peripheral inflammatory injury is not explained by decreases in α4β2 nicotinic receptor number or binding affinity

(304) The decreased antinociceptive efficacy of epibatidine in the rostral ventromedial medulla after peripheral inflammatory injury is not explained by decreases in α4β2 nicotinic receptor number or binding affinity

Nicotine shifts the temporal activation of hippocampal protein kinase A and extracellular signal-regulated kinase 1/2 to enhance long-term, but not short-term, hippocampus-dependent memory

Acute nicotine enhances hippocampus-dependent learning through nicotine binding to β2-containing nicotinic acetylcholine receptors (nAChRs), but it is unclear if nicotine is targeting processes involved in short-term memory (STM) leading to a strong long-term memory (LTM) or directly targeting LTM. In addition, the molecular mechanisms involved in the effects of nicotine on learning are unknown. Previous research indicates that protein kinase A (PKA), extracellular signal-regulated kinase 1/2 (ERK1/2), and protein synthesis are crucial for LTM. Therefore, the present study examined the effects of nicotine on STM and LTM and the involvement of PKA, ERK1/2, and protein synthesis in the nicotine-induced enhancement of hippocampus-dependent contextual learning in C57BL/6J mice. The protein synthesis inhibitor anisomycin impaired contextual conditioning assessed at 4h but not 2h post-training, delineating time points for STM (2h) and LTM (4h and beyond). Nicotine enhanced contextual conditioning at 4, 8, and 24h but not 2h post-training, indicating nicotine specifically enhances LTM but not STM. Furthermore, nicotine did not rescue deficits in contextual conditioning produced by anisomycin, suggesting that the nicotine enhancement of contextual conditioning occurs through a protein synthesis-dependent mechanism. In addition, inhibition of dorsal hippocampal PKA activity blocked the effect of acute nicotine on learning, and nicotine shifted the timing of learning-related PKA and ERK1/2 activity in the dorsal and ventral hippocampus. Thus, the present results suggest that nicotine specifically enhances LTM through altering the timing of PKA and ERK1/2 signaling in the hippocampus, and suggests that the timing of PKA and ERK1/2 activity could contribute to the strength of memories.

Varenicline: the risks of over-the-counter sales in Brazil

Brazil faces a knowledge gap concerning the factors associated with smoking patterns. In their large survey, Opaleye et al. report that approximately 16% of the 7,921 interviewees reported daily use of cigarettes, of whom 65% stated a wish to reduce or quit smoking. Varenicline, a partial agonist of the a4b2 nicotinic acetylcholine receptor, is considered one of the first-line treatments for tobacco dependence, which also include nicotine replacement therapy and bupropion. The efficacy of varenicline as a smoking cessation treatment is attributable to its binding and activation of the receptor at a significantly lower concentration than nicotine, thus preventing nicotine binding. During post-marketing surveillance studies, varenicline use was found to be associated with neuropsychiatric side effects, such as changes in behavior, depressed mood, and suicidal thoughts or actions. These side effects can occur in patients with or without preexisting neuropsychiatric disorders. Since some of these effects are related to nicotine withdrawal, it is difficult to distinguish whether the cause is the drug or the abstinence. In 2009, the Food and Drug Administration (FDA) required manufacturers of varenicline to add a boxed warning to its label. According to Rigotti, varenicline represents an important treatment for tobacco dependence, but the decision to prescribe it requires careful consideration of the risk–benefit balance and close monitoring by clinicians. The medication should be discontinued immediately if symptoms that concern the clinician, caregivers, family, or patient are observed. In Brazil, although varenicline is approved for sale ‘‘by prescription only,’’ it is readily available for purchase over the counter. No data are available on the number of side effects associated with the use of this drug in the country. The Brazilian Health Surveillance Agency (ANVISA) states that the release of this information was not authorized. Moore et al. assert that varenicline use is associated with an increased risk of suicidal/self-injury behavior and depression in smokers attempting to quit. Data for this study were drawn from case reports of adverse drug events received by the FDA from 1998 to 2010. The authors identified 3,249 cases of suicidal behavior and depression, with varenicline accounting for 90% of reports. The increased risk associated with this medication remained disproportionate even after comparison with other medications for smoking cessation, such as bupropion and nicotine replacement therapy, and after exclusion of case reports involving concomitant therapy with drugs carrying boxed warnings or precautions for suicidality in their prescribing information. Given Letters to the Editor 442

PET imaging of acetylcholinesterase inhibitor-induced effects on α4β2 nicotinic acetylcholine receptor binding

Acetylcholinesterase inhibitors (AChEIs) are drugs that increase synaptic acetylcholine (ACh) concentrations and are under investigation as treatments for symptoms accompanying Alzheimer's disease. The goal of this work was to use PET imaging to evaluate alterations of in vivo α4β2 nicotinic acetylcholine receptor (nAChR) binding induced by the AChEIs physostigmine (PHY) and galanthamine (GAL). The α4β2 nAChR-specific radioligand [(18)F]nifene was used to examine the effects of 0.1-0.2 mg/kg PHY, 5 mg/kg GAL, and saline in three separate experiments all performed on each of two rat subjects. A 60-min bolus-infusion protocol was used with drug administered after 30 min. Data from the thalamus and cortex were analyzed with a graphical model accounting for neurotransmitter activation using the cerebellum as a reference region to test for transient competition with bound [(18) F]nifene. Significant [(18) F]nifene displacement was detected in both regions during one PHY and both GAL studies, while no significant competition was observed in both saline studies. This preliminary work indicates the viability of [(18) F]nifene in detecting increases in synaptic ACh induced by AChEIs.

Alterations in α4β2 nicotinic receptors in cognitive decline in Alzheimer’s aetiopathology

Nicotinic acetylcholine receptor subtype α4β2 is considered important in the regulation of attention and memory, and cholinergic degeneration is known as one pathophysiology of Alzheimer's disease. Brain amyloid-β protein deposition is also a key pathological marker of Alzheimer's disease. Recent amyloid-β imaging has shown many cognitively normal subjects with amyloid-β deposits, indicating a missing link between amyloid-β deposition and cognitive decline. To date, the relationship between the α4β2 nicotinic acetylcholine receptor and amyloid-β burden has not been elucidated in vivo. In this study we investigated the relation between α4β2 nicotinic acetylcholine receptor availability in the brain, cognitive functions and amyloid-β burden in 20 non-smoking patients with Alzheimer's disease at an early stage and 25 age-matched non-smoking healthy elderly adults by measuring levels of α4β2 nicotinic acetylcholine receptor binding estimated from a simplified ratio method (BPRI) and Logan plot-based amyloid-β accumulation (BPND) using positron emission tomography with α4β2 nicotinic acetylcholine receptor tracer (18)F-2FA-85380 and (11)C-Pittsburgh compound B. The levels of tracer binding were compared with clinical measures for various brain functions (general cognition, episodic and spatial memory, execution, judgement, emotion) using regions of interest and statistical parametric mapping analyses. Between-group statistical parametric mapping analysis showed a significant reduction in (18)F-2FA-85380 BPRI in the cholinergic projection region in patients with Alzheimer's disease with a variety of (11)C-Pittsburgh compound B accumulation. Spearman rank correlation analyses showed positive correlations of (18)F-2FA-85380 BPRI values in the medial frontal cortex and nucleus basalis magnocellularis region with scores of the Frontal Assessment Battery (a test battery for executive functions and judgement) in the Alzheimer's disease group (P < 0.05 corrected for multiple comparison), and also positive correlations of the prefrontal and superior parietal (18)F-2FA-85380 BPRI values with the Frontal Assessment Battery score in the normal group (P < 0.05 corrected for multiple comparison). These positive correlations indicated an in vivo α4β2 nicotinic acetylcholine receptor role in those specific functions that may be different from memory. Both region of interest-based and voxelwise regression analyses showed a negative correlation between frontal (11)C-Pittsburgh compound B BPND and (18)F-2FA-85380 BPRI values in the medial frontal cortex and nucleus basalis magnocellularis region in patients with Alzheimer's disease (P < 0.05 corrected for multiple comparison). These findings suggest that an impairment of the cholinergic α4β2 nicotinic acetylcholine receptor system with the greater amount of amyloid deposition in the system plays an important role in the pathophysiology of Alzheimer's disease.

3H-Deprenyl and 3H-PIB autoradiography show different laminar distributions of astroglia and fibrillar β-amyloid in Alzheimer brain

BackgroundThe pathological features in Alzheimer’s disease (AD) brain include the accumulation and deposition of β-amyloid (Aβ), activation of astrocytes and microglia and disruption of cholinergic neurotransmission. Since the topographical characteristics of these different pathological processes in AD brain and how these relate to each other is not clear, this motivated further exploration using binding studies in postmortem brain with molecular imaging tracers. This information could aid the development of specific biomarkers to accurately chart disease progression.ResultsIn vitro binding assays demonstrated increased [3H]-PIB (fibrillar Aβ) and [3H]-PK11195 (activated microglia) binding in the frontal cortex (FC) and hippocampus (HIP), as well as increased binding of [3H]-l-deprenyl (activated astrocytes) in the HIP, but a decreased [3H]-nicotine (α4β2 nicotinic acetylcholine receptor (nAChR)) binding in the FC of AD cases compared to age-matched controls. Quantitative autoradiography binding studies were also performed to investigate the regional laminar distributions of [3H]-l-deprenyl, [3H]-PIB as well as [125I]-α-bungarotoxin (α7 nAChRs) and [3H]-nicotine in hemisphere brain of a typical AD case. A clear lamination pattern was observed with high [3H]-PIB binding in all layers and [3H]-deprenyl in superficial layers of the FC. In contrast, [3H]-PIB showed low binding to fibrillar Aβ, but [3H]-deprenyl high binding to activated astrocytes throughout the HIP. The [3H]-PIB binding was also low and the [3H]-deprenyl binding high in all layers of the medial temporal gyrus and insular cortex in comparison to the frontal cortex. Low [3H]-nicotine binding was observed in all layers of the frontal cortex in comparison to layers in the medial temporal gyrus, insular cortex and hippocampus. Immunohistochemical detection in the AD case revealed abundant glial fibrillary acidic protein positive (GFAP+) reactive astrocytes and α7 nAChR expressing GFAP+ astrocytes both in the vicinity and surrounding Aβ neuritic plaques in the FC and HIP. Although fewer Aβ plaques were observed in the HIP, some hippocampal GFAP+ astrocytes contained Aβ-positive (6 F/3D) granules within their somata.ConclusionsAstrocytosis shows a distinct regional pattern in AD brain compared to fibrillar Aβ, suggesting that different types of astrocytes may be associated with the pathophysiological processes in AD.

Rescue of Amyloid-Beta-Induced Inhibition of Nicotinic Acetylcholine Receptors by a Peptide Homologous to the Nicotine Binding Domain of the Alpha 7 Subtype

Alzheimer's disease (AD) is characterized by brain accumulation of the neurotoxic amyloid-β peptide (Aβ) and by loss of cholinergic neurons and nicotinic acetylcholine receptors (nAChRs). Recent evidence indicates that memory loss and cognitive decline in AD correlate better with the amount of soluble Aβ than with the extent of amyloid plaque deposits in affected brains. Inhibition of nAChRs by soluble Aβ40 is suggested to contribute to early cholinergic dysfunction in AD. Using phage display screening, we have previously identified a heptapeptide, termed IQ, homologous to most nAChR subtypes, binding with nanomolar affinity to soluble Aβ40 and blocking Aβ-induced inhibition of carbamylcholine-induced currents in PC12 cells expressing α7 nAChRs. Using alanine scanning mutagenesis and whole-cell current recording, we have now defined the amino acids in IQ essential for reversal of Aβ40 inhibition of carbamylcholine-induced responses in PC12 cells, mediated by α7 subtypes and other endogenously expressed nAChRs. We further investigated the effects of soluble Aβ, IQ and analogues of IQ on α3β4 nAChRs recombinantly expressed in HEK293 cells. Results show that nanomolar concentrations of soluble Aβ40 potently inhibit the function of α3β4 nAChRs, and that subsequent addition of IQ or its analogues does not reverse this effect. However, co-application of IQ makes the inhibition of α3β4 nAChRs by Aβ40 reversible. These findings indicate that Aβ40 inhibits different subtypes of nAChRs by interacting with specific receptor domains homologous to the IQ peptide, suggesting that IQ may be a lead for novel drugs to block the inhibition of cholinergic function in AD.

Toxic effects of pure anatoxin-a on biomarkers of rainbow trout, Oncorhynchus mykiss

Anatoxin-a is a neurotoxin produced by various bloom-forming cyanobacteria. Although it shows widespread occurrence and is highly toxic to rodents, its mechanisms of action and biotransformation, and effects in fish species are still poorly understood. The main aim of this study was, thus, to investigate sub-lethal effects of anatoxin-a on selected biochemical markers in rainbow trout fry in order to get information about the mechanisms of toxicity and biotransformation of this toxin in fish. Trout fry were administered sub-lethal doses of anatoxin-a (0.08–0.31 μg g−1) intraperitoneally. Livers and muscle tissue were collected 72 h later for quantification of key enzyme activities as biochemical markers. Enzymes assessed in muscle tissues were related to cholinergic transmission (acetylcholinesterase [AChE]), energy metabolism (lactate dehydrogenase [LDH] and NADP+-dependent isocitrate dehydrogenase [IDH]). Enzymes assessed in the liver were involved in biotransformation (ethoxyresorufin-O-deethylase [EROD] and glutathione S-transferases [GST]). The results indicated a significant increasing trend for AChE activity with the dose of anatoxin-a, possibly representing an attempt to cope with overstimulation of muscle activity by the toxin, which competes with acetylcholine for nicotinic receptors binding. Anatoxin-a was also found to significantly induce the activities of liver EROD and GST, indicating the involvement of phase I and II biotransformation in its detoxification. Likewise, lactate dehydrogenase activity recorded in fry muscle increased significantly with the dose of anatoxin-a, suggesting an induction of the anaerobic pathway of energy production to deal with toxic stress induced by the exposure. Altogether, the results suggest that under continued exposure in the wild fish may experience motor difficulties, possibly becoming vulnerable to predators, and be at increased metabolic demand to cope with energetic requirements imposed by anatoxin-a biotransformation mechanisms.