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Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell-surface immunoreceptor expressed on microglia, osteoclasts, dendritic cells andmacrophages. Heterozygous loss-of-function mutations in TREM2, including mutations enhancing shedding form the cell-surface, have been associated with myelin/neuronal loss and neuroinflammation in neurodegenerative diseases like Alzheimer`s disease and Frontotemporal Dementia. Using the cuprizone model, we investigated the involvement of soluble and cleavage-reduced TREM2 on central myelination processes in cleavage-reduced (TREM2-IPD), soluble-only (TREM2-sol), knock-out (TREM2-KO) and wildtype (WT) mice. In the acute cuprizone model consisting of a five-week intoxication by the copper chelator followed by a four-week recovery all genotypes showed demyelination at the peak of disease reflected by a T2-weighted signal intensity increase in magnetic resonance imaging (MRI), most prominently in the external capsule (EC). During the recovery phase a partial signal reduction in WT and TREM2-IPD was observed, while TREM2-sol and TREM2-KO showed an additional increase in MRI signal in the EC. At week 9 histology revealed no recovery of neuroinflammation as well as of the lysosomal marker LAMP-1 in TREM2-IPD animals, which also displayed enhanced cytokine/chemokine levels in the brain. Despite presenting reduced levels of some cytokines/chemokines, persistent microgliosis and astrocytosis at week 9 was likewise observed in TREM2-sol and to a much lesser extent in TREM2-KO, with both homeostatic (TMEM119) as well as activated (LAMP-1) microglia markers increased. This was accompanied, specifically in the EC, by no myelin recovery, with appearance of myelin debris and axonal pathology, while oligodendrocytes recovered. In the chronic model consisting of 12-week cuprizone administration followed by 3-week recovery TREM2-IPD displayed sustained microgliosis and enhanced remyelination in the recovery phase. Taken together, our data suggest that sustained microglia activation led to increased remyelination, whereas microglia with only soluble TREM2 had reduced phagocytic activity despite efficient lysosomal function leading to a dysfunctional phenotype with improper myelin debris removal, lack of remyelination and axonal pathology following cuprizone intoxication.

Triggering receptor expressed on myeloid cells 2 (TREM2) is a cell-surface immunoreceptor expressed on microglia, osteoclasts and monocytes/macrophages. In humans, homozygous loss-of-function mutation carriers exhibit bone cysts and fractures with an early-onset frontal lobe syndrome and progressive presenile dementia known as Nasu-Hakola. Neuropathological findings include myelin/neuronal loss and neuroinflammation. Heterozygous loss-of-function mutation in TREM2 has been associated with several neurodegenerative diseases like Alzheimer`s disease and Frontotemporal Dementia. We investigated the involvement of soluble and cleavage-reduced TREM2 on central myelination processes by using different gene-modified animals expressing (cleavage-reduced (TREM2-IPD), soluble-only (TREM2-sol), knock-out (TREM2-KO) TREM2and wildtype (WT) mice in the cuprizone model.. In the acute cuprizone model all genotypes showed demyelination detected by T2-weighted signal intensity increase in Magnetic resonance imaging (MRI), most prominent in the external capsule (EC) and less in the corpus callosum. In the 4-week recovery phase a partial signal reduction in WT and TREM2-IPD was observed, while in TREM2-sol and TREM2-KO showed an increase in MRI signal in the EC. In TREM2-IPD histology revealed no recovery of neuroinflammation as well as of the lysosomal marker LAMP-1, which was also evidenced in enhanced cytokine/chemokine levels. This persistent microgliosis and astrocytosis was also observed in TREM2-sol, to a lesser extent in TREM2-KO during recovery with both homeostatic (TMEM119) as well as activated (LAMP-1) microglia markers increased. An increase in cytokines/chemokines was not evident. This was accompanied, specifically in the EC with no myelin recovery, appearance of myelin debris and axonal pathology, while oligodendrocytes recovered. In the chronic cuprizone model TREM2-IPD displayed sustained microgliosis and enhanced remyelination in the recovery phase. Taken together, our data suggest that sustained microglia activation leads to increased remyelination, whereas microglia without membrane-functional or complete loss of TREM2 results in a dysfunctional phenotype with improper myelin debris removal, lack of remyelination and axonal pathology.

The alkaloids aristoteline (1), aristoquinoline (2), and aristone (3) were purified from the leaves of the Maqui tree Aristotelia chilensis and chemically characterized by NMR spectroscopy. The pharmacological activity of these natural compounds was evaluated on human (h) α3β4, α4β2, and α7 nicotinic acetylcholine receptors (AChRs) by Ca2+ influx measurements. The results suggest that these alkaloids do not have agonistic, but inhibitory, activity on each receptor subtype. The obtained IC50 values indicate the following receptor selectivity: hα3β4 > hα4β2 ≫ hα7. In the particular case of hα3β4 AChRs, 1 (0.40 ± 0.20 μM) and 2 (0.96 ± 0.38 μM) show higher potencies compared with 3 (167 ± 3 μM). Molecular docking and structure-activity relationship results indicate that ligand lipophilicity is important for the interaction with the luminal site located close to the cytoplasmic side of the hα3β4 ion channel between positions -2' and -4'. Compound 1 could be used as a molecular scaffold for the development of more potent noncompetitive inhibitors with higher selectivity for the hα3β4 AChR that could serve for novel addiction and depression therapies.

SummaryTREM2 is a transmembrane protein expressed exclusively in microglia in the brain that regulates inflammatory responses to pathological conditions. Proteolytic cleavage of membrane TREM2 affects microglial function and is associated with Alzheimer’s disease, but the consequence of reduced TREM2 proteolytic cleavage has not been determined. We generated a transgenic mouse model of reduced TREM2 shedding (Trem2-IPD) through amino acid substitution of ADAM-protease recognition site. We found that Trem2-IPD mice displayed increased TREM2 cell surface receptor load, survival and function in myeloid cells. Using single cell transcriptomic profiling of mouse cortex we show that sustained TREM2 stabilization induces a shift of fate in microglial maturation and accelerates microglial responses to Aβ pathology in a mouse model of Alzheimer’s disease. Our data indicate that reduction of TREM2 proteolytic cleavage aggravates neuroinflammation during the course of AD pathology suggesting that TREM2 shedding is a critical regulator of microglial activity in pathological states.

TREM2 is a transmembrane protein expressed exclusively in microglia in the brain that regulates inflammatory responses to pathological conditions. Proteolytic cleavage of membrane TREM2 affects microglial function and is associated with Alzheimer's disease, but the consequence of reduced TREM2 proteolytic cleavage has not been determined. Here, we generate a transgenic mouse model of reduced Trem2 shedding (Trem2-Ile-Pro-Asp [IPD]) through amino-acid substitution of an ADAM-protease recognition site. We show that Trem2-IPD mice display increased Trem2 cell-surface-receptor load, survival, and function in myeloid cells. Using single-cell transcriptomic profiling of mouse cortex, we show that sustained Trem2 stabilization induces a shift of fate in microglial maturation and accelerates microglial responses to Aβ pathology in a mouse model of Alzheimer's disease. Our data indicate that reduction of Trem2 proteolytic cleavage aggravates neuroinflammation during the course of Alzheimer's disease pathology, suggesting that TREM2 shedding is a critical regulator of microglial activity in pathological states.

Alpha-7-nicotine-acetylcholine-receptor (α7-nAChRs) agonists modulate the cholinergic antiinflammatory pathway to attenuate proinflammatory signals and reduce dopaminergic neuronal cell loss in toxin-induced experimental murine models of Parkinson’s disease (PD). The protein α-synuclein (αSyn) is considered to represent the major pathogenic component in the etiology and progression of sporadic PD. However, no research has been performed to evaluate the effect of α7-nAChR agonists in human αSyn mediated models of PD. We, therefore, investigated the effect of the compound JN403, an α7-nAChR specific agonist, in αSyn treatedin vitromicroglia culture and in a human αSyn overexpressionin vivomouse model. In primary mouse microglia cells, αSyn fragment 61-140 treatment increased the release of nitric oxide (NO), tumor necrosis factor (TNF)-α and interleukin (IL)-6, and decreased cell viability. In contrast, 100 nM or 1 μM of JN403 co-incubation significantly reduced the level of NO and TNF-α release in the microglial cells. For in-vivo testing of JN403, a recombinant adeno-associated viral vector (rAAV)-mediated unilateral intranigral overexpression of human wild-type-αSyn (WT-αSyn) or of the control protein luciferase (luc) was induced via stereotactic delivery in C57/BL6N mice. Targeted WT-αSyn overexpression reduced 20% of the number of tyrosine hydroxylase (TH) positive (+) nigral neurons after 10 weeks. Subcutaneous daily treatment of 30 mg/kg JN403 over 9 weeks starting at postoperative week 1 did not alter the decrease of TH+ neuronal numbers, and microglial density in WT-αSyn overexpression mouse model. The reduced density of TH+ striatal terminals in the WT-αSyn groups was also not recovered by the JN403 treatment. In summary, JN403, an α7-nAChR specific agonist shows a beneficial effect on ameliorating proinflammatory signals in αSyn exposed microglia cells. However, no significant in-vivo treatment effect was found in an intranigral WT-αSyn overexpression mouse model of PD.

The activity of tricyclic antidepressants (TCAs) at α7 and α9α10 nicotinic acetylcholine receptors (AChRs) as well as at hippocampal α7-containing (i.e., α7*) AChRs is determined by using Ca 2+ influx and electrophysiological recordings. To determine the inhibitory mechanisms, additional functional tests and molecular docking experiments are performed. The results established that TCAs (a) inhibit Ca 2+ influx in GH3-α7 cells with the following potency (IC 50 in μM) rank: amitriptyline (2.7 ± 0.3) > doxepin (5.9 ± 1.1) ∼ imipramine (6.6 ± 1.0). Interestingly, imipramine inhibits hippocampal α7* AChRs (42.2 ± 8.5 μM) in a noncompetitive and voltage-dependent manner, whereas it inhibits α9α10 AChRs (0.53 ± 0.05 μM) in a competitive and voltage-independent manner, and (b) inhibit [ 3 H]imipramine binding to resting α7 AChRs with the following affinity rank (IC 50 in μM): imipramine (1.6 ± 0.2) > amitriptyline (2.4 ± 0.3) > doxepin (4.9 ± 0.6), whereas imipramine's affinity was no significantly different to that for the desensitized state. The molecular docking and functional results support the notion that imipramine noncompetitively inhibits α7 AChRs by interacting with two overlapping luminal sites, whereas it competitively inhibits α9α10 AChRs by interacting with the orthosteric sites. Collectively our data indicate that TCAs inhibit α7, α9α10, and hippocampal α7* AChRs at clinically relevant concentrations and by different mechanisms of action. Fil: Arias, Hugo Rubén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. California Northstate University College of Medicine; Estados Unidos Fil: Vázquez Gómez, Elizabeth. Universidad Nacional Autónoma de México; México Fil: Hernández Abrego, Andy. Universidad Nacional Autónoma de México; México Fil: Gallino, Sofia Ludmila. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: Feuerbach, Dominik. Novartis Institutes for Biomedical Research; Suiza Fil: Ortells, Marcelo Oscar. Universidad de Morón. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Universidad de Buenos Aires. Facultad de Medicina; Argentina Fil: García Colunga, Jesús. Universidad Nacional Autónoma de México; México

The inhibitory activity of (±)-citalopram on human (h) α3β4, α4β2, and α7 nicotinic acetylcholine receptors(AChRs) was determined by Ca2+ influx assays, whereas its effect on rat α9α10 and mouse habenular α3β4*AChRs by electrophysiological recordings. The Ca2+ influx results clearly establish that (±)-citalopram inhibits(IC50´s in μM) hα3β4 AChRs (5.1 ± 1.3) with higher potency than that for hα7 (18.8 ± 1.1) and hα4β2(19.1 ± 4.2) AChRs. This is in agreement with the [3H]imipramine competition binding results indicating that(±)-citalopram binds to imipramine sites at desensitized hα3β4 with>2-fold higher affinity than that forhα4β2. The electrophysiological, molecular docking, and in silico mutation results indicate that (±)-citalopramcompetitively inhibits rα9α10 AChRs (7.5 ± 0.9) in a voltage-independent manner by interacting mainly withorthosteric sites, whereas it inhibits a homogeneous population of α3β4* AChRs at MHb (VI) neurons(7.6 ± 1.0) in a voltage-dependent manner by interacting mainly with a luminal site located in the middle ofthe ion channel, overlapping the imipramine site, which suggests an ion channel blocking mechanism. In conclusion,(±)-citalopram inhibits α3β4 and α9α10 AChRs with higher potency compared to other AChRs but bydifferent mechanisms. (±)-Citalopram also inhibits habenular α3β4*AChRs, supporting the notion that thesereceptors are important endogenous targets related to their anti-addictive activities. Fil: Arias, Hugo Rubén. Oklahoma State University; Estados Unidos Fil: Jin, Xiao Tao. Northwestern University; Estados Unidos Fil: Gallino, Sofia Ludmila. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Peng, Can. Northwestern University; Estados Unidos Fil: Feuerbach, Dominik. Novartis Institutes for Biomedical Research; Suiza Fil: García Colunga, Jesús. Universidad Nacional Autónoma de México; México Fil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina. Universidad de Buenos Aires. Facultad de Medicina. Instituto de Farmacologia; Argentina Fil: Drenan, Ryan M.. Northwestern University; Estados Unidos Fil: Ortells, Marcelo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Morón; Argentina

The alkaloids aristoteline (

Enhancement of α7 nicotinic receptor (nAChR) function by positive allosteric modulators (PAMs) is a promising therapeutic strategy to improve cognitive deficits. PAMs have been classified only on the basis of their macroscopic effects as type I, which only enhance agonist-induced currents, and type II, which also decrease desensitization and reactivate desensitized nAChRs. To decipher the molecular basis underlying these distinct activities, we explored the effects on single-α7 channel currents of representative members of each type and of less characterized compounds. Our results reveal that all PAMs enhance open-channel lifetime and produce episodes of successive openings, thus indicating that both types affect α7 kinetics. Different PAM types show different sensitivity to temperature, suggesting different mechanisms of potentiation. By using a mutant α7 receptor that is insensitive to the prototype type II PAM (PNU-120596), we show that some though not all type I PAMs share the structural determinants of potentiation. Overall, our study provides novel information on α7 potentiation, which is key to the ongoing development of therapeutic compounds. Fil: Andersen, Natalia Denise. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Nielsen, Beatriz Elizabeth. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Corradi, Jeremias. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Tolosa, María Fernanda. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina Fil: Feuerbach, Dominik. Novartis Institutes For Biomedical Research; Suiza Fil: Arias, Hugo Rubén. California Northstate University College Of Medicine; Estados Unidos Fil: Bouzat, Cecilia Beatriz. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Bahía Blanca. Instituto de Investigaciones Bioquímicas de Bahía Blanca. Universidad Nacional del Sur. Instituto de Investigaciones Bioquímicas de Bahía Blanca; Argentina

Macrophages and their counterparts in the central nervous system, the microglia, detect and subsequently clear microbial pathogens and injured tissue. These phagocytic cells alter and adapt their phenotype depending on their prime activity, i.e., whether they participate in acute defence against pathogenic organisms ('M1'-phenotype) or in clearing damaged tissues and performing repair activities ('M2'-phenotype). Stimulation of pattern recognition receptors by viruses (vaccines), bacterial membrane components (e.g., LPS), alcohol, or long-chain saturated fatty acids promotes M1-polarization. Vaccine or LPS administration to healthy human subjects can result in sickness symptoms and low mood. Alcohol abuse and abdominal obesity are recognized as risk factors for depression. In the M1-polarized form, microglia and macrophages generate reactive oxygen and nitrogen radicals to eradicate microbial pathogens. Inadvertently, also tetrahydrobiopterin (BH4) may become oxidized. This is an irreversible reaction that generates neopterin, a recognized biomarker for depression. BH4 is a critical cofactor for the synthesis of dopamine, noradrenaline, and serotonin, and its loss could explain some of the symptoms of depression. Based on these aspects, the suppression of M1-polarization would limit the inadvertent catabolism of BH4. In the current review, we evaluate the evidence that antidepressant treatments (monoamine reuptake inhibitors, PDE4 inhibitors, lithium, valproate, agomelatine, tianeptine, electroconvulsive shock, and vagus nerve stimulation) inhibit LPS-induced microglia/macrophage M1-polarization. Consequently, we propose that supplementation with BH4 could limit the reduction in central monoamine synthesis and might represent an effective treatment for depressed mood.

tThe activity of positive allosteric modulators (PAMs) of 7 nicotinic acetylcholine receptors (AChRs),including 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), 3-furan-2-yl-N-o-tolylacrylamide (PAM-3), and3-furan-2-yl-N-phenylacrylamide (PAM-4), was tested on a variety of ligand- [i.e., human (h) 7, rat(r) 910, h3-containing AChRs, mouse (m) 5-HT3AR, and several glutamate receptors (GluRs)] andvoltage-gated (i.e., sodium and potassium) ion channels, as well as on acetylcholinesterase (AChE) and-amyloid (A) content. The functional results indicate that PAM-2 inhibits h3-containing AChRs(IC50= 26 ± 6 M) with higher potency than that for NR1aNR2B and NR1aNR2A, two NMDA-sensitiveGluRs. PAM-2 affects neither the activity of m5-HT3ARs, GluR5/KA2 (a kainate-sensitive GluR), nor AChE,and PAM-4 does not affect agonist-activated r910 AChRs. Relevant clinical concentrations of PAM-2?4 do not inhibit Nav1.2 and Kv3.1 ion channels. These PAMs slightly enhance the activity of GluR1 andGluR2, two AMPA-sensitive GluRs. PAM-2 does not change the levels of A42in an Alzheimer?s diseasemouse model (i.e., 5XFAD). The molecular docking and dynamics results using the h7 model suggest thatthe active sites for PAM-2 include the intrasubunit (i.e., PNU-120596 locus) and intersubunit sites. Theseresults support our previous study showing that these PAMs are selective for the 7 AChR, and clarifythat the procognitive/promnesic/antidepressant activity of PAM-2 is not mediated by other targets. Fil: Arias, Hugo R.. California Northstate University; Estados Unidos Fil: Ravazzini, Federica. Università Degli Studi Di Modena e Reggio Emilia; Argentina Fil: Targowska Duda, Katarzyna M.. Medical University of Lublin; Polonia Fil: Kaczor, Agnieszka A.. Medical University of Lublin; Polonia Fil: Feuerbach, Dominik. Novartis Institutes for Biomedical Research; Suiza Fil: Boffi, Juan Carlos. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Draczkowski, Piotr. Medical University of Lublin; Polonia Fil: Montag, Dirk. Leibniz Institute for Neurobiology; Alemania Fil: Brown, Brandon M.. University of California; Estados Unidos Fil: Elgoyhen, Ana Belen. Consejo Nacional de Investigaciones Científicas y Técnicas. Instituto de Investigaciones en Ingeniería Genética y Biología Molecular "Dr. Héctor N. Torres"; Argentina Fil: Jozwiak, Krzysztof. Medical University of Lublin; Polonia Fil: Puia, Giulia. Università Degli Studi Di Modena e Reggio Emilia; Argentina

The Alzheimer's Association's Research roundtable met in April 2015 to explore the role of neuroinflammatory mechanisms in the progression of Alzheimer's disease (AD). The ability of innate immune cells, particularly microglia and astrocytes, to mediate neuroinflammation in AD has been implicated as a significant contributor to disease pathogenesis. Adaptive immunity, which plays an important role in responding to injury and some diseases of the central nervous system, may contribute to neuroinflammation in AD as well. Communication between the central and peripheral immune systems may also be important in AD. An increased understanding of the physiology of the innate immune system may aid the identification of new therapeutic targets or mechanisms. The development of predictive animal models and translatable neuroinflammation biomarkers for AD would also facilitate the advancement of novel treatments for innate immunity. Important challenges impeding the advancement of new therapeutic agents and strategies to overcome them were discussed.

Background This phase 2 randomized, double-blind, placebo-controlled study evaluated the efficacy and safety of the nicotinic acetylcholine receptor α7 agonist AQW051 in patients with Parkinson's disease and levodopa-induced dyskinesia. Methods Patients with idiopathic Parkinson's disease and moderate to severe levodopa-induced dyskinesia were randomized to AQW051 10 mg (n = 24), AQW051 50 mg (n = 24), or placebo (n = 23) once daily for 28 days. Coprimary end points were change in Modified Abnormal Involuntary Movement Scale and Unified Parkinson's Disease Rating Scale part III scores. Secondary outcomes included pharmacokinetics. Results In total, 67 patients completed the study. AQW051-treated patients experienced no significant improvements in Modified Abnormal Involuntary Movement Scale or Unified Parkinson's Disease Rating Scale part III scores by day 28. AQW051 was well tolerated; the most common adverse events were dyskinesia, fatigue, nausea, and falls. Conclusions AQW051 did not significantly reduce dyskinesia or parkinsonian severity. © 2016 International Parkinson and Movement Disorder Society

The clinical development of selective alpha-7 nicotinic acetylcholine receptor (α7 nAChR) agonists has hitherto been focused on disorders characterized by cognitive deficits (e.g., Alzheimer’s disease, schizophrenia). However, α7 nAChRs are also widely expressed by cells of the immune system and by cells with a secondary role in pathogen defense. Activation of α7 nAChRs leads to an anti-inflammatory effect. Since sterile inflammation is a frequently observed phenomenon in both psychiatric disorders (e.g., schizophrenia, melancholic and bipolar depression) and neurological disorders (e.g., Alzheimer’s disease, Parkinson’s disease, and multiple sclerosis), α7 nAChR agonists might show beneficial effects in these central nervous system disorders. In the current review, we summarize information on receptor expression, the intracellular signaling pathways they modulate and reasons for receptor dysfunction. Information from tobacco smoking, vagus nerve stimulation, and cholinesterase inhibition is used to evaluate the therapeutic potential of selective α7 nAChR agonists in these inflammation-related disorders.

In this work, the interaction of (±)-bupropion [(±)-BP] and (±)-2-(N-tert-butylamino)-3’-iodo-4’- azidopropiophenone [(±)-SADU-3-72] with the human (h) α3β4 nicotinic acetylcholine receptor (AChR) is determined by functional and structural approaches. The Ca2+ influx results indicate that (±)-BP inhibits hα3β4 AChRs with ~2-fold lower potency than that for (±)-SADU-3-72, indicating that this photoreactive analog can be used to further characterize the (±)-BP binding sites. The competition binding results show that (±)-BP binds to the [3 H]imipramine sites at desensitized hα3β4 AChRs with ~4-fold higher affinity compared to the resting state. Molecular docking results indicate that both enantiomers of BP and SADU-3-72, in the protonated state, interact with luminal and non-luminal sites. BP interacts with a luminal site which overlaps that for imipramine, and with non-luminal sites located in the transmembrane domain (TMD) at interfacial (+α3/-β4) and α3 intrasubunit sites, and in the TMD-ECD (extracellular domain) junction at the +β4/-α3 and +β4/-β4 interfaces. Our results are consistent with a hα3β4 model where BP and SADU-3-72 bind to overlapping non-luminal sites as well as non-overlapping luminal sites, probably in physiological conditions. This work expands on previous studies supporting the notion that structurally different antidepressants produce their clinical effects by inhibiting distinct AChR subtypes. Fil: Arias, Hugo Rubén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Morón; Argentina Fil: Feuerbach, D. Novartis Institutes for Biomedical Research; Suiza Fil: Ortells, Marcelo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Morón; Argentina

The drimane sesquiterpenoids drimenin, cinnamolide, dendocarbin A, and polygodial were purified from the Canelo tree (Drimys winteri) and chemically characterized by spectroscopic methods. The pharmacological activity of these natural compounds were determined on hα4β2, hα3β4, and hα7 nicotinic acetylcholine receptors (AChRs) by Ca2+ influx measurements. The results established that drimane sesquiterpenoids inhibit AChRs with the following selectivity: hα4β2 > hα3β4 > hα7. In the case of hα4β2 AChRs, the following potency rank order was determined (IC50's in μM): drimenin (0.97 ± 0.35) > cinnamolide (1.57 ± 0.36) > polygodial (62.5 ± 19.9) dendocarbin A (no activity). To determine putative structural features underlying the differences in inhibitory potency at hα4β2 AChRs, additional structure-activity relationship and molecular docking experiments were performed. The Ca2+ influx and structural results supported a noncompetitive mechanism of inhibition, where drimenin interacted with luminal and nonluminal (TMD-β2 intrasubunit) sites. The structure-activity relationship results, i.e., the lower the ligand polarity, the higher the inhibitory potency, supported the nonluminal interaction. Ligand binding to both sites might inhibit the hα4β2 AChR by a cooperative mechanism, as shown experimentally (nH > 1). Drimenin could be used as a molecular scaffold for the development of more potent inhibitors with higher selectivity for the hα4β2 AChR. Fil: Arias, Hugo Rubén. California Northstate University; Estados Unidos. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina Fil: Feuerbach, Dominik. Novartis Institutes for Biomedical Research; Suiza Fil: Schmidt, Bernd. Universitat Potsdam; Alemania Fil: Heydenreich, Matthias. Universitat Potsdam; Alemania Fil: Paz, Cristian. Universidad de La Frontera; Chile Fil: Ortells, Marcelo Oscar. Universidad de Morón; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

To characterize the interaction of coronaridine congeners with human (h) α3β4 nicotinic acetylcholine receptors (AChRs), structural and functional approaches were used. The Ca(2+) influx results established that coronaridine congeners noncompetitively inhibit hα3β4 AChRs with the following potency (IC50's in μM) sequence: (-)-ibogamine (0.62±0.23)∼(+)-catharanthine (0.68±0.10)>(-)-ibogaine (0.95±0.10)>(±)-18-methoxycoronaridine [(±)-18-MC] (1.47±0.21)>(-)-voacangine (2.28±0.33)>(±)-18-methylaminocoronaridine (2.62±0.57 μM)∼(±)-18-hydroxycoronaridine (2.81±0.54)>(-)-noribogaine (6.82±0.78). A good linear correlation (r(2)=0.771) between the calculated IC50 values and their polar surface area was found, suggesting that this is an important structural feature for its activity. The radioligand competition results indicate that (±)-18-MC and (-)-ibogaine partially inhibit [(3)H]imipramine binding by an allosteric mechanism. Molecular docking, molecular dynamics, and in silico mutation results suggest that protonated (-)-18-MC binds to luminal [i.e., β4-Phe255 (phenylalanine/valine ring; position 13'), and α3-Leu250 and β4-Leu251 (leucine ring; position 9')], non-luminal, and intersubunit sites. The pharmacophore model suggests that nitrogens from the ibogamine core as well as methylamino, hydroxyl, and methoxyl moieties at position 18 form hydrogen bonds. Collectively our data indicate that coronaridine congeners inhibit hα3β4 AChRs by blocking the ion channel's lumen and probably by additional negative allosteric mechanisms by interacting with a series of non-luminal sites.

Atopic diseases are frequently co-morbid with autism spectrum disorders (ASD). Allergic responses are associated with an activation of mast cells, innate lymphoid cells, and Th2 cells. These cells produce type-2 cytokines (IL4 and IL13), which stimulate microglia and macrophages to adopt a phenotype referred to as 'alternative activation' or 'M2A'. M2A-polarized macrophages and microglia play a physiological role in tissue repair by secreting growth factors such as brain-derived neurotrophic factor (BDNF) and insulin-like growth factor-1. In ASD there is evidence for increased type-2 cytokines, microglia activation, M2A polarization, and increased levels of growth factors. In neurons, these growth factors drive a signal transduction pathway that leads to activation of the enzyme mammalian Target of Rapamycin (mTOR), and thereby to the inhibition of autophagy. Activation of mTOR is an effect that is also common to several of the genetic forms of autism. In the central nervous system, redundant synapses are removed via an autophagic process. Activation of mTOR would diminish the pruning of redundant synapses, which in the context of ASD is likely to be undesired. Based on this line of reasoning, atopic diseases like food allergy, eczema or asthma would represent risk factors for autism spectrum disorders.

The inhibitory activity of coronaridine congeners on human (h) α4β2 and α7 nicotinic acetylcholine receptors (AChRs) is determined by Ca2+ influx assays, whereas their effects on neurons in the ventral inferior (VI) aspect of the mouse medial habenula (MHb) are determined by patch-clamp recordings. The Ca2+ influx results clearly establish that coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to hα4β2 and hα7 subtypes, and with the following potency sequence, for hα4β2: (±)-18-methoxycoronaridine [(±)-18-MC] > (+)-catharanthine > (±)-18-methylaminocoronaridine [(±)-18-MAC] ∼ (±)-18-hydroxycoronaridine [(±)-18-HC]; and for hα7: (+)-catharanthine > (±)-18-MC > (±)-18-HC > (±)-18-MAC. Interestingly, the inhibitory potency of (+)-catharanthine (27 ± 4 μM) and (±)-18-MC (28 ± 6 μM) on MHb (VI) neurons was lower than that observed on hα3β4 AChRs, suggesting that these compounds inhibit a variety of endogenous α3β4* AChRs. In addition, the interaction of bupropion with (−)-ibogaine sites on hα3β4 AChRs is tested by [3H]ibogaine competition binding experiments. The results indicate that bupropion binds to ibogaine sites at desensitized hα3β4 AChRs with 2-fold higher affinity than at resting receptors, suggesting that these compounds share the same binding sites. In conclusion, coronaridine congeners inhibit hα3β4 AChRs with higher selectivity compared to other AChRs, by interacting with the bupropion (luminal) site. Coronaridine congeners also inhibit α3β4*AChRs expressed in MHb (VI) neurons, supporting the notion that these receptors are important endogenous targets for their anti-addictive activities.

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Here, the question was addressed at which position sheddases cleave TREM2 and what are the proteases involved in this process. Using both pharmacological and genetic approaches we report that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNFα converting enzyme) while ADAM10 plays a minor role. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimers disease. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered.

Triggering receptor expressed in myeloid cells (TREM2) is a member of the immunoglobulin superfamily and is expressed in macrophages, dendritic cells, microglia, and osteoclasts. TREM2 plays a role in phagocytosis, regulates release of cytokine, contributes to microglia maintenance, and its ectodomain is shed from the cell surface. Using both pharmacological and genetic approaches we report here that the main protease contributing to the release of TREM2 ectodomain is ADAM17, (a disintegrin and metalloproteinase domain containing protein, also called TACE, TNFα converting enzyme) while ADAM10 plays a minor role. Using mutational analysis, we demonstrate that the main cleavage site of the sheddases is located within the stalk region of TREM2 proximal to the plasma membrane. Complementary biochemical experiments reveal that cleavage occurs between histidine 157 and serine 158. Shedding is not altered for the R47H-mutated TREM2 protein that confers an increased risk for the development of Alzheimers disease. O-glycosylation is detected within the stalk region, but distant to the cleavage site. These findings reveal a link between shedding of TREM2 and its regulation during inflammatory conditions or chronic neurodegenerative disease like AD in which activity or expression of sheddases might be altered.

The interaction of (−)-reboxetine, a non-tricyclic norepinephrine selective reuptake inhibitor, with muscle-type nicotinic acetylcholine receptors (AChRs) in different conformational states was studied by functional and structural approaches. The results established that (−)-reboxetine: (a) inhibits (±)-epibatidine-induced Ca2+ influx in human (h) muscle embryonic (hα1β1γδ) and adult (hα1β1εδ) AChRs in a non-competitive manner and with potencies IC50 = 3.86 ± 0.49 and 1.92 ± 0.48 μM, respectively, (b) binds to the [3H]TCP site with ∼13-fold higher affinity when the Torpedo AChR is in the desensitized state compared to the resting state, (c) enhances [3H]cytisine binding to the resting but activatableTorpedo AChR but not to the desensitized AChR, suggesting desensitizing properties, (d) overlaps the PCP luminal site located between rings 6′ and 13′ in the Torpedo but not human muscle AChRs. In silico mutation results indicate that ring 9′ is the minimum structural component for (−)-reboxetine binding, and (e) interacts to non-luminal sites located within the transmembrane segments from the Torpedo AChR γ subunit, and at the α1/ε transmembrane interface from the adult muscle AChR. In conclusion, (−)-reboxetine non-competitively inhibits muscle AChRs by binding to the TCP luminal site and by inducing receptor desensitization (maybe by interacting with non-luminal sites), a mechanism that is shared by tricyclic antidepressants. Fil: Arias, Hugo Rubén. California Northstate University College of Medicine; Estados Unidos Fil: Ortells, Marcelo Oscar. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Morón. Facultad de Medicina; Argentina Fil: Feuerbach, Dominik. Novartis Institutes for Biomedical Research; Suiza

Introduction AQW051, an α7-nicotinic acetylcholine receptor partial agonist, enhanced cognitive function in rodent models of learning and memory. This study evaluated brain activation during performance of a working memory task (WMT) and an episodic memory task (EMT), and the effect of AQW051 on task-related brain activation and performance in subjects with schizophrenia. Methods This was a double-blind, randomized, placebo-controlled, multicenter, 2-period cross-over trial ( NCT00825539 ) in participants with chronic, stable schizophrenia. Participants, stratified according to smoking status, were randomized (1:1:1:1:1:1) to 1 of 6 sequence groups that determined the study drug dose (AQW051 7.5 mg, 50 mg or 100 mg) and order of administration versus placebo. The primary outcome was brain activation in a priori target regions of interest (ROIs) during performance of the WMT and EMT, measured using functional magnetic resonance imaging. The effect of AQW051 on task-related (EMT and WMT) brain activation and performance was also assessed, as were safety and tolerability. Results Overall, 60 of 68 enrolled participants completed the study (AQW051 then placebo: 7.5 mg n = 9; 50 mg n = 11; 100 mg n = 10. Placebo then AQW051: 7.5 mg n = 10; 50 mg n = 11; 100 mg n = 9). Significant task-related brain activation (5% significance level) was observed with placebo. During the WMT, a medium effect size was observed in the inferior prefrontal cortex with AQW051 100 mg versus placebo (0.431; p = 0.105). During the EMT encoding phase, a large effect size was observed in the anterior hippocampus (0.795; p = 0.007) and a medium effect size in the posterior hippocampus (0.476; p = 0.079) with AQW051 7.5 mg. No other medium/large effect sizes were observed with any dose on either task. Effects on brain activation were generally not associated with changes in cognitive performance. AQW051 was well tolerated with an acceptable safety profile. Conclusions Overall, no consistent effects of AQW051 on brain regions involved in the performance of a WMT or EMT were observed; however, this study presents a model for evaluating potential response to pharmacological interventions for cognitive impairment in schizophrenia.

The interaction of (±)-bupropion [(±)-BP] with the human (h) α4β2 nicotinic acetylcholine receptor (AChR) was compared to that for its photoreactive analog (±)-2-(N-tert-butylamino)-3′-iodo-4′-azidopropiophenone [(±)-SADU-3-72]. Ca2+ influx results indicated that (±)-SADU-3-72 and (±)-BP inhibit hα4β2 AChRs with practically the same potency. However, (±)-SADU-3-72 binds to the [3H]imipramine sites at resting and desensitized hα4β2 AChRs with 3-fold higher affinity compared to that for (±)-BP, which is supported by molecular docking results. The docking results also indicate that each isomer of BP and SADU-3-72, in the protonated state, interacts with luminal and non-luminal sites. In the channel lumen, both ligands bind to two overlapping subsites, one that overlaps the imipramine site, and another much closer to the cytoplasmic side. The results suggest, for the first time, three differentiated non-luminal domains, including the transmembrane (TMD), extracellular (ECD), and ECD-TMD junction. In the ECD-TMD junction, BP and SADU-3-72 bind to overlapping sites. Interestingly, only SADU-3-72 binds to intrasubunit and intersubunit sites in the TMD, and to additional sites in the ECD. Our results are consistent with a model where BP and SADU-3-72 bind to overlapping sites in the luminal and ECD-TMD junctional domains of the hα4β2, whereas only SADU-3-72 binds to additional non-luminal sites. The BP junctional site opens the door for additional inhibitory mechanisms. The pharmacological properties of (±)-SADU-3-72 showed in this work support further photolabeling studies to mapping the BP binding sites in the hα4β2 AChR. Fil: Arias, Hugo Rubén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. California Northstate University College of Medicine; Estados Unidos Fil: Feuerbach, Dominik. Novartis Institutes for Biomedical Research; Suiza Fil: Ortells, Marcelo Oscar. Universidad de Morón. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina

This phase 2 randomized, double-blind, placebo-controlled study evaluated the efficacy and safety of the nicotinic acetylcholine receptor α7 agonist AQW051 in patients with Parkinson's disease and levodopa-induced dyskinesia.Patients with idiopathic Parkinson's disease and moderate to severe levodopa-induced dyskinesia were randomized to AQW051 10 mg (n = 24), AQW051 50 mg (n = 24), or placebo (n = 23) once daily for 28 days. Coprimary end points were change in Modified Abnormal Involuntary Movement Scale and Unified Parkinson's Disease Rating Scale part III scores. Secondary outcomes included pharmacokinetics.In total, 67 patients completed the study. AQW051-treated patients experienced no significant improvements in Modified Abnormal Involuntary Movement Scale or Unified Parkinson's Disease Rating Scale part III scores by day 28. AQW051 was well tolerated; the most common adverse events were dyskinesia, fatigue, nausea, and falls.AQW051 did not significantly reduce dyskinesia or parkinsonian severity. © 2016 International Parkinson and Movement Disorder Society.

The pharmacological properties of (±)-2-(N-tert-butylamino)-3'-iodo-4'-azidopropiophenone [(±)-SADU-3-72], a photoreactive analog of bupropion (BP), were characterized at different muscle nicotinic acetylcholine receptors (AChRs) by functional and structural approaches. Ca²⁺ influx results indicate that (±)-SADU-3-72 is 17- and 6-fold more potent than BP in inhibiting human (h) embryonic (hα1β1γδ) and adult (hα1β1εδ) muscle AChRs, respectively. (±)-SADU-3-72 binds with high affinity to the [³H]TCP site within the resting or desensitized Torpedo AChR ion channel, whereas BP has higher affinity for desensitized AChRs. Molecular docking results indicate that both SADU-3-72 enantiomers interact with the valine (position 13') and serine (position 6') rings. However, an additional domain, between the outer (position 20') and valine rings, is observed in Torpedo AChR ion channels. Our results indicate that the azido group of (±)-SADU-3-72 may enhance its interaction with polar groups and the formation of hydrogen bonds at AChRs, thus supporting the observed higher potency and affinity of (±)-SADU-3-72 compared to BP. Collectively our results are consistent with a model where BP/SADU-3-72 and TCP bind to overlapping sites within the lumen of muscle AChR ion channels. Based on these results, we believe that (±)-SADU-3-72 is a promising photoprobe for mapping the BP binding site, especially within the resting AChR ion channel.

The pharmacological activity of a series of novel amide derivatives was characterized on several nicotinic acetylcholine receptors (AChRs). Ca(2+) influx results indicate that these compounds are not agonists of the human (h) α4β2, α3β4, α7, and α1β1γδ AChRs; compounds 2-4 are specific positive allosteric modulators (PAMs) of hα7 AChRs, whereas compounds 1-4, 7, and 12 are noncompetitive antagonists of the other AChRs. Radioligand binding results indicate that PAMs do not inhibit binding of [(3)H]methyllycaconitine but enhance binding of [(3)H]epibatidine to hα7 AChRs, indicating that these compounds do not directly, but allosterically, interact with the hα7 agonist sites. Additional competition binding results indicate that the antagonistic action mediated by these compounds is produced by direct interaction with neither the phencyclidine site in the Torpedo AChR ion channel nor the imipramine and the agonist sites in the hα4β2 and hα3β4 AChRs. Molecular dynamics and docking results suggest that the binding site for compounds 2-4 is mainly located in the inner β-sheet of the hα7-α7 interface, ∼12 Å from the agonist locus. Hydrogen bond interactions between the amide group of the PAMs and the hα7 AChR binding site are found to be critical for their activity. The dual PAM and antagonistic activities elicited by compounds 2-4 might be therapeutically important.

The interaction of ibogaine and phencyclidine (PCP) with human (h) alpha3beta4-nicotinic acetylcholine receptors (AChRs) in different conformational states was determined by functional and structural approaches including, radioligand binding assays, Ca2+ influx detections, and thermodynamic and kinetics measurements. The results established that (a) ibogaine inhibits (+/-)-epibatidine-induced Ca2+ influx in h(alpha)3beta4 AChRs with approximately 9-fold higher potency than that for PCP, (b) [3H]ibogaine binds to a single site in the h(alpha)3beta4 AChR ion channel with relatively high affinity (Kd = 0.46 +/- 0.06 microM), and ibogaine inhibits [3H]ibogaine binding to the desensitized h(alpha)3beta4 AChR with slightly higher affinity compared to the resting AChR. This is explained by a slower dissociation rate from the desensitized ion channel compared to the resting ion channel, and (c) PCP inhibits [3H]ibogaine binding to the h(alpha)3beta4 AChR, suggesting overlapping sites. The experimental results correlate with the docking simulations suggesting that ibogaine and PCP interact with a binding domain located between the serine (position 6') and valine/phenylalanine (position 13') rings. This interaction is mediated mainly by van der Waals contacts, which is in agreement with the observed enthalpic contribution determined by non-linear chromatography. However, the calculated entropic contribution also indicates local conformational changes. Collectively our data suggest that ibogaine and PCP bind to overlapping sites located between the serine and valine/phenylalanine rings, to finally block the AChR ion channel, and in the case of ibogaine, to probably maintain the AChR in the desensitized state for longer time.

We compared the interaction of fluoxetine and paroxetine, two selective serotonin reuptake inhibitors (SSRIs), with the human (h) alpha4beta2, alpha3beta4, and alpha7 nicotinic acetylcholine receptors (AChRs) in different conformational states, using Ca(2+) influx, radioligand binding, and molecular docking approaches. The results established that (1) fluoxetine was more potent than paroxetine in inhibiting agonist-activated Ca(2+) influx on halpha4beta2 and halpha7 AChRs, whereas the potency of both SSRIs was practically the same in the halpha3beta4 AChR. [corrected] (2) SSRIs bind to the [(3)H]imipramine locus with a [corrected] higher affinity when the AChRs are in the desensitized states compared to the resting states. (3) The different receptor specificity for fluoxetine determined by their inhibitory potencies or binding affinities suggests different modes of interaction when the AChR is in the closed or activated state. (4) Neutral and protonated fluoxetine interacts with a binding domain located in the middle of the AChR ion channel. In conclusion, SSRIs inhibit the most important neuronal AChRs with potencies and affinities that are clinically relevant by binding to a luminal site that is shared with tricyclic antidepressants.

The interaction of the agonist JN403 with the human (h) alpha7 nicotinic acetylcholine receptor (AChR) was compared to that for the competitive antagonist methyllycaconitine (MLA). The receptor selectivity of JN403 was studied on the halpha7, halpha3beta4, and halpha4beta2 AChRs. The results established that the cationic center and the hydrophobic group found in JN430 and MLA are important for the interaction with the AChRs. MLA preincubation inhibits JN403-induced Ca(2+) influx in GH3-halpha7 cells with a potency 160-fold higher than that when MLA is co-injected with JN403. The most probable explanation, based on our dynamics results, is that MLA (more specifically the 3-methyl-2,5-dioxopyrrole ring and the B-D rings) stabilizes the resting conformational state. The order of receptor specificity for JN403 is as follows: halpha7halpha3beta4 ( approximately 40-fold)halpha4beta2 ( approximately 500-fold). This specificity is based on a larger number of hydrogen bonds between the carbamate group (another pharmacophore) of JN403 and the halpha7 sites, the electrostatic repulsion between the positively charged residues around the halpha3beta4 sites and the cationic center of JN403, fewer hydrogen bonds for the interaction of JN403 with the halpha3beta4 AChR, and an unfavorable van der Waals interaction between JN403 and the alpha4-beta2 interface. The higher receptor specificity for JN403 could be important for the treatment of alpha7-related disorders, including dementias, pain-related ailments, depression, anxiety, and wound healing.

The interaction of tricyclic antidepressants (TCAs) with the human (h)alpha3beta4 nicotinic acetylcholine receptor (AChR) in different conformational states was compared with that for mecamylamine by using functional and structural approaches including, Ca(2+) influx, radioligand binding, and molecular docking. The results established that: (a) [(3)H]imipramine binds to a single site with relatively high affinity (K(d) = 0.41 +/- 0.04 microM), (b) imipramine inhibits [(3)H]imipramine binding to the resting/kappa-bungarotoxin-bound AChR (K(i) = 0.68 +/- 0.08 microM) with practically the same affinity as to the desensitized/epibatidine-bound AChR (K(i) = 0.83 +/- 0.08 microM), suggesting that TCAs do not discriminate between these conformational states, and (c) although TCAs (IC(50) approximately 1.8-2.7 microM) and mecamylamine (IC(50) = 3.3 +/- 0.4 microM) inhibit (+/-)-epibatidine-induced Ca(2+) influx with potencies in the same concentration range, TCAs (K(i) approximately 1-3.6 microM), but not mecamylamine (apparent IC(50) approximately 0.2 mM), inhibit [(3)H]imipramine binding to halpha3beta4 AChRs in different conformational states. This is explained by our docking results where imipramine, in the neutral and protonated states, interacts with the leucine (position 9') and valine/phenylalanine (position 13') rings, whereas protonated mecamylamine (99% at physiological pH) interacts with the outer ring (position 20'). Our data indicate that TCAs bind to overlapping sites located between the serine and valine/phenylalanine rings in the halpha3beta4 AChR ion channel, whereas protonated mecamylamine can be attracted to the channel mouth before blocking ion flux by interacting with a luminal site in its neutral state.

To determine the pharmacologic activity of (-)-lobeline between human (h)α4β2 and hα4β4 nicotinic acetylcholine receptors (AChRs), functional and structural experiments were performed. The Ca2+ influx results established that (-)-lobeline neither actives nor enhances the function of the studied AChR subtypes, but competitively inhibits hα4β4 AChRs with potency ∼10-fold higher than that for hα4β2 AChRs. This difference is due to a higher binding affinity for the [3H]cytisine sites at hα4β4 compared to hα4β2 AChRs, which, in turn, can be explained by our molecular dynamics (MD) results: (1) higher stability of (-)-lobeline and its hydrogen bonds within the α4β4 pocket compared to the α4β2 pocket, (2) (-)-lobeline promotes Loop C to cap the binding site at the α4β4 pocket, but forces Loop C to get apart from the α4β2 pocket, precluding the gating process elicited by agonists, and (3) the orientation of (-)-lobeline within the α4β4, but not the α4β2, subpocket, promoted by the t-(or t+) rotameric state of α4-Tyr98, remains unchanged during the whole MD simulation. This study gives a detailed view of the molecular and dynamics events evoked by (-)-lobeline supporting the differential binding affinity and subsequent inhibitory potency between hα4β2 and hα4β4 AChRs, and supports the possibility that the latter subtype is also involved in its activity. Fil: Arias, Hugo Rubén. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. California Northstate University. College of Medicine. Department of Medical Education; Estados Unidos Fil: Feuerbach, Dominik. Novartis Institutes for Biomedical Research. Neuroscience Research; Suiza Fil: Ortells, Marcelo Oscar. Universidad de Buenos Aires. Facultad de Medicina; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Universidad de Morón; Argentina

The current study compares the antidepressant-like effect elicited by nicotine between wild-type (β4+/+) and knockout (β4-/-) mice, and subsequently, the effect of 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic receptors, on the previously determined activity of nicotine. Mice from each sex were injected daily with nicotine base (0.2 mg/kg; s.c.) or co-administered with PAM-2 (1.0 mg/kg; i.p.) for 3 weeks. Forced swim tests were performed to determine the acute (day 1), subchronic (day 7), and chronic (days 14 and 21) effects of the drugs, as well as their residual effects after treatment cessation (days 28 and 35). Our results indicate that nicotine mediates antidepressant-like activity after acute, subchronic, and chronic treatments in β4+/+, but not β4-/-, mice, and that these effects are not mediated by unspecific locomotor stimulation. Nicotine co-administered with PAM-2 produces antidepressant-like activity in both β4+/+ and β4-/- mice, except after the acute treatment of β4-/- mice, and decreases locomotor activity. This suggests that although the β4 subunit regulates the antidepressant-like activity of nicotine it does not affect the activity elicited by PAM-2 when is co-administered with nicotine. The residual antidepressant-like activity of PAM-2 + nicotine was observed only in female mice, suggesting gender-specific differences. Our findings clearly indicate that β4-containing nAChRs play an important role in the antidepressant-like activity elicited by nicotine but they are not essential for the modulatory activity of PAM-2. In fact, PAM-2 inhibits α4β4 and α3β4 AChRs at higher concentration ranges compared to that for the PAM activity previously found at the α7 AChR.

Nicotine, a component of tobacco, is highly addictive but possesses beneficial properties such as cognitive improvements and memory maintenance. Involved in these processes is the neuronal nicotinic acetylcholine receptor (nAChR) α7, whose activation triggers depolarization, intracellular signaling cascades, and synaptic plasticity underlying addiction and cognition. It is therefore important to investigate intracellular mechanisms by which a cell regulates α7 nAChR activity. We have examined the role of phosphorylation by combining molecular biology, biochemistry, and electrophysiology in SH-SY5Y neuroblastoma cells,Xenopusoocytes, rat hippocampal interneurons, and neurons from the supraoptic nucleus, and we found tyrosine phosphorylation of α7 nAChRs. Tyrosine kinase inhibition by genistein decreased α7 nAChR phosphorylation but strongly increased acetylcholine-evoked currents, whereas tyrosine phosphatase inhibition by pervanadate produced opposite effects. Src-family kinases (SFKs) directly interacted with the cytoplasmic loop of α7 nAChRs and phosphorylated the receptors at the plasma membrane. SFK inhibition by PP2 [4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine] or SU6656 (2,3-dihydro-N,N-dimethyl-2-oxo-3-[(4,5,6,7-tetrahydro-1H-indol-2-yl)methylene]-1H-indole-5-sulfonamide) increased α7 nAChR-mediated responses, whereas expression of active Src reduced α7 nAChR activity. Mutant α7 nAChRs lacking cytoplasmic loop tyrosine residues because of alanine replacement of Tyr-386 and Tyr-442 were more active than wild-type receptors and insensitive to kinase or phosphatase inhibition. Because the amount of surface α7 receptors was not affected by kinase or phosphatase inhibitors, these data show that functional properties of α7 nAChRs depend on the tyrosine phosphorylation status of the receptor and are the result of a balance between SFKs and tyrosine phosphatases. These findings reveal novel regulatory mechanisms that may help to understand nicotinic receptor-dependent plasticity, addiction, and pathology.

Serotonin 5-HT(7) receptors are present in astrocytes. Understanding their role in this type of cell would greatly benefit from the identification of astroglial cell lines expressing this receptor type. The aim of the present study was to assess the expression of native 5-HT(7) receptors and 5-HT(7) receptor mRNA in a number of human glioblastoma cell lines, by means of cAMP measurements, Western blot analysis and reverse transcriptase-polymerase chain reaction (RT-PCR) analysis. 5-Hydroxytryptamine (5-HT), 5-carboxamidotryptamine (5-CT), 5-methoxytryptamine (5-MeOT) and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT) induced concentration-dependent stimulations of cAMP accumulation in the human glioblastoma cell lines, U-373 MG, U-138 MG, U-87 MG, DBTRG-05MG, T98G, H4, CCF-STTG1 and Hs 683. The rank order of potency was 5-CT>5-HT=5-MeOT>>8-OH-DPAT. The effect of 5-CT was inhibited in a concentration-dependent manner by the selective 5-HT(7) receptor antagonist SB-269970 in all human glioblastoma cells. Schild analyses yielded slope factors close to unity (0.89-1.13) and pA(2) values of 8.69-9.05. Western blot analysis revealed the presence of immunoreactive bands corresponding to the human 5-HT(7) receptor in extracts of all human glioblastoma cell lines. The presence of the three splice variants of the 5-HT(7) receptor (5-HT(7(a/b/d))) was visualized by RT-PCR analysis with specific primers in all human glioblastoma cell lines. In conclusion, human glioblastoma cell lines express functional 5-HT(7) receptors and the three splice variants of the corresponding mRNA. These cell lines could serve as model systems of native 5-HT(7) receptors in glial cells to investigate their putative role in processes like release of neurotrophic factors or inflammatory cytokines.

Recombinant 5-hydroxytryptamine 5-HT7 receptors are known to express constitutive, i.e., agonist-independent activity. Nonselective ligands, like methiothepin, ritanserin or clozapine behave as full inverse agonists at 5-HT7 receptors. The aim of the present study was to evaluate the degree of inverse agonist activity of three selective 5-HT7 receptor antagonists ((R)-3,N-dimethyl-N-[1-methyl-3-(4-methyl-piperidin-1-yl)propyl]benzene sulfonamide or SB-258719, R-(+)-1-(toluene-3-sulfonyl)-2-[2-(4-methylpiperidin-1-yl)ethyl]-pyrrolidine or SB-258741 and (R)-3-(2-(2-(4-methylpiperidin-1-yl)ethyl)-pyrrolidine-1-sulfonyl)-phenol or SB-269970) in the same model. cAMP accumulation was measured in intact Chinese hamster ovary (CHO) cells expressing human recombinant 5-HT7a receptors. In these cells, 5-HT stimulated cAMP levels and a series of ligands antagonized the effect of 5-HT with a 5-HT7 receptor-like profile. SB-258719 had no inverse agonist activity, SB-258741 behaved as a partial inverse agonist and SB-269970 was a quasi-full inverse agonist (as compared to methiothepin). The inverse agonist effect of SB-269970 was antagonized in a concentration-dependent manner by SB-258719. The widespread spectrum of inverse agonist activities shown by these compounds should help assessing the physiological relevance of constitutive 5-HT7 receptor activity in native tissues.

In this study three major subtypes of nicotinic acetylcholine receptors were characterized pharmacologically using the calcium influx through the ion channel as a robust functional assay system. Human α3β4 receptors and α4β2 receptors were cloned and stably expressed in HEK293 cells. [125I]epibatidine saturation binding yielded a B max of 4420±840 fmol/mg protein for the α4β2 receptor (n=4) and 518±15 fmol/mg protein for the α3β4 receptor (n=4). As a source for muscle type of nicotinic receptor, the TE671 cell line was used which expresses endogenously the human fetal α1β1γδ subtype of nicotinic receptor. Stimulation of these nicotinic receptor subtypes in the different cell lines led to calcium transients that peaked 5–10 s after agonist application and declined thereafter. Eleven agonists were tested in this study and their efficacy and potency at the three nicotinic receptor subtypes were determined (epibatidine, ABT594, anatoxin, ABT418, nicotine, DMPP, cytisine, ABT089, choline, GTS21, AAR17779). This pharmacological characterization of agonist-induced elevation of intracellular free Ca2+ revealed a distinct rank order of agonist potency for each receptor subtype. Epibatidine showed at all three subtypes the highest potency and was a full agonist. The agonist-elicited response could be blocked by co-incubation of different antagonists from which mecamylamine did not display a strong subtype specificity. These data illustrate that the assessment of calcium transients upon receptor stimulation is a powerful tool for rapid examination of the functional properties of nicotinic receptors.

Somatostatin (somatotropin release inhibiting factor, SRIF), exerts its effects via specific G protein coupled receptors of which five subtypes have been cloned (sst 1–5 ). Recently, SRIF receptors have also been cloned from fish tissues. In this study, goldfish sst 5 receptors (gfsst 5 ) were expressed and characterised in the Chinese hamster lung fibroblast cell line, that harbours the luciferase reporter gene driven by the serum responsive element (CCL39-SRE-Luci). The agonist radioligands [ 125 I]-LTT-SRIF-28 ([Leu 8 , d Trp 22 , 125 I-Tyr 25 ]SRIF-28) and [ 125 I][Tyr 10 ]cortistatin-14 labelled similar receptor densities with high affinity and in a saturable manner (p K d : 9.99–9.71; B max : 300–350 fmol mg −1 ). 5′-Guanylyl-imidodiphosphate inhibited radioligand binding to some degree (38.5–57.9%). In competition binding studies, the pharmacological profile of SRIF binding sites defined with [ 125 I]LTT-SRIF-28 and [ 125 I][Tyr 10 ]cortistatin-14 correlated significantly ( r 2 =0.97, n =20). Pharmacological profiles of human and mouse sst 5 receptors expressed in CCL39 cells correlated markedly less with those of the gfsst 5 profile ( r 2 =0.52–0.78, n ≥16). Functional expression of the gfsst 5 receptor was examined by measurement of agonist-induced luciferase expression and stimulation of [ 35 S]GTPγS ([ 35 S]guanosine 5′- O -(3-thiotriphosphate) binding. Profiles were similar to those achieved in radioligand binding studies ( r 2 =0.81–0.93, n =20), although relative potency (pEC 50 ) was reduced compared to p K d values. Relative efficacy profiles of luciferase expression and [ 35 S]GTPγS binding, were rather divergent ( r 2 =0.48, n =20) with peptides showing full agonism at one pathway and absence of agonism at the other. BIM 23056 ( d -Phe-Phe-Tyr- d -Trp-Lys-Val-Phe- d -Nal-NH 2 ) acted as an antagonist on the effects of SRIF-14 (p K B =6.74±0.23) on stimulation of [ 35 S]GTPγS binding. Pertussis toxin abolished the effect of SRIF-14 on luciferase expression and [ 35 S]GTPγS binding suggesting coupling of the receptor to G i /G o proteins. In summary, the present studies demonstrate that the gfsst 5 receptor has a similar pharmacological profile and transductional properties to mammalian sst 5 receptors. The difference in efficacy profiles defined using different functional assays suggests numerous, agonist specific, conformational receptor states, and/or ligand-dependent receptor trafficking.

Ergolines were recently identified as a novel class of H3 receptor (H3R) inverse agonists. Although their optimization led to drug candidates with encouraging properties for the treatment of narcolepsy, brain penetration remained low. To overcome this issue, ergoline 1 ((6aR,9R,10aR)-4-(2-(dimethylamino)ethyl)-N-phenyl-9-(pyrrolidine-1-carbonyl)-6,6a,8,9,10,10a-hexahydroindolo[4,3-fg]quinoline-7(4H)-carboxamide)) was transformed into a series of indole derivatives with high H3R affinity. These new molecules were profiled by simultaneous determination of their brain receptor occupancy (RO) levels and pharmacodynamic (PD) effects in mice. These efforts culminated in the discovery of 15 m ((R)-1-isopropyl-5-(1-(2-(2-methylpyrrolidin-1-yl)ethyl)-1H-indol-4-yl)pyridin-2(1H)-one), which has an ideal profile showing a strong correlation of PD effects with RO, and no measurable safety liabilities. Its desirably short duration of action was confirmed by electroencephalography (EEG) measurements in rats.

Ergoline derivative (6aR,9R)-4-(2-(dimethylamino)ethyl)-N-phenyl-9-(pyrrolidine-1-carbonyl)-6,6a,8,9-tetrahydroindolo[4,3-fg]quinoline-7(4H)-carboxamide (1), a CXCR3 antagonist, also inhibits human histamine H3 receptors (H3R) and represents a structurally novel H3R inverse agonist chemotype. It displays favorable pharmacokinetic and in vitro safety profiles, and served as a lead compound in a program to explore ergoline derivatives as potential drug candidates for the treatment of narcolepsy. A key objective of this work was to enhance the safety and efficacy profiles of 1, while minimizing its duration of action to mitigate the episodes of insomnia documented with previously reported clinical candidates during the night following administration. Modifications to the ergoline core at positions 1, 6 and 8 were systematically investigated, and derivative 23 (1-((4aR,8R,9aR)-8-(hydroxymethyl)-1-(2-((R)-2-methylpyrrolidin-1-yl)ethyl)-4,4a,7,8,9,9a-hexahydroindolo[1,14-fg]quinolin-6(1H)-yl)ethanone) was identified as a promising lead compound. Derivative 23 has a desirable pharmacokinetic profile and demonstrated efficacy by enhancing brain concentrations of tele-methylhistamine, a major histamine metabolite. This validates the potential of the ergoline scaffold to serve as a template for the development of H3R inverse agonists.

The metabotropic glutamate receptor subtype 7 (mGlu7) is an important presynaptic regulator of neurotransmission in the mammalian CNS. mGlu7 function has been linked to autism, drug abuse, anxiety, and depression. Despite this, it has been difficult to develop specific blockers of native mGlu7 signaling in relevant brain areas such as amygdala and limbic cortex. Here, we present the mGlu7-selective antagonist 7-hydroxy-3-(4-iodophenoxy)-4H-chromen-4-one (XAP044), which inhibits lateral amygdala long term potentiation (LTP) in brain slices from wild type mice with a half-maximal blockade at 88 nm. There was no effect of XAP044 on LTP of mGlu7-deficient mice, indicating that this pharmacological effect is mGlu7-dependent. Unexpectedly and in contrast to all previous mGlu7-selective drugs, XAP044 does not act via the seven-transmembrane region but rather via a binding pocket localized in mGlu7's extracellular Venus flytrap domain, a region generally known for orthosteric agonist binding. This was shown by chimeric receptor studies in recombinant cell line assays. XAP044 demonstrates good brain exposure and wide spectrum anti-stress and antidepressant- and anxiolytic-like efficacy in rodent behavioral paradigms. XAP044 reduces freezing during acquisition of Pavlovian fear and reduces innate anxiety, which is consistent with the phenotypes of mGlu7-deficient mice, the results of mGlu7 siRNA knockdown studies, and the inhibition of amygdala LTP by XAP044. Thus, we present an mGlu7 antagonist with a novel molecular mode of pharmacological action, providing significant application potential in psychiatry. Modeling the selective interaction between XAP044 and mGlu7's Venus flytrap domain, whose three-dimensional structure is already known, will facilitate future drug development supported by computer-assisted drug design.

Nicotinic acetylcholine receptor (nAChR)-mediated signaling has been implicated in levodopa ( l -Dopa)-induced dyskinesias (LID). This study investigated the novel selective α7 nAChR partial agonist (R)-3-(6-ρ-Tolyl-pyridin-3-yloxy)-1-aza-bicyclo(2.2.2)octane (AQW051) for its antidyskinetic activity in l -Dopa-treated 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned cynomolgus monkeys. Six MPTP monkeys were repeatedly treated with l -Dopa to develop reproducible dyskinesias. AQW051 (2, 8, and 15 mg/kg) administered 1 h before l -Dopa treatment did not affect their parkinsonian scores or locomotor activity, but did significantly extend the duration of the l -Dopa antiparkinsonian response, by 30 min at the highest AQW051 dose (15 mg/kg). Dyskinesias were significantly reduced for the total period of l -Dopa effect following treatment with 15 mg/kg; achieving a reduction of 60% in median values. Significant reductions in 1 h peak dyskinesia scores and maximal dyskinesias were also observed with AQW051 (15 mg/kg). To understand the exposure–effect relationship and guide dose selection in clinical trials, plasma concentration-time data for the 15 mg/kg AQW051 dose were collected from three of the MPTP monkeys in a separate pharmacokinetic experiment. No abnormal behavioral or physiological effects were reported following AQW051 treatment. Our results show that AQW051 at a high dose can reduce LID without compromising the benefits of l -Dopa and extend the duration of the l -Dopa antiparkinsonian response in MPTP monkeys. This supports the clinical testing of α7 nAChR agonists to modulate LID and extend the duration of the therapeutic effect of l -Dopa.

The pharmacological activity of bupropion was compared between α7 nicotinic acetylcholine receptors expressed in heterologous cells and hippocampal and dorsal raphe nucleus neurons. The inhibitory activity of bupropion was studied on GH3-α7 cells by Ca2+ influx, as well as on neurons from the dorsal raphe nucleus and interneurons from the stratum radiatum of the hippocampal CA1 region by using a whole-cell voltage-clamp technique. In addition, the interaction of bupropion with the α7 nicotinic acetylcholine receptor was determined by [3H]imipramine competition binding assays and molecular docking. The fast component of acetylcholine- and choline-induced currents from both brain regions was inhibited by methyllycaconitine, indicating the participation of α7-containing nicotinic acetylcholine receptors. Choline-induced currents in hippocampal interneurons were partially inhibited by 10 µM bupropion, a concentration that could be reached in the brain during clinical administration. Additionally, both agonist-induced currents were reversibly inhibited by bupropion at concentrations that coincide with its inhibitory potency (IC50=54 µM) and binding affinity (Ki=63 µM) for α7 nicotinic acetylcholine receptors from heterologous cells. The [3H]imipramine competition binding and molecular docking results support a luminal location for the bupropion binding site(s). This study may help to understand the mechanisms of actions of bupropion at neuronal and molecular levels related with its therapeutic actions on depression and for smoking cessation.

Osteoblastic cells have been shown to be involved in osteoclast formation through cell to cell contacts. This study was designed to examine the possible function of vascular cell adhesion molecule 1 (VCAM-1) during osteoclastogenesis. As a source for stromal cells we used the recently established mouse bone marrow stromal cell line mBMS-B1 which has the ability to support osteoclastogenesis when used in co-culture with a crude spleen cell suspension. mBMS-B1 cells express a single approximately 3.9 kb VCAM-1 mRNA species. Expression was low under basal culture conditions and a 5-10-fold increase was observed in the presence of 1,25(OH)2D3. Cell surface expression of VCAM-1 examined by FACS analysis was increased about 2-fold after 1,25(OH)2D3 treatment. Immunoprecipitation of cell surface expressed VCAM-1 or total VCAM-1 protein using the anti-VCAM-1 monoclonal antibody MK2.7 resulted in a single approximately 110 kDa protein on SDS-PAGE. Induction by 1,25(OH)2D3 was about 2-5-fold on day 3. The stromal cell-osteoclast precursor cell interaction was investigated in a co-culture of the mBMS-B1 and mouse spleen cells in the presence of 1,25(OH)2D3. The monoclonal antibody MK2.7 which is known to block hemopoietic-stromal cell recognition inhibited the formation of osteoclasts when added to the co-culture at day 2 but not day 4. These data suggest that VCAM-1 is involved in the interaction between stromal cells and osteoclastic precursor cells during osteoclastogenesis presumably most important during early stages of the formation of osteoclasts.

The objective of the current study is to determine whether 3-furan-2-yl-N-p-tolyl-acrylamide (PAM-2), a positive allosteric modulator of α7 nicotinic receptors (AChRs), produces antidepressant-like behavior in mice, and reactivates desensitized α7 AChRs expressed in CH3-α7 cells. Mice from both sexes were injected (i.p.) with PAM-2 (1.0mg/kg) on a daily basis for three weeks. Forced swim tests (FSTs) were performed on Day 1 and Day 7 to determine the acute and subchronic effects of PAM-2, respectively, and on Days 14 and 21 to determine its chronic activity. To examine the residual effects after drug treatment, a withdrawal period of two more weeks was continued with FSTs performed on Day 28 and 35. Our results indicate that: (1) PAM-2 does not induce acute antidepressant effects in male or female mice, (2) PAM-2 induces antidepressant effects in mice from both sexes after one (subchronic) and two (chronic) weeks, whereas at the third week (chronic), the antidepressant effect is decreased in male and increased in female mice. Since PAM-2 does not influence the locomotor activity of mice, the observed antidepressant activity is not driven by nonspecific motor-stimulant actions, (3) the residual antidepressant effect mediated by PAM-2 after one week of treatment cessation is observed only in female mice, and finally the Ca(2+) influx results indicate that (4) PAM-2 can reactivate desensitized α7 AChRs. Our results clearly indicate that PAM-2 elicits antidepressant activity, probably by enhancing the activity of the endogenous neurotransmitter acetylcholine on α7 AChRs, without inducing receptor desensitization, and that this activity is gender-dependent. This is the first time that an antidepressant activity is described for an α7 PAM, supporting further studies as potential therapeutic medications for depressive states.

The differential action of the novel agonist JN403 at neuronalα7 and muscle nicotinic receptors (AChRs)was explored by using a combination of functional and structural approaches. Single-channel recordings reveal that JN403 is a potent agonist of α7 but a very low-efficacy agonist of muscle AChRs. JN403 elicits detectable openings of α7 and muscle AChRs at concentrations∼1000-fold lower and∼20-fold higher, respectively, than that for ACh. Single-channel activity elicited by JN403 is very similar to that elicited by ACh in α7 but profoundly different in muscle AChRs, where openings are brief and infrequent and do not appear in clusters at any concentration. JN403 elicits single-channel activity of muscle AChRs lacking theεsubunit, with opening events being more frequent and prolonged than those of wild-type AChRs. This finding is in line with the molecular docking studies predicting that JN403 may form a hydrogen bond required for potent activation at the α−δ but not at the α−ε binding site. JN403 does not elicit detectable Ca2+ influx in muscle AChRs but inhibits (±)-epibatidine-elicited influx mainly by a noncompetitive mechanism. Such inhibition is compatible with single-channel recordings revealing that JN403 produces open-channel blockade and early termination of ACh-elicited clusters, and it is therefore also a potent desensitizing enhancer of muscle AChRs. The latter mechanism is supported by the JN403-induced increase in the level of binding of [3H]cytisine and [3H]TCP to resting AChRs. Elucidation of the differences in activity of JN403 between neuronal α7 and muscle AChRs provides further insights into mechanisms underlying selectivity for α7 AChRs. Fil: Arias, Hugo Ruben. California Northstate University College of Medicine; Estados Unidos Fil: de Rosa, Maria Jose. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico CONICET Bahía Blanca. Instituto de Investigaciones Bioquímicas Bahía Blanca (i); Argentina Fil: Bergé, Ignacio. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnol.conicet - Bahia Blanca. Instituto de Invest.bioquimicas Bahia Blanca (i); Argentina Fil: Feuerbach, Dominik. Novartis Institutes for Biomedical Research; Suiza Fil: Bouzat, Cecilia Beatriz. Consejo Nacional de Investigaciones Cientificas y Tecnicas. Centro Cientifico Tecnol.conicet - Bahia Blanca. Instituto de Invest.bioquimicas Bahia Blanca (i); Argentina