Receptor Inactivation Research Articles

Photoactivatable Dopamine and Sulpiride to Explore the Function of Dopaminergic Neurons and Circuits.

Kinetic analysis of dopamine receptor activation and inactivation and the study of dopamine-dependent signaling requires precise simulation of the presynaptic release of the neurotransmitter dopamine and tight temporal control over the release of dopamine receptor antagonists. The 8-cyano-7-hydroxyquinolinyl (CyHQ) photoremovable protecting group was conjugated to dopamine and the dopamine receptor antagonist sulpiride to generate “caged” versions of these neuromodulators (CyHQ-O-DA and CyHQ-sulpiride, respectively) that could release their payloads with 365 or 405 nm light or through 2-photon excitation (2PE) at 740 nm. These compounds are stable under physiological conditions in the dark, yet photolyze rapidly and cleanly to yield dopamine or sulpiride and the caging remnant CyHQ–OH. CyHQ-O-DA mediated the light activation of dopamine-1 (D1) receptors on the breast cancer cell line MDA-MB-231 in culture. In mouse brain slice from the substantia nigra pars compacta, localized flash photolysis of CyHQ-O-DA accurately mimicked the natural presynaptic release of dopamine and activation of dopamine-2 (D2) receptors, causing a robust, concentration-dependent, and repeatable G protein-coupled inwardly rectifying potassium channel-mediated outward current in whole-cell voltage clamp recordings that was amplified by cocaine and blocked by sulpiride. Photolysis of CyHQ-sulpiride rapidly blocked synaptic activity, enabling measurement of the unbinding rates of dopamine and quinpirole, a D2 receptor agonist. These tools will enable more detailed study of dopamine receptors, their interactions with other GPCRs, and the physiology of dopamine signaling in the brain.

TLR9 and beclin1 crosstalk regulates muscle AMPK activation in exercise.

The activation of adenosine monophosphate-activated protein kinase (AMPK) in skeletal muscle coordinates systemic metabolic responses to exercise1. Autophagy, a lysosomal degradation pathway that maintains cellular homeostasis2, is upregulated during exercise, and a core autophagy protein, beclin 1, is required for skeletal muscle AMPK activation3. Herein, we describe a role for the innate immune sensing molecule, toll-like-receptor 9 (Tlr9)4, and its interaction with beclin 1 in exercise-induced muscle AMPK activation. Mice lacking Tlr9 are deficient in exercise-induced skeletal muscle AMPK activation and GLUT4 plasma membrane localization but not autophagy. Tlr9 binds beclin 1, and this interaction is increased by energy stress (glucose starvation and endurance exercise) and decreased by a BCL2 mutation3, 5 that blocks disruption of BCL2/beclin 1 binding. Tlr9 regulates the assembly of the endolysosomal beclin 1/UVRAG-containing phosphatidylinositol 3-kinase complex (PI3KC3-C2) in skeletal muscle during exercise, and knockout of beclin 1 or UVRAG inhibits glucose starvation-induced cellular AMPK activation. Moreover, Tlr9 functions in a muscle-autonomous fashion in ex vivo contraction-induced AMPK activation, glucose uptake, and beclin 1/UVRAG assembly. These findings reveal a heretofore undescribed role of a toll-like receptor in muscle AMPK activation and glucose metabolism during exercise, as well as unexpected cross-talk between this innate immune sensor and autophagy proteins.

Conformational plasticity of ligand-bound and ternary GPCR complexes studied by 19F NMR of the \u03b21-adrenergic receptor

G-protein-coupled receptors (GPCRs) are allosteric signaling proteins that transmit an extracellular stimulus across the cell membrane. Using 19F NMR and site-specific labelling, we investigate the response of the cytoplasmic region of transmembrane helices 6 and 7 of the β1-adrenergic receptor to agonist stimulation and coupling to a Gs-protein-mimetic nanobody. Agonist binding shows the receptor in equilibrium between two inactive states and a pre-active form, increasingly populated with higher ligand efficacy. Nanobody coupling leads to a fully active ternary receptor complex present in amounts correlating directly with agonist efficacy, consistent with partial agonism. While for different agonists the helix 6 environment in the active-state ternary complexes resides in a well-defined conformation, showing little conformational mobility, the environment of the highly conserved NPxxY motif on helix 7 remains dynamic adopting diverse, agonist-specific conformations, implying a further role of this region in receptor function. An inactive nanobody-coupled ternary receptor form is also observed.

12 A FUNCTIONAL TL1A/DR3 SYSTEM IS CRUCIAL FOR OPTIMAL GENERATION OF PATHOGENIC TH9 CELLS IN MURINE CROHN’S DISEASE-LIKE ILEITIS

Abstract Background Despite numerous therapeutic advancements, inflammatory bowel disease (IBD) remains a major health burden due to the inefficiency of conventional therapies. We recently demonstrated the role of death receptor 3 (DR3), a member of the tumor necrosis factor receptor (TNFR) superfamily and receptor for the cytokine TL1A, in regulating the balance between effector [T helper type 1 (TH)1,TH2,TH17] and regulatory T cells during murine Crohn’s disease (CD)-like ileitis. New evidence suggests a potential role of IL-9-secreting TH9 cells in the pathogenesis of IBD. However, the role of the TL1A/DR3 system in the differentiation and function of TH9 cells in CD is not fully understood. Thus, here, we investigated the role of a functional DR3 receptor in disease progression and activation of downstream signaling pathways relevant to TH9 cell differentiation and pathogenicity. Methods Ileal tissues were collected from 20-wk SAMP/YitFc (SAMP,WT) and DR3×SAMP (KO) mice, and analyzed by H&E, RT-qPCR and ELISA. CD4+ cells were purified by MACS sorting from mesenteric lymph nodes (MLNs) and spleens of WT and KO mice. TH9 cell were generated in medium supplemented with IL-4, TGF-β and TL1A. TH9 cells were analyzed by flow cytometry and cell supernatants by ELISA. TH9 cells (5x105) were adoptively transferred by i.p. injection into Rag2-/- mice, which were euthanized after 6-weeks. Colons were analyzed by H&E and MLNs were immunophenotyped by flow cytometry. Results Lack of DR3 ameliorated ileitis in SAMP (histologic score:5.2±1.3vs17.2±3.1, P≤0.0001, n=6), reduced of ∼ 50% the expression of Baft3, PU.1, il-9 mRNAs and IL-9 protein (P≤0.003, n=12), and 3-fold increased Id3 mRNA (P<0.0007,n=12) in small intestinal tissues. CD4+-enriched TH9 cells from KO mice secreted 2-fold lower IL-9 and TNF (P≤0.0002,n=8), and 5-fold higher IL-10 protein level (P<0.0001, n=8) than those from WT mice. Using the Rag2-/- T-cell transfer model of chronic colitis, we found that recipients of TH9 cells from KO donors developed less severe colitis (histologic score: 0.9±0.1vs3.9±0.8, P=0.005, n=5) and retained only half of the frequency of circulating TH9 cells in MLNs (P≤0.02,n=5) compared to those receiving WT TH9 cells, at 6-week after adoptive transfer. A similar experiment is currently ongoing to verify the impact of WT and KO TH9 cells on ileo-colitis development in SAMP×Rag2-/- mice. Conclusions The TL1A/DR3 system heavily contributes to IL-9-signaling pathway activation and it is required for the secretion of higher amount of IL-9 protein by TH9 cells in SAMP mice. KO TH9 cells were less colitogenic that those isolated from WT mice, indicating a crucial role of functional DR3 receptor in TH9 pathogenicity. Collectively, our results hold great translational significance by showing that modulation of DR3 signaling may be a novel therapeutic target for the treatment of CD.

Enhancing the Signaling of GPCRs via Orthosteric Ions.

G protein-coupled receptors play essential roles in cellular processes such as neuronal signaling, vision, olfaction, tasting, and metabolism. As GPCRs are the most important drug targets, understanding their interactions with ligands is of utmost importance for discovering related new medicines. In many GPCRs, an allosteric sodium ion next to the highly conserved residue D2.50 has been proposed to stabilize the inactive receptor state by mediating interactions between transmembrane helices. Here, we probed the existence of internal and functionally important sodium ions in the dopamine D2 receptor, using molecular dynamics simulations. Besides a new sodium ion at the allosteric ligand binding site, we discovered an additional sodium ion, located close to the orthosteric ligand binding site. Through cell-based activation assays, the signaling of D2 receptor with site-specific mutations was tested against a series of chemically modified agonists. We concluded an important structural role of this newly discovered orthosteric sodium ion in modulating the receptor signaling: It enables the coordination of a polar residue in the ligand binding site with an appropriately designed agonist molecule. An identical interaction was also observed in a recently released high-resolution crystal structure of mu-opioid receptor, which was reresolved in this work. Probably because of similar interactions, various metal ions have been found to increase the signaling of many other GPCRs. This unique principle and strategy could be used to optimize the drug activity of GPCR. Our findings open a new mechanistic opportunity of GPCR signaling and help design the next generation of drugs targeting GPCRs.

Juglone Attenuates Sepsis-Induced Acute Lung Injury via Suppression of the TLR4/Nf-κb Pathway

Sepsis, with high mortality, induces deleterious organ dysfunction and acute lung injury. Natural compounds show protective effect against sepsis-induced acute lung injury. Juglone, a natural naphthoquinone, demonstrates pharmacological actions as a pro-apoptotic substrate in tumor treatment and anti-inflammation substrate in organ injury. In this study, the influence of juglone on sepsis-induced acute lung injury was investigated. First, a septic mice model was established via cecal ligation and puncture, and then verified via histopathological analysis of lung tissues, the wet/dry mass ratio and myeloperoxidase activity was determined. Cecal ligation and puncture could induce acute lung injury in septic mice, as demonstrated by alveolar damage and increase of wet/dry mass ratio and myeloperoxidase activity. However, intragastric administration juglone attenuated cecal ligation and puncture-induced acute lung injury. Secondly, cecal ligation and puncture-induced increase of inflammatory cells in bronchoalveolar lavage fluid was also alleviated by the administration of juglone. Similarly, the protective effect of juglone against cecal ligation and puncture-induced acute lung injury was accompanied by a reduction of pro-inflammatory factor secretion in bronchoalveolar lavage fluid and lung tissues. Cecal ligation and puncture could activate toll-like receptor 4/nuclear factor-kappa B signaling pathway, and administration of juglone suppressed toll-like receptor 4/nuclear factor-kappa B activation. In conclusion, juglone attenuated cecal ligation and puncture-induced lung damage and inflammatory response through inactivation of toll-like receptor 4/nuclear factor-kappa B, suggesting a potential therapeutic strategy in the treatment of sepsis-induced acute lung injury.

The continuing arc toward phototropic enlightenment.

Phototropism represents a simple physiological mechanism—differential growth across the growing organ of a plant—to respond to gradients of light and maximize photosynthetic light capture (in aerial tissues) and water/nutrient acquisition (in roots). The phototropin blue light receptors, phot1 and phot2, have been identified as the essential sensors for phototropism. Additionally, several downstream signal/response components have been identified, including the phot-interacting proteins NON-PHOTOTROPIC HYPOCOTYL 3 (NPH3) and PHYTOCHROME SUBSTRATE 4 (PKS4). While the structural and photochemical properties of the phots are quite well understood, much less is known about how the phots signal through downstream regulators. Recent advances have, however, provided some intriguing clues. It appears that inactive receptor phot1 is found dispersed in a monomeric form at the plasma membrane in darkness. Upon light absorption dimerizes and clusters in sterol-rich microdomains where it is signal active. Additional studies showed that the phot-regulated phosphorylation status of both NPH3 and PKS4 is linked to phototropic responsiveness. While PKS4 can function as both a positive (in low light) and a negative (in high light) regulator of phototropism, NPH3 appears to function solely as a key positive regulator. Ultimately, it is the subcellular localization of NPH3 that appears crucial, an aspect regulated by its phosphorylation status. While phot1 activation promotes dephosphorylation of NPH3 and its movement from the plasma membrane to cytoplasmic foci, phot2 appears to modulate relocalization back to the plasma membrane. Together these findings are beginning to illuminate the complex biochemical and cellular events, involved in adaptively modifying phototropic responsiveness under a wide varying range of light conditions.

Chemoreceptors with C-terminal pentapeptides for CheR and CheB binding are abundant in bacteria that maintain host interactions

Chemosensory pathways represent a major prokaryotic signal transduction mechanism that is based on signal sensing by chemoreceptors. An essential feature of chemosensory pathways is the CheR and CheB mediated control of chemoreceptor methylation causing pathway adaptation. At their C-terminal extension the Tar and Tsr model chemoreceptors contain a pentapeptide that acts as an additional CheR and CheB binding site. The relevance of this pentapeptide is poorly understood since pentapeptide removal from Tar/Tsr causes receptor inactivation, whereas many other chemoreceptors do not require this pentapeptide for correct function. We report here a bioinformatic analysis of pentapeptide containing chemoreceptors. These receptors were detected in 11 bacterial phyla and represent approximately 10% of all chemoreceptors. Pentapeptide containing chemoreceptors are mainly found in Gram-negative bacteria, are of low abundance in Gram-positive species and almost absent from archaea. Almost 50% of TarH (Tar homologue) ligand binding domain containing chemoreceptors possess pentapeptides, whereas chemoreceptor families with other ligand binding domains are devoid of pentapeptides. The abundance of chemoreceptors with C-terminal pentapeptides correlated negatively with the number of chemoreceptor genes per genome. The consensus sequence reveals a negative net charge for many pentapeptides. Pentapeptide containing chemoreceptors are very abundant in the order Enterobacterales, particularly in the families Pectobacterium and Dickeya, where they represent about 50% of the total number. In contrast, bacteria with primarily free living lifestyles have a reduced number of pentapeptides, such as approximately 1% for Pseudomonadales. It is proposed that pentapeptide function is related to mechanisms that permit host interaction.

Mechanical force regulation of YAP by F-actin and GPCR revealed by super-resolution imaging.

The Hippo signaling pathway plays critical roles in many biological processes including mechanotransduction. The key activator YAP of this pathway is considered as a central component of mechanotransduction signaling sensing the extracellular mechanical microenvironment changes, such as different cell density, the architecture of tissues and matrix stiffness. Although it has been largely studied that YAP is involved in these processes, the underlying mechanism of mechanical force-induced YAP regulation remains unclear. Here we exerted pressure on cell surfaces and investigated how YAP senses the extracellular mechanical force change using one of the super-resolution imaging techniques, dSTORM. We demonstrated that pressure promoted F-actin depolymerization, RhoA down-regulation, and LPAR1 (Gα12/13-coupled receptor) inactivation, which led to YAP cytoplasmic translocation and decreased clustering. Our work uncovers the role of GPCRs and F-actin in pressure-controlled YAP inactivation, and provides new insights into the mechanisms of mechanical regulation of the Hippo signaling pathway.

Nerve growth factor activates autophagy in Schwann cells to enhance myelin debris clearance and to expedite nerve regeneration.

Rationale: Autophagy in Schwann cells (SCs) is crucial for myelin debris degradation and clearance following peripheral nerve injury (PNI). Nerve growth factor (NGF) plays an important role in reconstructing peripheral nerve fibers and promoting axonal regeneration. However, it remains unclear if NGF effect in enhancing nerve regeneration is mediated through autophagic clearance of myelin debris in SCs.Methods: In vivo, free NGF solution plus with/without pharmacological inhibitors were administered to a rat sciatic nerve crush injury model. In vitro, the primary Schwann cells (SCs) and its cell line were cultured in normal medium containing NGF, their capable of swallowing or clearing degenerated myelin was evaluated through supplement of homogenized myelin fractions.Results: Administration of exogenous NGF could activate autophagy in dedifferentiated SCs, accelerate myelin debris clearance and phagocytosis, as well as promote axon and myelin regeneration at early stage of PNI. These NGF effects were effectively blocked by autophagy inhibitors. In addition, inhibition of the p75 kD neurotrophin receptor (p75NTR) signal or inactivation of the AMP-activated protein kinase (AMPK) also inhibited the NGF effect as well.Conclusions: NGF effect on promoting early nerve regeneration is closely associated with its accelerating autophagic clearance of myelin debris in SCs, which probably regulated by the p75NTR/AMPK/mTOR axis. Our studies thus provide strong support that NGF may serve as a powerful pharmacological therapy for peripheral nerve injuries.

Involvement of P2 receptors in hematopoiesis and hematopoietic disorders, and as pharmacological targets.

Several reports have shown the presence of P2 receptors in hematopoietic stem cells (HSCs). These receptors are activated by extracellular nucleotides released from different sources. In the hematopoietic niche, the release of purines and pyrimidines in the milieu by lytic and nonlytic mechanisms has been described. The expression of P2 receptors from HSCs until maturity is still intriguing scientists. Several reports have shown the participation of P2 receptors in events associated with modulation of the immune system, but their participation in other physiological processes is under investigation. The presence of P2 receptors in HSCs and their ability to modulate this population have awakened interest in exploring the involvement of P2 receptors in hematopoiesis and their participation in hematopoietic disorders. Among the P2 receptors, the receptor P2X7 is of particular interest, because of its different roles in hematopoietic cells (e.g., infection, inflammation, cell death and survival, leukemias and lymphomas), making the P2X7 receptor a promising pharmacological target. Additionally, the role of P2Y12 receptor in platelet activation has been well-documented and is the main example of the importance of the pharmacological modulation of P2 receptor activity. In this review, we focus on the role of P2 receptors in the hematopoietic system, addressing these receptors as potential pharmacological targets.

Low acetylcholine during early sleep is important for motor memory consolidation.

The synaptic homeostasis theory of sleep proposes that low neurotransmitter activity in sleep optimizes memory consolidation. We tested this theory by asking whether increasing acetylcholine levels during early sleep would weaken motor memory consolidation. We trained separate groups of adult mice on the rotarod walking task and the single pellet reaching task, and after training, administered physostigmine, an acetylcholinesterase inhibitor, to increase cholinergic tone in subsequent sleep. Post-sleep testing showed that physostigmine impaired motor skill acquisition of both tasks. Home-cage video monitoring and electrophysiology revealed that physostigmine disrupted sleep structure, delayed non-rapid-eye-movement sleep onset, and reduced slow-wave power in the hippocampus and cortex. Additional experiments showed that: (1) the impaired performance associated with physostigmine was not due to its effects on sleep structure, as 1 h of sleep deprivation after training did not impair rotarod performance, (2) a reduction in cholinergic tone by inactivation of cholinergic neurons during early sleep did not affect rotarod performance, and (3) stimulating or blocking muscarinic and nicotinic acetylcholine receptors did not impair rotarod performance. Taken together, the experiments suggest that the increased slow wave activity and inactivation of both muscarinic and nicotinic receptors during early sleep due to reduced acetylcholine contribute to motor memory consolidation.

Nicotine directly affects milk production in lactating mammary epithelial cells concurrently with inactivation of STAT5 and glucocorticoid receptor in vitro

Nicotine from tobacco smoke is absorbed into the bloodstream and transferred into breast milk in breastfeeding mothers. Smoking causes a decrease in breast milk volume, adverse changes to the milk composition, and a shortened lactation period. Breast milk is produced by mammary epithelial cells (MECs) in mammary glands during lactation. However, it remains unclear whether nicotine directly affects milk production in lactating MECs. To address this issue, we prepared a culture model with high milk production ability and less-permeable tight junctions (TJs) by seeding mouse MECs on a cell culture insert. Lactating MECs showed expression of α2, α3, β2, and β4 of nicotinic acetylcholine receptors. The high concentration of nicotine at 10–100 μM inhibited β-casein secretion and caused abnormal localization of TJ proteins. We subsequently investigated whether nicotine at a physiological concentration could affect lactating MECs. Nicotine at 1.0 μM directly inhibited α- and β-casein secretion in lactating MECs concurrently with inactivation of STAT5 and glucocorticoid receptor without affecting the TJ barrier. Nicotine treatment also induced MEC apoptosis concurrently with inactivation of Akt. These results support the adverse effects of nicotine on breastfeeding in smoking mothers.

Ginsenoside Rg1 Exerts Anti-inflammatory Effects via G Protein-Coupled Estrogen Receptor in Lipopolysaccharide-Induced Microglia Activation.

Neuroinflammation plays a pivotal role in the pathogenesis of Parkinson’s disease. Ginsenoside Rg1, the most active ingredient of ginseng, has been reported to exert neuroprotective effects via estrogen and glucocorticoid receptors. The present study evaluated the involvement of the G protein-coupled estrogen receptor (GPER) in the anti-inflammatory effects of ginsenoside Rg1 against lipopolysaccharide (LPS)-induced microglia activation in the BV2 microglial cell line and ventral mesencephalic primary microglial culture. The pharmacological blockade and lentivirus-mediated small interfering RNA (siRNA) knockdown of GPER were used to study the underlying mechanism. Rg1 attenuated LPS-induced upregulation of tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), inducible nitric oxide synthase (iNOS), and cyclooxygenase-2 (COX-2) mRNA and protein levels. The GPER antagonist G15 blocked the inhibitory effects of Rg1 and the GPER-specific agonist G1 on LPS-induced microglia activation. Rg1 mimicked the effects of G1 by inhibiting the LPS-induced activation of nuclear transcription factor-kappa B (NF-κB) and mitogen activated protein kinase signaling pathways, which was also blocked by G15. Moreover, lentivirus-mediated siRNA knockdown of GPER inhibited the anti-inflammatory effects of Rg1. Taken together, our results indicate that GPER is involved in the anti-inflammatory effects of Rg1 against LPS-induced microglia activation. These findings provide a new biological target of Rg1 for the treatment of neuroinflammatory disorders.

Innate Immune Signaling Contributes to Tubular Cell Senescence in the Glis2 Knockout Mouse Model of Nephronophthisis

Nephronophthisis (NPHP), the leading genetic cause of end-stage renal failure in children and young adults, is a group of autosomal recessive diseases characterized by kidney-cyst degeneration and fibrosis for which no therapy is currently available. To date, mutations in >25 genes have been identified as causes of this disease that, in several cases, result in chronic DNA damage in kidney tubular cells. Among such mutations, those in the transcription factor-encoding GLIS2 cause NPHP type 7. Loss of function of mouse Glis2 causes senescence of kidney tubular cells. Senescent cells secrete proinflammatory molecules that induce progressive organ damage through several pathways, among which NF-κB signaling is prevalent. Herein, we show that the NF-κB signaling is active in Glis2 knockout kidney epithelial cells and that genetic inactivation of the toll-like receptor (TLR)/IL-1 receptor or pharmacologic elimination of senescent cells (senolytic therapy) reduces tubule damage, fibrosis, and apoptosis in the Glis2 mouse model of NPHP. Notably, in Glis2, Tlr2 double knockouts, senescence was also reduced and proliferation was increased, suggesting that loss of TLR2 activity improves the regenerative potential of tubular cells in Glis2 knockout kidneys. Our results further suggest that a combination of TLR/IL-1 receptor inhibition and senolytic therapy may delay the progression of kidney disease in NPHP type 7 and other forms of this disease.

28P - GLPG 1790, a new selective EPHA2 inhibitor, is active in colorectal cancer cell lines belonging to the CMS4/mesenchymal-like subtype

BackgroundEPHA2 tyrosine kinase receptor is implicated in tumor progression, stemness phenotype and resistance to treatment in a wide range of cancers. We investigated the effects of GLPG1790, a new selective Eph receptor inhibitor, in colorectal cancer (CRC) across the 4 consensus molecular subtypes (CMS). MethodsWe tested the antiproliferative effect of GLPG 1790 used alone or in combination with either 5-fluorouracil, oxaliplatin or SN38 (the active metabolite of irinotecan) in a panel of 11 CRC cell lines encompassing the 4 consensus molecular subtypes (CMS). Cell cycle analysis was performed in order to understand possible cell cycle perturbation after treatment. Pathway analysis using western blot (WB) was also performed. We then evaluated the expression of stemness genes upon treatment using qRT-PCR. ResultsGLPG 1790 is active in CRC cell lines, with the strongest activity in the cell lines from the CMS4/mesenchymal-like cluster. Combination with chemotherapeutics is not synergistic according to Chou-Talalay model. The selective inhibitor elicits a persistent inactivation of EPHA2 receptor, associated to G0-G1 cell cycle block in the sensitive cell lines. Furthermore, GLPG 1790 is able to decrease the expression of cancer stem cell genes in cell lines belonging to the CMS4 group. ConclusionsEPHA2 blockade using the selective inhibitor GLPG 1790 has a strong antiproliferative effect in the chemorefractory subgroup of CMS4/mesenchymal-like CRC cell lines, associated to a G0-G1 cell cycle arrest.The stronger efficacy of GLPG1790 on the mesenchymal-like subtype is probably due to the impairment of cancer cell stemness and induction of cell differentiation after treatment. Legal entity responsible for the studyThe authors. FundingUniversità della Campania "Luigi Vanvitelli"; Galapagos NV (drug supply); Associazione Italiana per la Ricerca sul Cancro (AIRC). DisclosureP.P. Vitiello: Travel/Accommodation/Expenses: Amgen; Research grant/Funding (institution): Bayer; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Roche; Research grant/Funding (institution): Servier; Research grant/Funding (institution): Ipsen; Travel/Accommodation/Expenses: BMS; Travel/Accommodation/Expenses: Sanofi. C. Cardone: Research grant/Funding (institution): Amgen; Research grant/Funding (institution): Bayer; Research grant/Funding (institution): Ipsen; Research grant/Funding (institution): Merck; Research grant/Funding (institution): Roche. D. Ciardiello: Travel/Accommodation/Expenses: Sanofi. L. Poliero: Travel/Accommodation/Expenses: BMS. C. Borrelli: Travel/Accommodation/Expenses: BMS. N. Zanaletti: Travel/Accommodation/Expenses: BMS. P. Vitale: Travel/Accommodation/Expenses: BMS. T. Troiani: Honoraria (self), Research grant/Funding (institution): Roche; Honoraria (self), Research grant/Funding (institution): Merck; Honoraria (self), Research grant/Funding (institution): Bayer; Honoraria (self), Travel/Accommodation/Expenses: Amgen; Travel/Accommodation/Expenses: Servier; Travel/Accommodation/Expenses: Sanofi; Travel/Accommodation/Expenses: Novartis. F. Ciardiello: Advisory/Consultancy, Research grant/Funding (institution): Amgen; Advisory/Consultancy, Research grant/Funding (institution): Bayer; Advisory/Consultancy, Research grant/Funding (institution): Roche; Advisory/Consultancy, Research grant/Funding (institution): Merck; Advisory/Consultancy: Servier; Advisory/Consultancy: Pfizer; Research grant/Funding (institution): Ipsen. E. Martinelli: Honoraria (self), Research grant/Funding (institution): Amgen; Honoraria (self), Research grant/Funding (institution): Merck; Honoraria (self), Research grant/Funding (institution): Bayer; Honoraria (self), Research grant/Funding (institution): Roche; Honoraria (self), Honoraria (institution): Servier. All other authors have declared no conflicts of interest.

MYASTHENIA GRAVIS: P.374Surrogacy prevents recurrence of fetal acetylcholine receptor inactivation syndrome

Fetal acetylcholine receptor inactivation syndrome (FARIS) is a recently recognized congenital myopathy due to in utero exposure to maternal acetylcholine receptor (AChR) antibodies directed against the fetal AChR. Unlike transient neonatal myasthenia gravis, FARIS extends well beyond the neonatal period. We review the literature, report the second case of FARIS born to an asymptomatic mother, and describe how surrogacy successfully prevented the development of FARIS. We describe a non-consanguineous couple whose first 3 pregnancies were complicated by FARIS. All 3 fetuses had polyhydramnios and arthrogryposis. Only one child survived the neonatal period, a term female born with weakness, respiratory and bulbar insufficiency. She improved markedly during childhood, but still has significant facial weakness. Extensive investigations in the siblings, including whole exome sequencing, were negative. The mother, who is completely asymptomatic for myasthenia gravis (MG), was then found to have elevated AChR antibodies, clinching the diagnosis of FARIS. This couple then had recourse to a surrogate, using their own ovum and sperm. Ultrasounds were normal. A female child was born at term, without any feature of FARIS. At 3 months, she is healthy and developing normally. We found 12 other FARIS cases from 6 different families. Only one other mother was asymptomatic for MG. Children who survive the neonatal period typically improve, but have persistent myopathic facies. The recurrence risk is extremely high. More aggressive MG treatment during pregnancy appears to lessen the severity, but does not prevent the development of FARIS. FARIS is a myopathy caused by in utero exposure to maternal AChR antibodies directed against the fetal AChR. Diagnosing FARIS is particularly challenging when the mother is not known for MG, but has important clinical implications for future pregnancies. Therefore, FARIS should be included in the differential diagnosis of congenital myopathies.

Catalytically inactive receptor tyrosine kinase PTK7 activates FGFR1 independent of FGF.

Protein tyrosine kinase 7 (PTK7), a catalytically defective receptor protein tyrosine kinase (RPTK), plays an oncogenic role by activating an unidentified TKI-258 (dovitinib)-sensitive RPTK in esophageal squamous cell carcinoma (ESCC) cells. Here, we demonstrate that among TKI-258–sensitive RPTKs, fibroblast growth factor receptor (FGFR) 1 is significantly up-regulated in ESCC tissues and cell lines. We show that PTK7 colocalizes with FGFR1 and binds it via its extracellular domain in human embryonic kidney 293 and ESCC TE-10 cells. PTK7 knockdown not only reduced ligand-free and fibroblast growth factor (FGF)-induced phosphorylation of FGFR1 but also the interaction of signaling adaptor proteins with FGFR1 and activation of downstream signaling proteins in TE-10 cells. In addition, PTK7 knockdown reduced FGF-induced oncogenic phenotypes including proliferation, anchorage-independent colony formation, wound healing, and invasion in ESCC cells. Taken together, our data demonstrate that PTK7 binds and activates FGFR1 independent of FGF and thus increases oncogenicity of PTK7- and FGFR1-positive cancers such as ESCC.—Shin, W.-S., Lee, H. W., Lee, S.-T. Catalytically inactive receptor tyrosine kinase PTK7 activates FGFR1 independent of FGF.

Modulation of lysophosphatidic acid (LPA) receptor activity: the key to successful neural regeneration?

Modulation of lysophosphatidic acid (LPA) receptor activity: the key to successful neural regeneration?

Ca2+-Dependent Inactivation of GluN2A and GluN2B NMDA Receptors Occurs by a Common Kinetic Mechanism

N-Methyl-d-aspartate (NMDA) receptors are Ca2+-permeable channels gated by glutamate and glycine that are essential for central excitatory transmission. Ca2+-dependent inactivation (CDI) is a regulatory feedback mechanism that reduces GluN2A-type NMDA receptor responses in an activity-dependent manner. Although CDI is mediated by calmodulin binding to the constitutive GluN1 subunit, prior studies suggest that GluN2B-type receptors are insensitive to CDI. We examined the mechanism of CDI subtype dependence using electrophysiological recordings of recombinant NMDA receptors expressed in HEK-293 cells. In physiological external Ca2+, we observed robust CDI of whole-cell GluN2A currents (0.42 ± 0.05) but no CDI in GluN2B currents (0.08 ± 0.07). In contrast, when Ca2+ was supplied intracellularly, robust CDI occurred for both GluN2A and GluN2B currents (0.75 ± 0.03 and 0.67 ± 0.02, respectively). To examine how the source of Ca2+ affects CDI, we recorded one-channel Na+ currents to quantify the receptor gating mechanism while simultaneously monitoring ionomycin-induced intracellular Ca2+ elevations with fluorometry. We found that CDI of both GluN2A and GluN2B receptors reflects receptor accumulation in long-lived closed (desensitized) states, suggesting that the observed subtype-dependent differences in macroscopic CDI reflect intrinsic differences in equilibrium open probabilities (Po). We tested this hypothesis by measuring substantial macroscopic CDI, in physiologic conditions, for high Po GluN2B receptors (GluN1A652Y/GluN2B). Together, these results show that Ca2+ flux produces activity-dependent inactivation for both GluN2A and GluN2B receptors and that the extent of CDI varies with channel Po. These results are consistent with CDI as an autoinhibitory feedback mechanism against excessive Ca2+ load during high Po activation.