Key role of the TM2-TM3 loop in calcium potentiation of the α9α10 nicotinic acetylcholine receptor.

The α9α10 nicotinic cholinergic receptor (nAChR) is a ligand-gated pentameric cation-permeable ion channel that mediates synaptic transmission between descending efferent neurons and mechanosensory inner ear hair cells. When expressed in heterologous systems, α9 and α10 subunits can assemble into functional homomeric α9 and heteromeric α9α10 receptors. One of the differential properties between these nAChRs is the modulation of their ACh-evoked responses by extracellular calcium (Ca2+). While α9 nAChRs responses are blocked by Ca2+, ACh-evoked currents through α9α10 nAChRs are potentiated by Ca2+ in the micromolar range and blocked at millimolar concentrations. Using chimeric and mutant subunits, together with electrophysiological recordings under two-electrode voltage-clamp, we show that the TM2-TM3 loop of the rat α10 subunit contains key structural determinants responsible for the potentiation of the α9α10 nAChR by extracellular Ca2+. Moreover, molecular dynamics simulations reveal that the TM2-TM3 loop of α10 does not contribute to the Ca2+ potentiation phenotype through the formation of novel Ca2+ binding sites not present in the α9 receptor. These results suggest that the TM2-TM3 loop of α10 might act as a control element that facilitates the intramolecular rearrangements that follow ACh-evoked α9α10 nAChRs gating in response to local and transient changes of extracellular Ca2+ concentration. This finding might pave the way for the future rational design of drugs that target α9α10 nAChRs as otoprotectants.

Detection of Autoantibodies Against the Acetylcholine Receptor, Evaluation of Commercially Available Methodologies: Fixed Cell-Based Assay, Radioimmunoprecipitation Assay and Enzyme-Linked Immunosorbent Assay1.

Background/Objective: Myasthenia Gravis (MG) is an autoimmune disorder characterized by pathogenic autoantibodies (AAbs) targeting nicotinic acetylcholine receptors (AChR), disrupting neuromuscular communication. RadioImmunoPrecipitation Assay (RIPA) is recommended to detect AChR AAbs, but its complexity and radioactive requirements limit widespread use. We compare non-RIPA anti-AChR immunoassays, including Cell-Based Assay (CBA) and two ELISA kits, against the gold standard RIPA. Methods/Results: 145 samples were included with medical indication for anti-AChR testing. By the RIPA method, 63 were negative (RIPA-Neg < 0.02 nmol/L), 18 were classified as Borderline (≥0.02 -1 nmol/L), and 64 were positive (RIPA-Pos > 1 nmol/L). The competitive ELISA showed poor agreement with RIPA (Kappa = 0.216). The indirect ELISA demonstrated substantial agreement with RIPA (Kappa = 0.652), with ∼76% sensitivity and ∼94% specificity for MG diagnostic. The CBA, where fixed cells expressing clustered AChR were used as substrate, exhibited almost perfect agreement with RIPA (Kappa = 0.984), yielding ∼98% sensitivity and 96% specificity for MG. In addition, a semiquantitative analysis showed a strong correlation between CBA titration, indirect ELISA, and RIPA levels (r = 0.793 and r = 0.789, respectively). Conclusions: The CBA displayed excellent analytical performance for MG diagnostic when compared to RIPA, making it a potential replacement for RIPA in clinical laboratories. Some solid-phase assays (such as the indirect ELISA applied here), as well as CBA titration, offer reliable options to estimate anti-AChR AAb levels after confirming positivity by the CBA.∥.

Carlos Gutiérrez-Merino: Synergy of Theory and Experimentation in Biological Membrane Research.

Professor Carlos Gutiérrez-Merino, a prominent scientist working in the complex realm of biological membranes, has made significant theoretical and experimental contributions to the field. Contemporaneous with the development of the fluid-mosaic model of Singer and Nicolson, the Förster resonance energy transfer (FRET) approach has become an invaluable tool for studying molecular interactions in membranes, providing structural insights on a scale of 1-10 nm and remaining important alongside evolving perspectives on membrane structures. In the last few decades, Gutiérrez-Merino's work has covered multiple facets in the field of FRET, with his contributions producing significant advances in quantitative membrane biology. His more recent experimental work expanded the ground concepts of FRET to high-resolution cell imaging. Commencing in the late 1980s, a series of collaborations between Gutiérrez-Merino and the authors involved research visits and joint investigations focused on the nicotinic acetylcholine receptor and its relation to membrane lipids, fostering a lasting friendship.

Molecular Docking Analysis at the Human α7-nAChR and Proliferative and Evoked-Calcium Changes in SH-SY5Y Cells by Imidacloprid and Acetamiprid Insecticides.

Acetamiprid (ACE) and Imidacloprid (IMI) are widely-used neonicotinoid insecticides (NNIs) with functional activity at human acetylcholine nicotinic receptors and, therefore, with putative toxic effects. The objective of this study was the evaluation of the interactions between NNIs and α7-nAChR, as this receptor keeps intracellular Ca2+ ([Ca2+]i) to an optimum for an adequate neuronal functioning. Possible interactions between NNIs and the cryo-EM structure of the human α-7 nAChR were identified by molecular docking. Additionally, NNI effects were analyzed in neuroblastoma SH-SY5Y cells, as they naturally express α-7 nAChRs. Functional studies included proliferative/cytotoxic effects (MTT test) in undifferentiated SH-SY-5Y cells and indirect measurements of [Ca2+]i transients in retinoic acid-differentiated SH-SY-5Y cells loaded with Fluo-4 AM. Docking analysis showed that the binding of IMI and ACE occurred at the same aromatic cage that the specific α-7 nAChR agonist EVP-6124. IMI showed a better docking strength than ACE. According to the MTT assays, low doses (10-50 µM) of IMI better than ACE stimulated neuroblastoma cell proliferation. At higher doses (250-500 µM), IMI also prevailed over ACE and dose-dependently triggered more abrupt fluorescence changes due to [Ca2+]i mobilization in differentiated SH-SY5Y neurons. Indeed, only IMI blunted nicotine-evoked intracellular fluorescence stimulation (i.e., nicotine cross-desensitization). Summarizing, IMI demonstrated a superior docking strength and more robust cellular responses compared to ACE, which were likely associated with a stronger activity at α-7nAChRs. Through the interaction with α-7nAChRs, IMI would demonstrate its high neurotoxic potential for humans. More research is needed for investigating the proliferative effects of IMI in neuroblastoma cells.

Alpha-7 Nicotinic Receptor Agonist Protects Mice Against Pulmonary Emphysema Induced by Elastase.

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

Hypothalamic inflammation and the development of an obese phenotype induced by high-fat diet consumption is exacerbated in alpha7 nicotinic cholinergic receptor knockout mice.

Hypothalamic inflammation and metabolic changes resulting from the consumption of high-fat diets have been linked to low grade inflammation and obesity. Inflammation impairs the hypothalamic expression of α7 nicotinic acetylcholine receptor (α7nAChR). The α7nAChR is described as the main component of the anti-inflammatory cholinergic pathway in different inflammation models. To assess whether the reduction in α7nAChR expression exacerbates hypothalamic inflammation induced by a high-fat diet (HFD), were used male and female global α7nAChR knockout mouse line in normal or high-fat diet for 4 weeks. Body weight gain, adiposity, glucose homeostasis, hypothalamic inflammation, food intake, and energy expenditure were evaluated. Insulin sensitivity was evaluated in neuronal cell culture. Consumption of an HFD for 4 weeks resulted in body weight gain and adiposity in male Chrna7-/- mice and the hypothalamus of male Chrna7-/- mice showed neuroinflammatory markers, with increased gene expression of pro-inflammatory cytokines and dysregulation in the nuclear factor kappa B pathway. Moreover, male Chrna7-/- mice consuming an HFD showed alterations in glucose homeostasis and serum of Chrna7-/- mice that consumed an HFD impaired insulin signalling in neuronal cell culture experiments. In general, female Chrna7-/- mice that consumed an HFD did not show the phenotypic and molecular changes found in male mice, indicating that there is sexual dimorphism in the analysed parameters. Thus, receptor deletion resulted in increased susceptibility to hypothalamic inflammation and metabolic damage associated with HFD consumption in male mice.

Hypothalamic α7 nicotinic acetylcholine receptor (α7nAChR) is downregulated by TNFα-induced Let-7 overexpression driven by fatty acids.

The α7nAChR is crucial to the anti-inflammatory reflex, and to the expression of neuropeptides that control food intake, but its expression can be decreased by environmental factors. We aimed to investigate whether microRNA modulation could be an underlying mechanism in the α7nAchR downregulation in mouse hypothalamus following a short-term exposure to an obesogenic diet. Bioinformatic analysis revealed Let-7 microRNAs as candidates to regulate Chrna7, which was confirmed by the luciferase assay. Mice exposed to an obesogenic diet for 3 days had increased Let-7a and decreased α7nAChR levels, accompanied by hypothalamic fatty acids and TNFα content. Hypothalamic neuronal cells exposed to fatty acids presented higher Let-7a and TNFα levels and lower Chrna7 expression, but when the cells were pre-treated with TLR4 inhibitor, Let-7a, TNFα, and Chrna7 were rescued to normal levels. Thus, the fatty acids overload trigger TNFα-induced Let-7 overexpression in hypothalamic neuronal cells, which negatively regulates α7nAChR, an event that can be related to hyperphagia and obesity predisposition in mice.

Comparative Analysis of Alpha-1 Orthosteric-Site Binding by a Clade of Central American Pit Vipers (Genera Atropoides, Cerrophidion, Metlapilcoatlus, and Porthidium).

The distribution and relative potency of post-synaptic neurotoxic activity within Crotalinae venoms has been the subject of less investigation in comparison with Elapidae snake venoms. No previous studies have investigated post-synaptic neurotoxic activity within the Atropoides, Metlapilcoatlus, Cerrophidion, and Porthidium clade. Given the specificity of neurotoxins to relevant prey types, we aimed to uncover any activity present within this clade of snakes that may have been overlooked due to lower potency upon humans and thus not appearing as a clinical feature. Using biolayer interferometry, we assessed the relative binding of crude venoms to amphibian, lizard, bird, rodent and human α-1 nAChR orthosteric sites. We report potent alpha-1 orthosteric site binding in venoms from Atropoides picadoi, Metlapilcoatlus occiduus, M. olmec, M. mexicanus, M. nummifer. Lower levels of binding, but still notable, were evident for Cerrophidion godmani, C. tzotzilorum and C. wilsoni venoms. No activity was observed for Porthidium venoms, which is consistent with significant alpha-1 orthosteric site neurotoxicity being a trait that was amplified in the last common ancestor of Atropoides/Cerrophidion/Metlapilcoatlus subsequent to the split by Porthidium. We also observed potent taxon-selective activity, with strong selection for non-mammalian targets (amphibian, lizard, and bird). As these are poorly studied snakes, much of what is known about them is from clinical reports. The lack of affinity towards mammalian targets may explain the knowledge gap in neurotoxic activity within these species, since symptoms would not appear in bite reports. This study reports novel venom activity, which was previously unreported, indicating toxins that bind to post-synaptic receptors may be more widespread in pit vipers than previously considered. While these effects appear to not be clinically significant due to lineage-specific effects, they are of significant evolutionary novelty and of biodiscovery interest. This work sets the stage for future research directions, such as the use of in vitro and in vivo models to determine whether the alpha-1 orthosteric site binding observed within this study confers neurotoxic venom activity.

Statin exposure during pregnancy promotes neuromuscular junction alterations in postpartum Wistar rats.

INTRODUCTION/AIMS: The mechanisms that underlie the pathogenesis of statin-associated muscle symptoms (SAMS) remain unclear. Pregnancy is associated with increased cholesterol levels. Statins may be useful during pregnancy, but their safety is uncertain. Hence, we investigated the postpartum effects of exposure to rosuvastatin and simvastatin during pregnancy in Wistar rats, targeting the neuromuscular structures. METHODS: Twenty-one pregnant Wistar rats were divided into three groups: control (C) treated with vehicle (dimethylsulfoxide + dH20), simvastatin (S) 62.5 mg/kg/day, and rosuvastatin (R) 10 mg/kg/day. Gavage was performed daily from the gestational days 8 to 20. At weaning, the postpartum mother tissues were collected and subjected to morphological and morphometric analysis of the soleus muscle, associated neuromuscular junctions (NMJs), and the sciatic nerve; protein quantification; quantification of the cholesterol and creatine kinase in the serum; and intramuscular collagen analysis. RESULTS: An increase in morphometric parameters (area, maximum and minimum diameters, Feret diameter, and minimum Feret) was observed in NMJs from the S and R groups in comparison with the C group, and there was also a loss of common NMJ circularity. The number of myofibers with central nuclei was higher in S (17 ± 3.9, P = .0083) and R (18.86 ± 14.42, P = .0498) than in C (6.8 ± 2.6). DISCUSSION: Gestational exposure to statins induced postpartum NMJ morphology alterations in soleus muscle, which may be caused by the remodeling of clusters of nicotinic acetylcholine receptors. This may be associated with the development and progression of SAMS observed in clinical practice.

Modulation of a rapid neurotransmitter receptor-ion channel by membrane lipids.

Membrane lipids modulate the proteins embedded in the bilayer matrix by two non-exclusive mechanisms: direct or indirect. The latter comprise those effects mediated by the physicochemical state of the membrane bilayer, whereas direct modulation entails the more specific regulatory effects transduced via recognition sites on the target membrane protein. The nicotinic acetylcholine receptor (nAChR), the paradigm member of the pentameric ligand-gated ion channel (pLGIC) superfamily of rapid neurotransmitter receptors, is modulated by both mechanisms. Reciprocally, the nAChR protein exerts influence on its surrounding interstitial lipids. Folding, conformational equilibria, ligand binding, ion permeation, topography, and diffusion of the nAChR are modulated by membrane lipids. The knowledge gained from biophysical studies of this prototypic membrane protein can be applied to other neurotransmitter receptors and most other integral membrane proteins.

Fluorescence microscopy imaging of a neurotransmitter receptor and its cell membrane lipid milieu.

Hampered by the diffraction phenomenon, as expressed in 1873 by Abbe, applications of optical microscopy to image biological structures were for a long time limited to resolutions above the ∼200 nm barrier and restricted to the observation of stained specimens. The introduction of fluorescence was a game changer, and since its inception it became the gold standard technique in biological microscopy. The plasma membrane is a tenuous envelope of 4 nm-10 nm in thickness surrounding the cell. Because of its highly versatile spectroscopic properties and availability of suitable instrumentation, fluorescence techniques epitomize the current approach to study this delicate structure and its molecular constituents. The wide spectral range covered by fluorescence, intimately linked to the availability of appropriate intrinsic and extrinsic probes, provides the ability to dissect membrane constituents at the molecular scale in the spatial domain. In addition, the time resolution capabilities of fluorescence methods provide complementary high precision for studying the behavior of membrane molecules in the time domain. This review illustrates the value of various fluorescence techniques to extract information on the topography and motion of plasma membrane receptors. To this end I resort to a paradigmatic membrane-bound neurotransmitter receptor, the nicotinic acetylcholine receptor (nAChR). The structural and dynamic picture emerging from studies of this prototypic pentameric ligand-gated ion channel can be extrapolated not only to other members of this superfamily of ion channels but to other membrane-bound proteins. I also briefly discuss the various emerging techniques in the field of biomembrane labeling with new organic chemistry strategies oriented to applications in fluorescence nanoscopy, the form of fluorescence microscopy that is expanding the depth and scope of interrogation of membrane-associated phenomena.

Membrane lipid organization and nicotinic acetylcholine receptor function: A two-way physiological relationship.

Nicotinic acetylcholine receptors (nAChRs) are involved in a great range of physiological and pathological conditions. Since they are transmembrane proteins, they interact strongly with the lipids surrounding them. Thus, the plasma membrane composition and heterogeneity play an essential role for the correct nAChR function, on the one hand, and the nAChR influences its immediate lipid environment, on the other hand. The aim of this work was to investigate in more detail the role of the biophysical properties of the membrane in nAChR function and vice versa, focusing on the relationship between Chol and nAChRs. To this end, we worked with different model systems which were treated either with (i) more Chol, (ii) cholesteryl hemisuccinate, or (iii) the enzyme cholesterol oxidase to generate different membrane sterol conditions and in the absence and presence of γTM4 peptide as a representative model of the nAChR. Fluorescence measurements with crystal violet and patch-clamp recordings were used to study nAChR conformation and function, respectively. Using confocal microscopy of giant unilamellar vesicles we probed the membrane phase state/order and organization (coexistence of lipid domains) and lipid-nAChR interaction. Our results show a feedback relationship between membrane organization and nAChR function, i.e. whereas the presence of a model of nAChRs conditions membrane organization, changing its lipid microenvironment, membrane organization and composition perturb nAChRs function. We postulate that nAChRs have a gain of function in disordered membrane environments but a loss of function in ordered ones, and that Chol molecules at the outer leaflet in annular sites and at the inner leaflet in non-annular sites are related to nAChR gating and desensitization, respectively. Thus, depending on the membrane composition, organization, and/or order, the nAChR adopts different conformations and locates in distinct lipid domains and this has a direct effect on its function.

Mapping the Nicotinic Acetylcholine Receptor Nanocluster Topography at the Cell Membrane with STED and STORM Nanoscopies.

The cell-surface topography and density of nicotinic acetylcholine receptors (nAChRs) play a key functional role in the synapse. Here we employ in parallel two labeling and two super-resolution microscopy strategies to characterize the distribution of this receptor at the plasma membrane of the mammalian clonal cell line CHO-K1/A5. Cells were interrogated with two targeted techniques (confocal microscopy and stimulated emission depletion (STED) nanoscopy) and single-molecule nanoscopy (stochastic optical reconstruction microscopy, STORM) using the same fluorophore, Alexa Fluor 647, tagged onto either α-bungarotoxin (BTX) or the monoclonal antibody mAb35. Analysis of the topography of nanometer-sized aggregates ("nanoclusters") was carried out using STORMGraph, a quantitative clustering analysis for single-molecule localization microscopy based on graph theory and community detection, and ASTRICS, an inter-cluster similarity algorithm based on computational geometry. Antibody-induced crosslinking of receptors resulted in nanoclusters with a larger number of receptor molecules and higher densities than those observed in BTX-labeled samples. STORM and STED provided complementary information, STED rendering a direct map of the mesoscale nAChR distribution at distances ~10-times larger than the nanocluster centroid distances measured in STORM samples. By applying photon threshold filtering analysis, we show that it is also possible to detect the mesoscale organization in STORM images.

A short framework-III (mini-M-2) conotoxin from the venom of a vermivorous species, Conus archon, inhibits human neuronal nicotinic acetylcholine receptors.

The venoms of Conus snails contain neuroactive peptides named conotoxins (CTXs). Some CTXs are nicotinic acetylcholine receptor (nAChRs) antagonists. nAChRs modulate the release of neurotransmitters and are implicated in several pathophysiologies. One venom peptide from Conus archon, a vermivorous species from the Mexican Pacific, was purified by RP-HPLC and its activity on human α7, α3ß2, and α7ß2 nAChRs was assessed by the two-electrode voltage clamp technique. At 36.3 µM the purified peptide (F27-1, renamed tentatively ArchIIIA) slowly reversibly inhibited the ACh-induced response of the hα7 subtype by 44.52 ± 5.83%, while it had low or no significant effect on the response of the hα3ß2 and hα7ß2 subtypes; the EC50 of the inhibiting effect was 45.7 µM on the hα7 subtype. This peptide has 15 amino acid residues and a monoisotopic mass of 1654.6 Da (CCSALCSRYHCLPCC), with three disulfide bridges and a free C-terminus. This sequence with a CC-C-C-CC arrangement (framework III) belongs to the M superfamily of conotoxins, corresponding to the mini-M´s (M-1-M-3) conotoxins; due to its size and inter-Cys spacings it is an M-2 conotoxin. This toxin is a novel mini-M conotoxin affecting ligand-gated ion channels, like the maxi-M CTX ψ-conotoxins and α-MIIIJ conotoxin (nAChRs blockers). This peptide seems to be homologous to the reg3b conotoxin (from Conus regius) with an identity of 93.3%, differing only in the third residue in the sequence, serine for threonine, both uncharged polar residues. We obtained, in silico, a probable 3D structure, which is consistent with its effect on neuronal subtypes.

Dendritic spine membrane proteome and its alterations in autistic spectrum disorder.

Dendritic spines are small protrusions stemming from the dendritic shaft that constitute the primary specialization for receiving and processing excitatory neurotransmission in brain synapses. The disruption of dendritic spine function in several neurological and neuropsychiatric diseases leads to severe information-processing deficits with impairments in neuronal connectivity and plasticity. Spine dysregulation is usually accompanied by morphological alterations to spine shape, size and/or number that may occur at early pathophysiological stages and not necessarily be reflected in clinical manifestations. Autism spectrum disorder (ASD) is one such group of diseases involving changes in neuronal connectivity and abnormal morphology of dendritic spines on postsynaptic neurons. These alterations at the subcellular level correlate with molecular changes in the spine proteome, with alterations in the copy number, topography, or in severe cases in the phenotype of the molecular components, predominantly of those proteins involved in spine recognition and adhesion, reflected in abnormally short lifetimes of the synapse and compensatory increases in synaptic connections. Since cholinergic neurotransmission participates in the regulation of cognitive function (attention, memory, learning processes, cognitive flexibility, social interactions) brain acetylcholine receptors are likely to play an important role in the dysfunctional synapses in ASD, either directly or indirectly via the modulatory functions exerted on other neurotransmitter receptor proteins and spine-resident proteins.

Cholinergic immunomodulation in inflammatory bowel diseases.

Inflammatory bowel diseases (IBD) are chronic intestinal disorders characterized by dysregulated immune responses to resident microbiota in genetically susceptible hosts. The activation of the cholinergic system has been proposed for the treatment of IBD patients according to its potential anti-inflammatory effect in vivo. The α-7-nicotinic-acetylcholine receptor (α7nAChR) is involved in the inhibition of inflammatory processes, modulating the production of cytokines, suppressing dendritic cells and macrophage activity, leading to the suppression of T cells. In this review, we address the most recent studies and clinical trials concerning cholinergic signaling and its therapeutic potential for inflammatory bowel diseases.

A phase II randomized trial to evaluate the long-term (12-week) efficacy and safety of OC-01 (varenicline solution) nasal spray for dry eye disease: The MYSTIC study.

PURPOSE: Dry eye disease is characterized by loss of tear film stability. OC-01 (varenicline solution) is a small-molecule nicotinic acetylcholine receptor agonist administered as a nasal spray that stimulates tear production. METHODS: In MYSTIC (NCT03873246) patients aged ≥22 years with dry eye disease were randomized 1:1:1 to OC-01 0.03 mg, OC-01 0.06 mg, or vehicle (n = 41 per group), administered twice daily via intranasal spray, for 12 weeks (84 days). Primary efficacy endpoint was mean change from baseline in anesthetized Schirmer's test score (STS) in study eye at day (D) 84. RESULTS: Patients receiving OC-01 0.03 and 0.06 mg had statistically significantly increased tear production at D84 versus vehicle; least squares mean changes from baseline in STS were 10.8 mm and 11.0 mm for OC-01 0.03 and 0.06 mg, respectively. A trend toward a higher proportion of patients experiencing ≥10-mm improvement in STS from baseline was observed with OC-01 0.03 mg (36.6%; p > 0.05), and was significant for OC-01 0.06 mg (48.8%; p = 0.024), versus vehicle (24.4%). Non-ocular treatment-emergent adverse events (TEAEs) were reported by 21 patients; the most common was sneezing (OC-01 0.03 mg, 2 [4.9%]; OC-01 0.06 mg, 3 [7.3%]), with similar frequencies between treatment groups. No severe or serious TEAEs were reported. CONCLUSIONS: OC-01 (varenicline solution) nasal spray improved tear production in patients with dry eye disease over a long-term (12-week) period, and represents a receptor neuro-activator with a nasal route of administration that spares the ocular surface to stimulate tear production.

Toxic mechanisms of cigarette smoke and heat-not-burn tobacco vapor inhalation on rheumatoid arthritis.

Tobacco combustion exposure worsens rheumatoid arthritis (RA). Non-combustible tobacco devices, as heat-not-burn tobacco (HNBT), are emerging as harm reduction to smokers by releasing nicotine and lower combustible tobacco products. Nevertheless, HNBT toxicity remains unclear. Hence, here we investigated the impacts of the tobacco combustible product (cigarette smoke; CS) or HNBT vapor exposures on antigen-induced arthritis (AIA) in C57BL/6 mice. Animals were exposed to airflow, HNBT vapor, or CS during 1 h/twice a day, under the Health Canada Intense (HCI) smoking regime, between days 14 to 20 after the first immunization. At day 21, 16 h after the last exposures, mice were i.a. challenged and the AIA effects were evaluated 24 h later. CS- or HNBT-exposed mice presented equivalent blood nicotine levels. CS exposure worsened articular symptoms, pulmonary inflammation, and expression of lung metallothioneins. Nevertheless, CS or HNBT exposures reduced lymphoid organs' cellularity, splenocyte proliferation and IL-2 secretion. Additional in vitro CS or HNBT exposures confirmed the harmful effects on splenocytes, which were partially mediated by the activation of nicotine/α7nAchR pathway. Associated, data demonstrate the toxic mechanisms of CS or HNBT inhalation at HCI regime on RA, and highlight that further investigations are fundamental to assure the toxicity of emerging tobacco products on the immune system during specific challenges.

Oxidative stress and decreased dopamine levels induced by imidacloprid exposure cause behavioral changes in a neurodevelopmental disorder model in Drosophila melanogaster.

Neurodevelopmental disorders, such as Autism Spectrum Disorder (ASD) and Attention Deficit Hyperactivity Disorder (ADHD) are responsible for behavioral deficits in children. Imidacloprid is a nicotinic acetylcholine receptor agonist, capable of causing behavioral changes in Drosophila melanogaster, similar to the ADHD-like phenotypes. We assess whether behavioral damage induced by imidacloprid exposure in Drosophila melanogaster is associated with neurochemical changes and whether these changes are similar to those observed in neurodevelopmental disorders such as ASD and ADHD. The fruit flies were divided into four groups, exposed to either a standard diet (control) or a diet containing imidacloprid (200, 400 or 600 ρM) and allowed to mate for 7 days. After hatching, the progeny was subjected to in vivo and ex vivo tests. The ones exposed to imidacloprid showed an increase in hyperactivity, aggressiveness, anxiety and repetitive movements, as well as, a decrease in social interaction. Furthermore, exposure to imidacloprid decreased dopamine levels, cell viability and increased oxidative stress in the flies' progeny. These results demonstrated that the behavioral damage induced by imidacloprid exposure involves a reduction in dopamine levels and oxidative stress and that these neurochemical changes are in line with the events that occur in ASD and ADHD-like phenotypes in other models.

Possible implications of dysregulated nicotinic acetylcholine receptor diffusion and nanocluster formation in myasthenia gravis.

Myasthenia gravis is a rare and invalidating disease affecting the neuromuscular junction of voluntary muscles. The classical form of this autoimmune disease is characterized by the presence of antibodies against the most abundant protein in the neuromuscular junction, the nicotinic acetylcholine receptor. Other variants of the disease involve autoimmune attack of non-receptor scaffolding proteins or enzymes essential for building or maintaining the integrity of this peripheral synapse. This review summarizes the participation of the above proteins in building the neuromuscular junction and the destruction of this cholinergic synapse by autoimmune aggression in myasthenia gravis. The review also covers the application of a powerful biophysical technique, superresolution optical microscopy, to image the nicotinic receptor in live cells and follow its motional dynamics. The hypothesis is entertained that anomalous nanocluster formation by antibody crosslinking may lead to accelerated endocytic internalization and elevated turnover of the receptor, as observed in myasthenia gravis.