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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.

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.

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.