Potent T cell-mediated anti-inflammatory role of the selective CB2 agonist lenabasum in multiple sclerosis.

BACKGROUND: Lenabasum is a synthetic cannabinoid receptor type-2 (CB2) agonist able to exert potent anti-inflammatory effects, but its role on T cells remains unknown. OBJECTIVES: The present study was undertaken to investigate anti-inflammatory mechanisms of lenabasum in T lymphocyte subsets and its in vivo therapeutic efficacy in experimental autoimmune encephalomyelitis (EAE). METHODS: Mononuclear cells from 17 healthy subjects (HS) and 25 relapsing-remitting multiple sclerosis (RRMS) patients were activated in presence or absence of lenabasum and analysed by flow cytometry and qRT-PCR. EAE mice were treated with lenabasum, and clinical score and neuroinflammation were evaluated. RESULTS: Lenabasum significantly reduced TNF-a production from CD4+ T cells and CD8+ T cells in a dose-dependent manner in both HS and RRMS patients. In MS patients, lenabasum also reduced activation marker CD25 and inhibited IL-2 production from both T cell subsets and IFN-γ and IL-17 from committed Th1 and Th17 cells, respectively. These effects were blocked by the pretreatment with selective CB2 inverse agonist SR144528. In vivo treatment of EAE mice with lenabasum significantly ameliorated disease severity, reduced neuroinflammation and demyelination in spinal cord. CONCLUSION: Lenabasum exerts potent T cell-mediated immunomodulatory effects, suggesting CB2 as a promising pharmacological target to counteract neuroinflammation in MS.

Anti-inflammatory effects of lenabasum, a cannabinoid receptor type 2 agonist, on macrophages from cystic fibrosis.

BACKGROUND: Lenabasum is an oral synthetic cannabinoid receptor type 2 agonist previously shown to reduce the production of key airway pro-inflammatory cytokines known to play a role in cystic fibrosis (CF). In a double-blinded, randomized, placebo-control phase 2 study, lenabasum lowered the rate of pulmonary exacerbation among patients with CF. The present study was undertaken to investigate anti-inflammatory mechanisms of lenabasum exhibits in CF macrophages. METHODS: We used monocyte-derived macrophages (MDMs) from healthy donors (n = 15), MDMs with CFTR inhibited with C-172 (n = 5) and MDMs from patients with CF (n = 4). Monocytes were differentiated to macrophages and polarized into classically activated (M1) macrophages by LPS or alternatively activated (M2) macrophages by IL-13 in presence or absence of lenabasum. RESULTS: Lenabasum had no effect on differentiation, polarization and function of macrophages from healthy individuals. However, in CF macrophages lenabasum downregulated macrophage polarization into the pro-inflammatory M1 phenotype and secretion of the pro-inflammatory cytokines IL-8 and TNF-α in a dose-dependent manner. An improvement in phagocytic activity was also observed following lenabasum treatment. Although lenabasum did not restore the impaired polarization of anti-inflammatory M2 macrophage, it reduced the levels of IL-13 and enhanced the endocytic function of CF MDMs. The effects of lenabasum on MDMs with CFTR inhibited by C-172 were not as obvious. CONCLUSION: In CF macrophages lenabasum modulates macrophage polarization and function in vitro in a way that would reduce inflammation in vivo. Further studies are warranted to determine the link between activating the CBR2 receptor and CFTR.

Distinct cortical and striatal actions of a ß-arrestin-biased dopamine D2 receptor ligand reveal unique antipsychotic-like properties.

The current dopamine (DA) hypothesis of schizophrenia postulates striatal hyperdopaminergia and cortical hypodopaminergia. Although partial agonists at DA D2 receptors (D2Rs), like aripiprazole, were developed to simultaneously target both phenomena, they do not effectively improve cortical dysfunction. In this study, we investigate the potential for newly developed ß-arrestin2 (ßarr2)-biased D2R partial agonists to simultaneously target hyper- and hypodopaminergia. Using neuron-specific ßarr2-KO mice, we show that the antipsychotic-like effects of a ßarr2-biased D2R ligand are driven through both striatal antagonism and cortical agonism of D2R-ßarr2 signaling. Furthermore, ßarr2-biased D2R agonism enhances firing of cortical fast-spiking interneurons. This enhanced cortical agonism of the biased ligand can be attributed to a lack of G-protein signaling and elevated expression of ßarr2 and G protein-coupled receptor (GPCR) kinase 2 in the cortex versus the striatum. Therefore, we propose that ßarr2-biased D2R ligands that exert region-selective actions could provide a path to develop more effective antipsychotic therapies.

Zebrafish behavioral profiling identifies multitarget antipsychotic-like compounds.

Many psychiatric drugs act on multiple targets and therefore require screening assays that encompass a wide target space. With sufficiently rich phenotyping and a large sampling of compounds, it should be possible to identify compounds with desired mechanisms of action on the basis of behavioral profiles alone. Although zebrafish (Danio rerio) behavior has been used to rapidly identify neuroactive compounds, it is not clear what types of behavioral assays would be necessary to identify multitarget compounds such as antipsychotics. Here we developed a battery of behavioral assays in larval zebrafish to determine whether behavioral profiles can provide sufficient phenotypic resolution to identify and classify psychiatric drugs. Using the antipsychotic drug haloperidol as a test case, we found that behavioral profiles of haloperidol-treated zebrafish could be used to identify previously uncharacterized compounds with desired antipsychotic-like activities and multitarget mechanisms of action.

ML314: A Biased Neurotensin Receptor Ligand for Methamphetamine Abuse.

Pharmacological treatment for methamphetamine addiction will provide important societal benefits. Neurotensin receptor NTR1 and dopamine receptor distributions coincide in brain areas regulating methamphetamine-associated reward, and neurotensin peptides produce behaviors opposing psychostimulants. Therefore, undesirable methamphetamine-associated activities should be treatable with druggable NTR1 agonists, but no such FDA-approved therapeutics exist. We address this limitation with proof-of-concept data for ML314, a small-molecule, brain penetrant, ß-arrestin biased, NTR1 agonist. ML314 attenuates amphetamine-like hyperlocomotion in dopamine transporter knockout mice, and in C57BL/6J mice it attenuates methamphetamine-induced hyperlocomotion, potentiates the psychostimulant inhibitory effects of a ghrelin antagonist, and reduces methamphetamine-associated conditioned place preference. In rats, ML314 blocks methamphetamine self-administration. ML314 acts as an allosteric enhancer of endogenous neurotensin, unmasking stoichiometric numbers of hidden NTR1 binding sites in transfected-cell membranes or mouse striatal membranes, while additionally supporting NTR1 endocytosis in cells in the absence of NT peptide. These results indicate ML314 is a viable, preclinical lead for methamphetamine abuse treatment and support an allosteric model of G protein-coupled receptor signaling.

G Protein and ß-arrestin signaling bias at the ghrelin receptor.

The G protein-coupled ghrelin receptor GHSR1a is a potential pharmacological target for treating obesity and addiction because of the critical role ghrelin plays in energy homeostasis and dopamine-dependent reward. GHSR1a enhances growth hormone release, appetite, and dopamine signaling through G(q/11), G(i/o), and G(12/13) as well as ß-arrestin-based scaffolds. However, the contribution of individual G protein and ß-arrestin pathways to the diverse physiological responses mediated by ghrelin remains unknown. To characterize whether a signaling bias occurs for GHSR1a, we investigated ghrelin signaling in a number of cell-based assays, including Ca(2+) mobilization, serum response factor response element, stress fiber formation, ERK1/2 phosphorylation, and ß-arrestin translocation, utilizing intracellular second loop and C-tail mutants of GHSR1a. We observed that GHSR1a and ß-arrestin rapidly form metastable plasma membrane complexes following exposure to an agonist, but replacement of the GHSR1a C-tail by the tail of the vasopressin 2 receptor greatly stabilizes them, producing complexes observable on the plasma membrane and also in endocytic vesicles. Mutations of the contiguous conserved amino acids Pro-148 and Leu-149 in the GHSR1a intracellular second loop generate receptors with a strong bias to G protein and ß-arrestin, respectively, supporting a role for conformation-dependent signaling bias in the wild-type receptor. Our results demonstrate more balance in GHSR1a-mediated ERK signaling from G proteins and ß-arrestin but uncover an important role for ß-arrestin in RhoA activation and stress fiber formation. These findings suggest an avenue for modulating drug abuse-associated changes in synaptic plasticity via GHSR1a and indicate the development of GHSR1a-biased ligands as a promising strategy for selectively targeting downstream signaling events.

Structural basis for Smoothened receptor modulation and chemoresistance to anticancer drugs.

The Smoothened receptor (SMO) mediates signal transduction in the hedgehog pathway, which is implicated in normal development and carcinogenesis. SMO antagonists can suppress the growth of some tumours; however, mutations at SMO have been found to abolish their antitumour effects, a phenomenon known as chemoresistance. Here we report three crystal structures of human SMO bound to the antagonists SANT1 and Anta XV, and the agonist, SAG1.5, at 2.6-2.8 Å resolution. The long and narrow cavity in the transmembrane domain of SMO harbours multiple ligand binding sites, where SANT1 binds at a deeper site as compared with other ligands. Distinct interactions at D473(6.54f) elucidated the structural basis for the differential effects of chemoresistance mutations on SMO antagonists. The agonist SAG1.5 induces a conformational rearrangement of the binding pocket residues, which could contribute to SMO activation. Collectively, these studies reveal the structural basis for the modulation of SMO by small molecules.

The role of arrestins in development.

ß-Arrestins are versatile scaffolding proteins that are involved in orchestrating a large number of signaling cascades. Because ß-arrestin 1 and ß-arrestin 2 are individually dispensable during development, it has long been assumed that ß-arrestins do not play an important role during embryogenesis. Nonetheless, there is growing evidence from both invertebrate and vertebrate animal models that ß-arrestins are integral regulators of developmental pathways. They are involved in diverse processes such as early hematopoiesis, establishment of axial development axes, cell convergence and extension movements leading to axial elongation, musculoskeletal and craniofacial development, cell proliferation, and neuronal development. Most of the developmental roles of arrestins involve interactions with or regulation of novel non-G protein-coupled receptor partners, such as atypical seven-transmembrane receptors, small G proteins, and nuclear transcription factors, revealing surprising diversity in arrestin function. In contrast to their largely overlapping roles in G protein-coupled receptor desensitization and internalization, study of the role of arrestins in development has demonstrated marked functional specialization of the ß-arrestin 1 and 2 isoforms.

GRK2: multiple roles beyond G protein-coupled receptor desensitization.

G protein-coupled receptor kinases (GRKs) regulate numerous G protein-coupled receptors (GPCRs) by phosphorylating the intracellular domain of the active receptor, resulting in receptor desensitization and internalization. GRKs also regulate GPCR trafficking in a phosphorylation-independent manner via direct protein-protein interactions. Emerging evidence suggests that GRK2, the most widely studied member of this family of kinases, modulates multiple cellular responses in various physiological contexts by either phosphorylating non-receptor substrates or interacting directly with signaling molecules. In this review, we discuss traditional and newly discovered roles of GRK2 in receptor internalization and signaling as well as its impact on non-receptor substrates. We also discuss novel exciting roles of GRK2 in the regulation of dopamine receptor signaling and in the activation and trafficking of the atypical GPCR, Smoothened (Smo).

Growth Arrest Specific 8 (Gas8) and G protein-coupled receptor kinase 2 (GRK2) cooperate in the control of Smoothened signaling.

The G protein-coupled receptor (GPCR)-like molecule Smoothened (Smo) undergoes dynamic intracellular trafficking modulated by the microtubule associated kinase GRK2 and recruitment of ß-arrestin. Of this trafficking, especially the translocation of Smo into primary cilia and back to the cytoplasm is essential for the activation of Hedgehog (Hh) signaling in vertebrates. The complete mechanism of this bidirectional transport, however, is not completely understood. Here we demonstrate that Growth Arrest Specific 8 (Gas8), a microtubule associated subunit of the Dynein Regulatory Complex (DRC), interacts with Smo to modulate this process. Gas8 knockdown in ciliated cells reduces Smo signaling activity and ciliary localization whereas overexpression stimulates Smo activity in a GRK2-dependent manner. The C terminus of Gas8 is important for both Gas8 interaction with Smo and facilitating Smo signaling. In zebrafish, knocking down Gas8 results in attenuated Hh transcriptional responses and impaired early muscle development. These effects can be reversed by the co-injection of Gas8 mRNA or by constitutive activation of the downstream Gli transcription factors. Furthermore, Gas8 and GRK2 display a synergistic effect on zebrafish early muscle development and some effects of GRK2 knockdown can be rescued by Gas8 mRNA. Interestingly, Gas8 does not interfere with cilia assembly, as the primary cilia architecture is unchanged upon Gas8 knock down or heterologous expression. This is in contrast to cells stably expressing both GRK2 and Smo, in which cilia are significantly elongated. These results identify Gas8 as a positive regulator of Hh signaling that cooperates with GRK2 to control Smo signaling.

Cholinergic status modulations in human volunteers under acute inflammation.

Cholinergic Status, the total soluble circulation capacity for acetylcholine hydrolysis, was tested for putative involvement in individual variabilities of the recruitment of immune cells in response to endotoxin challenge. Young (average age 26) and elderly (average age 70) volunteers injected with either Escherichia coli endotoxin or saline on two different occasions were first designated Enhancers and Suppressors if they showed increase or decrease, respectively, in plasma acetylcholinesterase (AChE) activity 1.5 h after endotoxin administration compared to saline. Enhancers showed significant co-increases in plasma butyrylcholinesterase (BChE) and paraoxonase (PON1) activities, accompanied by rapid recovery of lymphocyte counts. Young Enhancers alone showed pronounced post-exposure increases in the pro-inflammatory cytokine interleukin-6 (IL-6), and upregulation of the normally rare, stress-induced AChE-R variant, suggesting age-associated exhaustion of the cholinergic effects on recruiting innate immune reactions to endotoxin challenge. Importantly, IL-6 injected to young volunteers or administered in vitro to primary mononuclear blood cells caused upregulation of AChE, but not BChE or PON1, excluding it from being the sole cause for this extended response. Interestingly, Suppressors but not Enhancers showed improved post-exposure working memory performance, indicating that limited cholinergic reactions may be beneficial for cognition. Our findings establish Cholinergic Status modulations as early facilitators and predictors of individual variabilities in the peripheral response to infection.

Plant-derived human acetylcholinesterase-R provides protection from lethal organophosphate poisoning and its chronic aftermath.

Therapeutically valuable proteins are often rare and/or unstable in their natural context, calling for production solutions in heterologous systems. A relevant example is that of the stress-induced, normally rare, and naturally unstable "read-through" human acetylcholinesterase variant, AChE-R. AChE-R shares its active site with the synaptic AChE-S variant, which is the target of poisonous organophosphate anticholinesterase insecticides such as the parathion metabolite paraoxon. Inherent AChE-R overproduction under organophosphate intoxication confers both short-term protection (as a bioscavenger) and long-term neuromuscular damages (as a regulator). Here we report the purification, characterization, and testing of human, endoplasmic reticulum-retained AChE-R(ER) produced from plant-optimized cDNA in Nicotiana benthamiana plants. AChE-R(ER) purified to homogeneity showed indistinguishable biochemical properties, with IC50 = 10(-7) M for the organophosphate paraoxon, similar to mammalian cell culture-derived AChE. In vivo titration showed dose-dependent protection by intravenously injected AChE-R(ER) of FVB/N male mice challenged with a lethal dose of paraoxon, with complete elimination of short-term clinical symptoms at near molar equivalence. By 10 days postexposure, AChE-R prophylaxis markedly limited postexposure increases in plasma murine AChE-R levels while minimizing the organophosphate-induced neuromuscular junction dismorphology. Our findings present plant-produced AChE-R(ER) as a bimodal agent, conferring both short- and long-term protection from organophosphate intoxication.

A novel isoform of acetylcholinesterase exacerbates photoreceptors death after photic stress.

PURPOSE: To study the involvement of stress-induced acetylcholinesterase (AChE) expression in light-induced retinal damage in albino rats. METHODS: Adult albino rats were exposed for 24 hours to bright, damaging light. AChE expression was monitored by in situ hybridization, by histochemistry for AChE activity, and by immunocytochemistry. An orphan antisense agent (Monarsen; Ester Neurosciences, Ltd., Herzlia Pituach, Israel) was administered intraperitoneally to minimize light-induced AChE expression. The electroretinogram (ERG) was recorded to assess retinal function. RESULTS: Twenty-four-hour exposure to bright light caused severe reduction in the ERG responses and augmented expression of mRNA for the "read-through" variant of AChE (AChE-R) in photoreceptor inner segments (IS), bipolar cells, and ganglion cells. AChE activity increased in IS. The expressed AChE protein was a novel variant, characterized by an extended N terminus (N-AChE). Systemic administration of the orphan antisense agent, Monarsen, reduced the photic induction of mRNA for AChE-R, and of the N-AChE protein. Rats exposed to bright, damaging light and treated daily with Monarsen exhibited larger ERG responses, relatively thicker outer nuclear layer (ONL), and more ONL nuclei than did rats exposed to the same damaging light but treated daily with saline. CONCLUSIONS: The findings indicate that the photic-induced novel variant of AChE (N-AChE-R) may be causally involved with retinal light damage and suggest the use of RNA targeting for limiting such damage.

Adaptive changes in acetylcholinesterase gene expression as mediators of recovery from chemical and biological insults.

Both organophosphate (OP) exposure and bacterial infection notably induce short- and long-term cholinergic responses. These span the central and peripheral nervous system, neuromuscular pathway and hematopoietic cells and involve over-expression of the "readthrough" variant of acetylcholinesterase, AChE-R, and its naturally cleavable C-terminal peptide ARP. However, the causal involvement of these changes with post-exposure recovery as opposed to apoptotic events remained to be demonstrated. Here, we report the establishment of stably transfected cell lines expressing catalytically active human "synaptic" AChE-S or AChE-R which are fully viable and non-apoptotic. In addition, intraperitoneally injected synthetic mouse ARP (mARP) elevated serum AChE levels post-paraoxon exposure. Moreover, mARP treatment ameliorated post-exposure increases in corticosterone and decreases in AChE gene expression and facilitated earlier retrieval of motor activity following both paraoxon and lipopolysaccharide (LPS) exposures. Our findings suggest a potential physiological role for overproduction of AChE-R and the ARP peptide following exposure to both chemical warfare agents and bacterial LPS.

Purification of transgenic plant-derived recombinant human acetylcholinesterase-R.

Nicotiana benthamiana plants were engineered to express a codon-optimized gene encoding the human acetylcholinesterase-R (AChE) isoform. The transgenic plants expressed the protein at >0.4% of total soluble protein, and the plant-produced enzyme was purified to homogeneity. Following lysis, procainamide affinity chromatography and anion-exchange chromatography, more than 400-fold purification was achieved and electrophoretic purity was obtained. This pure protein is kinetically indistinguishable from the only commercially available source of human acetylcholinesterase, which is produced in mammalian cell culture. Thus, we have demonstrated a model system for the production of acetylcholinesterase, which is not susceptible to the quantitative limitations or mammalian pathogens associated with purification from mammalian cell culture or human serum.

RNA-targeted suppression of stress-induced allostasis in primate spinal cord neurons.

Peripheral acetylcholine levels notably control the synthesis in macrophages of pro-inflammatory cytokines; however, it remains unclear whether this peripheral regulatory pathway affects central nervous system neurons. To explore the interrelationship between neuronal cholinergic homeostasis and peripheral inflammatory responses in primates, we used spinal cord sections from cynomolgus monkeys after 7 days oral or intravenous treatment with Monarsen oligonucleotide. Monarsen is an antisense oligonucleotide 3'-protected by 2'-oxymethylation, which was proved to induce selective destruction of the stress-induced acetylcholinesterase splice variant AChE-R mRNA. Handling stress predictably suppressed neuronal choline acetyl transferase (ChAT) and the vesicular acetylcholine transporter (VAChT) in all treated monkeys. In Monarsen-treated animals, we further observed suppression of stress-induced increases in plasma AChE activities. Corresponding decreases in AChE-R mRNA were seen in spinal cord neurons, associated with parallel decline patterns in the mRNA encoding for the splice factor SC35 (the levels of which co-increase with those of AChE-R) as well as in the neuronal pro-inflammatory interleukins IL-1beta and IL-6. The antisense effects showed direct dose dependence and were inversely associated with neuronal cell size. These findings suggest a causal association between neuronal cholinergic allostasis and inflammatory reactions in primates and support the peripheral use of RNA-targeted intervention with AChE-R accumulation for the management of both stress and inflammatory responses.

Tissue distribution of cholinesterases and anticholinesterases in native and transgenic tomato plants.

Acetylcholinesterase, a major component of the central and peripheral nervous systems, is ubiquitous among multicellular animals, where its main function is to terminate synaptic transmission by hydrolyzing the neurotransmitter, acetylcholine. However, previous reports describe cholinesterase activities in several plant species and we present data for its presence in tomato plants. Ectopic expression of a recombinant form of the human enzyme and the expression pattern of the transgene and the accumulation of its product in transgenic tomato plants are described. Levels of acetylcholinesterase activity in different tissues are closely effected by and can be separated from alpha-tomatine, an anticholinesterase steroidal glycoalkaloid. The recombinant enzyme can also be separated from the endogenous cholinesterase activity by its subcellular localization and distinct biochemical properties. Our results provide evidence for the co-existence in tomato plants of both acetylcholinesterase activity and a steroidal glycoalkaloid with anticholinesterase activity and suggest spatial mutual exclusivity of these antagonistic activities. Potential functions, including roles in plant-pathogen interactions are discussed.

The role of readthrough acetylcholinesterase in the pathophysiology of myasthenia gravis.

Alternative splicing induces, under abnormal cholinergic neurotransmission, overproduction of the rare "readthrough" acetylcholinesterase variant AChE-R. We explored the pathophysiological relevance of this phenomenon in patients with myasthenia gravis (MG) and rats with experimental autoimmune MG (EAMG), neuromuscular junction diseases with depleted acetylcholine receptors. In MG and EAMG, we detected serum AChE-R accumulation. In EAMG, we alleviated electromyographic abnormalities by nanomolar doses of EN101, an antisense oligonucleotide that selectively lowers AChE-R in blood and muscle yet leaves unaffected the synaptic variant AChE-S. Whereas animals treated with placebo or conventional anticholinesterases continued to deteriorate, a 4 wk daily oral administration of EN101 improved survival, neuromuscular strength and clinical status in moribund EAMG rats. The efficacy of targeting only one AChE splicing variant highlights potential advantages of mRNA-targeted therapeutics for chronic cholinergic malfunctioning.