DESCRIPTIF DU TRAVAIL DE RECHERCHE
Our expertise in development of a large variety of screening methods (BRET, TR-FRET, Alpha screen, RILES) allows us to identify potent pharmacological tools and novel biological targets involved in cancer and neurological disorders. We are currently extending our efforts in these fields of investigation and focus our attention on dysregulated cell membrane receptors and microRNA expression that are hallmarks of these pathologies.
Pharmacology of neuroreceptors
The first axe of our researches is focused on LINGO-1, a receptor belonging to the LRIG (Leucine Rich Immunoglobulin) family which is considered as a promising therapeutic target in the field of neurodegenerative and demyelinating diseases. We notably demonstrated that LINGO-1 is endowed with cis-dimerization properties that can be modulated by small molecule protein-protein interaction modulators (SMPPIM) targeting LINGO-1. To modulate LINGO-1 activity in vivo, we are deciphering the molecular bases of its activation by studying LINGO-1 associated cell signalling and by identifying potent ligands from chemical compounds libraries. In addition, we focus our activity on the serotonin 5-HT7 receptor, which is known to regulate important neuronal functions. We will use potent and selective biased ligands that we recently identified for basic and translational researches. The team also takes advantage of its expertise in genetics of Drosophila melanogaster to develop animal models to better understand the physiopathological role of these receptors (5-HT7R and LINGO-1). Our unique expertise in the application of localised HR-MAS 1H NMR spectroscopy to analyse metabolites in living drosophila will be applied to identify metabolite biomarkers in brain tumours in Drosophila and to analyse their variation in models expressing human neuroreceptors.
miRNA biology and therapeutics
Increasing number of studies indicate that the underestimation of the dynamic and spatiotemporal resolution of miRNA expression result in loss of important information connecting miRNA expression and function. Recently we develop an engineered genetic switch expression system, called RILES for RNAi-inducible Expression System, to track expression of miRNA during development of pathologies in small animal models. As the RILES is a non-invasive luciferase reporting system, it offers a temporal dimension analysis of miRNA expression that conventional monitoring methods could not do. As a result, better clues about miRNA expression and function can be collected and then exploited for miRNA based therapeutics. We are currently exploiting the RILES system to (1) study impact of the dynamic regulation of miRNA in cancer development including neurofibromatosis, (2) identify novel biomarkers and (3) screen for small pharmacological molecules capable to modulate expression of tumour associated miRNA. Our objective is to better understand the molecular basis of miRNA expression and to exploit their mode of regulation for image-guided cancer miRNA therapy.
- Deraredj Nadim W., Chaumont-Dubel S., Madouri F., Cobret L., De Tauzia M.-L., Zajdel P., Bénédetti H., Marin P. and Morisset-Lopez S. Physical Interaction between Neurofibromin and Serotonin 5-HT6 Receptor Promotes Receptor Constitutive Activity. Proc Natl Acad Sci U S A (2016) in press - doi : 10.1073/pnas.1600914113
- Baril P. and Pichon C. Positive Bioluminescence Imaging of MicroRNA Expression in Small Animal Models Using an Engineered Genetic-Switch Expression System, RILES. Methods in Molecular Biology (2016) 1372, 193-208.
- Baril P., Ezzine S. and Pichon C. Monitoring the Spatiotemporal Activities of miRNAs in Small Animal Models Using Molecular Imaging Modalities. International Journal of Molecular Science (2015) 16 (3) 4947-4972.
- Cobret L., De Tauzia M.-L., Ferent J., Traiffort E., Hénaoui I., Godin F., Kellenberger E., Rognan D., Pantel J., Bénédetti H. and Morisset-Lopez S. Targeting the cis-dimerization of LINGO-1 with low MW compounds affects its downstream signalling. British Journal of Pharmacology (2015) 172 (3) 841-56.
- Deau E., Robin E., Voinea R., Percina N., Satała G., Fînaru A. L., Chartier A., Tamagnan G. D., Alagille D., Bojarski A. J., Morisset-Lopez S., Suzenet F. and Guillaumet G. Rational Design, Pharmacomodulation, Synthesis of Dual 5- Hydroxytryptamine 7 (5-HT7) / 5-Hydroxytryptamine 2A (5-HT2A) Receptors Antagonists and Evaluation by [(18)F]-PET Imaging in a Primate Brain. Journal of Medicinal Chemistry (2015) 58 (20) 8066-96.
- Sarou-Kanian V., Joudiou N., Louat F., Yon M., Szeremeta F., Même S., Massiot D., Decoville M., Fayon F. and Beloeil J.-C. Metabolite localization in living drosophila using High Resolution Magic Angle Spinning NMR. Scientific Reports (2015) 5, 9872.
- Ezzine S., Vassaux G., Pitard P., Barteau B., Malinge J.-M., Midoux P., Pichon C. and Baril P. RILES, a novel method for temporal analysis of the in vivo regulation of miRNA expression. Nucleic Acids Research (2013) 41 (20) e192.
Nadim W. D., Simion V., Bénédetti H., Pichon C., Baril P. and Morisset-Lopez S. (2016)
Neurodegenerative and cognitive disorders are multifactorial diseases (i.e., involving neurodevelopmental, genetic, age or environmental factors) that are characterized by an abnormal development affecting neuronal function and integrity. Recently, an increasing number of studies revealed that the dysregulation of microRNAs (miRNAs) may be involved in the etiology of cognitive disorders such as Alzheimer, Parkinson, and Huntington’s diseases, Schizophrenia and Autism spectrum disorders. MiRNAs are a class of small non-coding RNAs that regulate gene expression through a base pairing mechanism with their target mRNAs, thereby inducing translational repression or mRNA degradation. Intriguingly, 70 % of known miRNAs are expressed in the brain while functional studies indicate that they play crucial roles in brain development by regulating key signaling pathways involved in synaptogenesis, neuronal plasticity, neurite outgrowth and memory processes. Further, the relevance of modulating miRNA expression levels to correct neurocognitive disorders is supported by the increasing number of studies demonstrating a link between miRNA dysregulation and the etiology and/or pathophysiology of cognitive dysfunction in several neurologic and neuropsychiatric disorders. This suggests that miRNAs might be used as diagnosis markers and/or could be exploited for therapeutic interventions. In this review, we first briefly summarize the basic molecular events involved in memory, a process inherent to cognitive disease, and then describe the role of miRNAs in neurodevelopment, synaptic plasticity and memory. Secondly, we provide an overview of the impact of miRNA dysregulation in the pathogenesis of different neurocognitive disorders, and lastly discuss the feasibility of miRNA-based therapeutics in the treatment of these cognitive disorders. This last part will be discussed in detail in the following review.
Physical Interaction between Neurofibromin and Serotonin 5-HT6 Receptor Promotes Receptor Constitutive ActivityProc Natl Acad Sci U S A (2016) 113 (43) 12310-12315 - doi : 10.1073/pnas.1600914113
Active G protein-coupled receptor (GPCR) conformations not only are promoted by agonists but also occur in their absence, leading to constitutive activity. Association of GPCRs with intracellular protein partners might be one of the mechanisms underlying GPCR constitutive activity. Here, we show that serotonin 5 hydroxytryptamine 6 (5-HT6) receptor constitutively activates the Gs/adenylyl cyclase pathway in various cell types, including neurons. Constitutive activity is strongly reduced by silencing expression of the Ras-GTPase activating protein (Ras-GAP) neurofibromin, a 5-HT6 receptor partner. Neurofibromin is a multidomain protein encoded by the NF1 gene, the mutation of which causes Neurofibromatosis type 1 (NF1), a genetic disorder characterized by multiple benign and malignant nervous system tumors and cognitive deficits. Disrupting association of 5-HT6 receptor with neurofibromin Pleckstrin Homology (PH) domain also inhibits receptor constitutive activity, and PH domain expression rescues 5-HT6 receptor-operated cAMP signaling in neurofibromin-deficient cells. Furthermore, PH domains carrying mutations identified in NF1 patients that prevent interaction with the 5-HT6 receptor fail to rescue receptor constitutive activity in neurofibromin-depleted cells. Further supporting a role of neurofibromin in agonist-independent Gs signaling elicited by native receptors, the phosphorylation of cAMP-responsive element-binding protein (CREB) is strongly decreased in prefrontal cortex of Nf1+/− mice compared with WT mice. Moreover, systemic administration of a 5-HT6 receptor inverse agonist reduces CREB phosphorylation in prefrontal cortex of WT mice but not Nf1+/− mice. Collectively, these findings suggest that disrupting 5-HT6 receptor–neurofibromin interaction prevents agonist-independent 5-HT6 receptor-operated cAMP signaling in prefrontal cortex, an effect that might underlie neuronal abnormalities in NF1 patients.
Pharmacomodulation of microRNA expression in neurocognitive diseases : obstacles and future opportunitiesCurrent Neuropharmacology (sous presse) Vassaux G., Angelova A. L., Rommelaere J., Baril P., Midoux P. and Cordelier P. (2016)
Unlike other digestive-cancer entities, chemotherapy, radiotherapy and targeted therapies have, so far, largely failed to improve patient survival in pancreatic adenocarcinoma (PDAC), which remains the fourth leading cause of cancer-related death in Europe and the United States. In this context, gene therapy may offer a new avenue for PDAC patients. In this review, we explore the research currently ongoing in French laboratories aimed at defeating PDAC using non-viral therapeutic gene delivery, targeted transgene expression or oncolytic virotherapy that recently or will soon bridge the gap between experimental models of cancer and clinical trials. These studies are likely to change clinical practice or thinking about PDAC management, as they represent a major advance not only for PDAC and may also largely influence the field of gene-based molecular treatment of cancer.
Enhanced responsiveness of Ghsr Q343X rats to ghrelin results in enhanced adiposity without increased appetiteScience signaling (2016) 9 (424) ra39 - doi : 10.1126/scisignal.aae0374
The ability of the gut hormone ghrelin to promote positive energy balance is mediated by the growth hormone secretagogue receptor (GHSR). GHSR is a G protein-coupled receptor (GPCR) that is found centrally and peripherally and that can signal in a ligand-independent manner basally or when heterodimerized with other GPCRs. However, currentGhsrknockout models cannot dissect ghrelin-dependent and ghrelin-independent signaling, precluding assessment of the physiological importance of these signaling pathways. An animal model carrying aGhsrmutation that preserves GHSR cell surface abundance, but selectively alters GHSR signaling, would be a useful tool to decipher GHSR signaling in vivo. We used rats with theGhsr(Q343X)mutation (Ghsr(M/M)), which is predicted to delete the distal part of the GHSR carboxyl-terminal tail, a domain critical for the signal termination processes of receptor internalization and beta-arrestin recruitment. In cells, the GHSR-Q343X mutant showed enhanced ligand-induced G protein-dependent signaling and blunted activity of processes involved in GPCR signal termination.Ghsr(M/M)rats displayed enhanced responses to submaximal doses of ghrelin or GHSR agonist. Moreover,Ghsr(M/M)rats had a more stable body weight under caloric restriction, a condition that increases endogenous ghrelin tone, whereas under standard housing conditions,Ghsr(M/M)rats showed increased body weight and adiposity and reduced glucose tolerance. Overall, our data stress the physiological role of the distal domain of GHSR carboxyl terminus as a suppressor of ghrelin sensitivity, and we propose using theGhsr(M/M)rat as a physiological model of gain of function inGhsrto identify treatments for obesity-related conditions.
Oat (Avena sativa L.) seed extracts exhibited a high degree of catalytic activity including amylase activities. Proteins in the oat seed extracts were optimized for their amylolytic activities. Oat extract with amylolytic activity was separated by SDS-PAGE and a major protein band with an apparent molecular mass of 53 kDa was subjected to tryptic digestion. The generated amino acid sequences were analyzed by liquid chromatography–tandem mass spectrometry (LC/ESI/MS/MS) and database searches. These sequences were used to identify a partial cDNA from expressed sequence tags (ESTs) of A. sativa L. Based upon EST sequences, a predicted full-length gene was identified, with an open reading frame of 1464 bp encoding a protein of 488 amino acid residues (AsBAMY), with a theoretical molecular mass of 55 kDa identified as a β-amylase belonging to the plant β-amylase family. Primary structure of oat β-amylase (AsBAMY) protein indicated high similarity with other β-amylase from other cereals such as wheat (Triticum aestivum), barley (Hordeum vulgare), and rye (Secale cereale) with two conserved Glu residues (E184 and E378) assigned as the “putative” catalytic residues which would act as an acid and base pair in the catalytic process. In addition, a 3D-model of AsBAMY was built from known X-ray structures and sequence alignments. A similar core (β/α)8-barrel architecture was found in AsBAMY like the other cereal β-amylases with a specific location of the active site in a pocket-like cavity structure made at one end of this core (β/α)8-barrel domain suggesting an accessibility of the non-reducing end of the substrate and thus confirming the results of AsBAMY exo-acting hydrolase.
Positive Bioluminescence Imaging of MicroRNA Expression in Small Animal Models Using an Engineered Genetic-Switch Expression System, RILESMethods in molecular biology (2016) 1372, 193-208 - doi : 10.1007/978-1-4939-3148-4_15
MicroRNAs (miRNAs) are a class of small, noncoding RNAs which regulate gene expression by directing their target mRNA for degradation or translational repression. Since their discovery in the early 1990s, miRNAs have emerged as key components in the posttranscriptional regulation of gene networks, shaping many biological processes from development, morphogenesis, differentiation, proliferation and apoptosis. Although understanding of the molecular basis of miRNA biology is improving, methods to monitor the dynamic and the spatiotemporal aspects of miRNA expression under physiopathological conditions are required. However, monitoring of miRNAs is difficult due to their small size, low abundance, high degree of sequence similarity, and their dynamic expression pattern which is subjected to tight transcriptional and post-transcriptional controls. Recently, we developed a miRNA monitoring system called RILES, standing for RNAi-inducible expression system, which relies on an engineered regulatable expression system, to switch on the expression of the luciferase gene when the targeted miRNA is expressed in cells. We demonstrated that RILES is a specific, sensitive, and robust method to determine the fine-tuning of miRNA expression during the development of an experimental pathological process in mice. Because RILES offers the possibility for longitudinal studies on individual subjects, sharper insights into miRNA regulation can be generated, with applications in physiology, pathophysiology and development of RNAi-based therapies. This chapter describes methods and protocols to monitor the expression of myomiR-206, -1, and -133 in the tibialis anterior muscle of mice. These protocols can be used and adapted to monitor the expression of other miRNAs in other biological processes.
Gaspar V. M., Baril P., Costa E. C., de Melo-Diogo D., Foucher F., Queiroz J. A., Sousa F., Pichon C. and Correia I. J. (2015)
Bioreducible poly(2-ethyl-2-oxazoline)-PLA-PEI-SS triblock copolymer micelles for co-delivery of DNA minicircles and DoxorubicinJournal of Controlled Release (2015) 213, 175-191 - doi : 10.1016/j.jconrel.2015.07.011
The co-delivery of minicircle DNA (mcDNA) and small anti-cancer drugs via stimuli-sensitive nanocarriers is a promising approach for combinatorial cancer therapy. However, the simultaneous loading of drugs and DNA in nanosized delivery systems is remarkably challenging. In this study we describe the synthesis of triblock copolymer micelles based on poly(2-ethyl-2-oxazoline)-poly(L-lactide) grafted with bioreducible polyethylenimine (PEOz-PLA-g-PEI-SS) for co-delivery of supercoiled (sc) mcDNA vectors and Doxorubicin (Dox). These amphiphilic carriers take advantage of non-fouling oxazolines to confer biological stability, of PLA to provide a hydrophobic core for drug encapsulation and of bioreducible PEI-SS to provide mcDNA complexation and an on-demand stimuli-responsive release. The obtained results show that mcDNA-loaded micelleplexes penetrate into in vitro tumor spheroid models with specific kinetics and exhibit a higher gene expression when compared to non-bioreducible nanocarriers. Moreover, in vivo bioluminescence imaging showed that gene expression is detected up to 8days following mcDNA-micelles intratumoral administration. Furthermore, drug-gene co-delivery in PEOz-PLA-g-PEI-SS carriers was verified by successful encapsulation of both Dox and mcDNA with high efficacy. Moreover, dual-loaded micelleplexes presented significant uptake and a cytotoxic effect in 2D cultures of cancer cells. The co-delivery of mcDNA-Dox to B16F10-Luciferase tumor bearing mice resulted in a reduction in tumor volume and cancer cells viability. Overall, such findings indicate that bioreducible triblock micelles are efficient for focal delivery in vivo and have potential for future application in combinatorial DNA-drug therapy.
Tendon injury is a major musculoskeletal disorder with a high public health impact. We propose a non-viral based strategy of gene therapy for the treatment of tendon injuries using histidylated vectors. Gene delivery of fibromodulin, a proteoglycan involved in collagen assembly was found to promote rat Achilles tendon repair in vivo and in vitro. In vivo liposome-based transfection of fibromodulin led to a better healing after surgical injury, biomechanical properties were better restored compared to untransfected control. These measures were confirmed by histological observations and scoring. To get better understandings of the mechanisms underlying fibromodulin transfection, an in vitro tendon healing model was developed. In vitro, polymer-based transfection of fibromodulin led to the best wound enclosure speed and a pronounced migration of tenocytes primary cultures was observed. These results suggest that fibromodulin non-viral gene therapy could be proposed as a new therapeutic strategy to accelerate tendon healing. FROM THE CLINICAL EDITOR : Tendon injury is relatively common and healing remains unsatisfactory. In this study, the effects of liposomal-based delivery of fibromodulin gene were investigated in a rat Achilles tendon injury model. The positive results observed would provide a new therapeutic strategy in clinical setting in the future.
Rational Design, Pharmacomodulation, and Synthesis of Dual 5-Hydroxytryptamine 7 (5-HT7)/5-Hydroxytryptamine 2A (5-HT2A) Receptor Antagonists and Evaluation by F-18 -PET Imaging in a Primate BrainJournal of Medicinal Chemistry (2015) 58 (20) 8066-8096 - doi : 10.1021/acs.jmedchem.5b00874
We report the synthesis of 46 tertiary amine-bearing N-alkylated benzo[d]imidazol-2(3H)-ones, imidazo-[4,5-b]pyridin-2(3H)-ones, irnidazo[4,5-c]pyridin-2(3H)-ones, benzo [d] oxazol-2 (3H) -ones, oxazolo [4,5- b]pyridin-2(3H)-ones and N,N’-dialkylate d benzo [d] imidazol-2 (3H)-ones. These compounds were evaluated against 5-HT7R, 5-HT2AR, 5-HT1AR, and 5-HT6R as potent dual 5-HT7/5-HT2A serotonin receptors ligands. A thorough study of the structure-activity relationship of the aromatic rings and their substituents, the alkyl chain length and the tertiary amine was conducted. 1-(4-(4-(4-Fluorobenzoyl)piperidin-1-yl)butyl)-1H-benzo[d]imidazol-2(3H)-one (79) and 1-(6-(4-(4-fluorobenzoyDpiperidin-1-yOhexyl)-1H-benzo[d]imidazol-2(3H)-one (81) were identified as full antagonist ligands on cyclic adenosine monophosphate (cAMP, K-B = 4.9 and 5.9 nM, respectively) and inositol monophosphate (IP1, K-B = 0.6 and 16 nM, respectively) signaling pathways of 5-HT7R and 5-HT2AR. Both antagonists crossed the blood-brain barrier as evaluated with [F-18] radiolabeled compounds [F-18]79 and [F-18]81 in a primate’s central nervous system using positron emission tomography. Both radioligands showed standard uptake values ranging from 0.8 to 1.1, a good plasmatic stability, and a distribution consistent with 5-HT7R and 5-HT2AR in the CNS.
We have developed new methods enabling in vivo localization and identification of metabolites through their (1)H NMR signatures, in a drosophila. Metabolic profiles in localized regions were obtained using HR-MAS Slice Localized Spectroscopy and Chemical Shift Imaging at high magnetic fields. These methods enabled measurement of metabolite contents in anatomic regions of the fly, demonstrated by a decrease in beta-alanine signals in the thorax of flies showing muscle degeneration.
Statistical approaches were employed for the optimisation of the extraction of amylolytic activity from oat (Avena sativa) seeds. The application of the response surface methodology allows us to determine a set of optimal conditions (ratio seed weight/buffer volume 0.1, germination days 10 days, temperature 20 degrees C and pH 5.6). Experiments carried out under these conditions led to amylase production yield of 91 U/g. Its maximal activity was in the pH 5.6 and at 55 degrees C. Study of the incorporation of the optimised oat extract into the bread formulation revealed an improvement of the sensory quality and the textural properties of fresh and stored bread. Three-dimensional elaborations of Confocal Laser Scanning Microscopy (CLSM) images were performed on crumb of the different breads to evaluate the influence of amylase activity on microstructure. The result showed improved baking characteristics as well as overall microscopic and macroscopic appearance.
Monitoring the Spatiotemporal Activities of miRNAs in Small Animal Models Using Molecular Imaging ModalitiesInternational Journal of Molecular Sciences (2015) 16 (3) 4947-4972 - doi : 10.3390/ijms16034947
MicroRNAs (miRNAs) are a class of small non-coding RNAs that regulate gene expression by binding mRNA targets via sequence complementary inducing translational repression and/or mRNA degradation. A current challenge in the field of miRNA biology is to understand the functionality of miRNAs under physiopathological conditions. Recent evidence indicates that miRNA expression is more complex than simple regulation at the transcriptional level. MiRNAs undergo complex post-transcriptional regulations such miRNA processing, editing, accumulation and re-cycling within P-bodies. They are dynamically regulated and have a well-orchestrated spatiotemporal localization pattern. Real-time and spatio-temporal analyses of miRNA expression are difficult to evaluate and often underestimated. Therefore, important information connecting miRNA expression and function can be lost. Conventional miRNA profiling methods such as Northern blot, real-time PCR, microarray, in situ hybridization and deep sequencing continue to contribute to our knowledge of miRNA biology. However, these methods can seldom shed light on the spatiotemporal organization and function of miRNAs in real-time. Non-invasive molecular imaging methods have the potential to address these issues and are thus attracting increasing attention. This paper reviews the state-of-the-art of methods used to detect miRNAs and discusses their contribution in the emerging field of miRNA biology and therapy.
Background and Purpose The transmembrane protein LINGO-1 is a negative regulator in the nervous system mainly affecting axonal regeneration, neuronal survival, oligodendrocyte differentiation and myelination. However, the molecular mechanisms regulating its functions are poorly understood. In the present study, we investigated the formation and the role of LINGO-1 cis-dimers in the regulation of its biological activity. Experimental Approach LINGO-1 homodimers were identified in both HEK293 and SH-SY5Y cells using co-immunoprecipitation experiments and BRET saturation analysis. We performed a hypothesis-driven screen for identification of small-molecule protein–protein interaction modulators of LINGO-1 using a BRET-based assay, adapted for screening. The compound identified was further assessed for effects on LINGO-1 downstream signalling pathways using Western blotting analysis and AlphaScreen technology. Key Results LINGO-1 was present as homodimers in primary neuronal cultures. LINGO-1 interacted homotypically in cis-orientation and LINGO-1 cis-dimers were formed early during LINGO-1 biosynthesis. A BRET-based assay allowed us to identify phenoxybenzamine as the first conformational modulator of LINGO-1 dimers. In HEK-293 cells, phenoxybenzamine was a positive modulator of LINGO-1 function, increasing the LINGO-1-mediated inhibition of EGF receptor signalling and Erk phosphorylation. Conclusions and Implications Our data suggest that LINGO-1 forms constitutive cis-dimers at the plasma membrane and that low MW compounds affecting the conformational state of these dimers can regulate LINGO-1 downstream signalling pathways. We propose that targeting the LINGO-1 dimerization interface opens a new pharmacological approach to the modulation of its function and provides a new strategy for drug discovery.
Duhr, F., Déléris, P., Raynaud, F., Séveno, M., Morisset-Lopez, S., Mannoury la Cour, C., Millan, M.J., Bockaert, J., Marin, P. and Chaumont-Dubel, S. (2014)
The serotonin6 receptor (5-HT6R) is a promising target for treating cognitive deficits of schizophrenia often linked to alterations of neuronal development. This receptor controls neurodevelopmental processes, but the signaling mechanisms involved remain poorly understood. Using a proteomic strategy, we show that 5-HT6Rs constitutively interact with cyclin-dependent kinase 5 (Cdk5). Expression of 5-HT6Rs in NG108-15 cells induced neurite growth and expression of voltage-gated Ca2+ channels, two hallmarks of neuronal differentiation. 5-HT6R–elicited neurite growth was agonist independent and prevented by the 5-HT6R antagonist SB258585, which behaved as an inverse agonist. Moreover, it required receptor phosphorylation at Ser350 by Cdk5 and Cdc42 activity. Supporting a role of native 5-HT6Rs in neuronal differentiation, neurite growth of primary neurons was reduced by SB258585, by silencing 5-HT6R expression or by mutating Ser350 into alanine. These results reveal a functional interplay between Cdk5 and a G protein–coupled receptor to control neuronal differentiation.
Lethal and sublethal effects of imidacloprid, after chronic exposure, on the insect model Drosophila melanogasterEnvironmental science and technology (201) 48 (7) 4096-4102 - doi : 10.1021/es405331c
Neonicotinoids are subjected to vigilance because of environmental contaminations and deleterious effects on bees. Imidacloprid (IMI) is one of the most representative insecticides of this family. At chronic exposure, concentration-effect relationships are non linear. An insect model should allow a better description of this toxicity. We compared the lethal concentration 50% (LC50) of IMI for a Drosophila-field strain, after acute and chronic exposure. Relative to the acute LC50, the chronic LC50 was lowered by a factor of 29 for males (1.3 mM/45 muM), 52 for larvae (157 muM/3 muM) and more than 172 for females (>3.1 mM/18 muM). Chronic exposure also revealed significant lethal and sublethal effects, at concentrations 3-5 orders of magnitude lower than the chronic LC50. Mean mortalities reached 28% (at 3.91 nM) and 27% (at 39.1 nM) for females and males, respectively. Fecundity decreased of 16% at 1.96 nM. Mating increased of 30% at 0.391 nM. The LOEC (lowest observed effect concentration : 0.391 nM) was 46 000 times lower than the chronic LC50 for males ; it was 115 000 times lower than the chronic LC50 for females. This study illuminates effects that neonicotinoids can induce at very low concentrations. This is of particular interest for nontarget insects and for insect dependent species.
Ezzine, S., Vassaux, G., Pitard, B., Barteau, B., Malinge, J. M., Midoux, P., Pichon, C. and Baril, P. (2013)
Novel methods are required to investigate the complexity of microRNA (miRNA) biology and particularly their dynamic regulation under physiopathological conditions. Herein, a novel plasmid-based RNAi-Inducible Luciferase Expression System (RILES) was engineered to monitor the activity of endogenous RNAi machinery. When RILES is transfected in a target cell, the miRNA of interest suppresses the expression of a transcriptional repressor and consequently switch-ON the expression of the luciferase reporter gene. Hence, miRNA expression in cells is signed by the emission of bioluminescence signals that can be monitored using standard bioluminescence equipment. We validated this approach by monitoring in mice the expression of myomiRs-133, -206 and -1 in skeletal muscles and miRNA-122 in liver. Bioluminescence experiments demonstrated robust qualitative and quantitative data that correlate with the miRNA expression pattern detected by quantitative RT-PCR (qPCR). We further demonstrated that the regulation of miRNA-206 expression during the development of muscular atrophy is individual-dependent, time-regulated and more complex than the information generated by qPCR. As RILES is simple and versatile, we believe that this methodology will contribute to a better understanding of miRNA biology and could serve as a rationale for the development of a novel generation of regulatable gene expression systems with potential therapeutic applications.
In preclinical research, genetic studies have made considerable progress as a result of the development of transgenic animal models of human diseases. Consequently, there is now a need for higher resolution MRI to provide finer details for studies of small animals (rats, mice) or very small animals (insects). One way to address this issue is to work with high-magnetic-field spectrometers (dedicated to small animal imaging) with strong magnetic field gradients. It is also necessary to develop a complete methodology (transmit/receive coil, pulse sequence, fixing system, air supply, anesthesia capabilities, etc.). In this study, we developed noninvasive protocols, both in vitro and in vivo (from coil construction to image generation), for drosophila MRI at 9.4 T. The 10*10*80-μm resolution makes it possible to visualize whole drosophila (head, thorax, abdomen) and internal organs (ovaries, longitudinal and transverse muscles, bowel, proboscis, antennae and optical lobes). We also provide some results obtained with a Drosophila model of muscle degeneration. This opens the way for new applications of structural genetic modification studies using MRI of drosophila.
Godin, F., Villette, S., Vallée, B., Doudeau, M., Morisset-Lopez, S., Ardourel, M., Hevor, T., Pichon, C. and Bénédetti, B. (2012)
A fraction of neurofibromin interacts with PML bodies in the nucleus of the CCF astrocytoma cell lineBiochemical and Biophysical Research Communications 418 (4) 689-694
Neurofibromatosis type 1 is a common genetic disease that causes nervous system tumors, and cognitive deficits. It is due to mutations within the NF1 gene, which encodes the Nf1 protein. Nf1 has been shown to be involved in the regulation of Ras, cAMP and actin cytoskeleton dynamics. In this study, using immunofluorescence experiments, we have shown a partial nuclear localization of Nf1 in the astrocytoma cell line : CCF and we have demonstrated that Nf1 partially colocalizes with PML (promyelocytic leukemia) nuclear bodies. A direct interaction between Nf1 and the multiprotein complex has further been demonstrated using ‘‘in situ’’ proximity ligation assay (PLA).
Baril, P. ; Touchefeu, Y. ; Cany, J. ; Cherel, Y. ; Thorne, S. H. ; Tran, L. ; Conchon, S. and Vassaux, G. (2011)
Differential biodistribution of oncolytic poxvirus administered systemically in an autochthonous model of hepatocellular carcinomaThe Journal of Gene Medicine (2011) 13 (12) 692-701
Background Preclinical studies have demonstrated that, unlike oncolytic adenoviruses, oncolytic vaccinia viruses can reach implanted tumors upon systemic injection. However, the biodistribution of this oncolytic agent in in situ autochthonous tumor models remains poorly characterized. In the present study, we assessed this biodistribution in a model of mouse hepatocellular carcinoma (HCC) obtained after injection of the carcinogen diethylnitrosamine (DEN). Methods Twelve months after DEN administration, histology, quantitative reverse transcription-polymerase chain reaction, in situ hybridization and viral titration were used to characterize tumors, as well as to assess the viral load of the livers upon either intravenous or intraperitoineal injection. Results The results obtained showed that the architecture of the liver was lost, with a noticeable absence of sinusoids, as well as the presence of steatosis and α-fetoprotein-positive HCC tumor nodules. Bioluminescence imaging and measures of the infective virus load demonstrated that intravenous injection of 108 plaque-forming units of the recombinant vaccinia virus led to a predominant transduction of the liver, whereas intraperitoneal injection resulted in a lower level of liver transduction accompanied by an increased infection of the lungs, spleen, kidneys and bowels. Immunohistochemical analysis of liver sections of animals injected intravenously with the virus revealed a preferential localization of vaccinia-specific immunoreactivity in the tumors. Conclusions The findings of the present study emphasize the importance of the route of administration of the vector and highlight the relevance of systemic injection of oncolytic vaccinia virus in the context of hepatocellular carcinoma.
The MAP kinase ERK and its scaffold protein MP1 interact with the chromatin regulator Corto during Drosophila wing tissue development.Bmc Developmental Biology 11 (1) 17
Background : Mitogen-activated protein kinase (MAPK) cascades (p38, JNK, ERK pathways) are involved in cell fate acquisition during development. These kinase modules are associated with scaffold proteins that control their activity. In Drosophila, dMP1, that encodes an ERK scaffold protein, regulates ERK signaling during wing development and contributes to intervein and vein cell differentiation. Functional relationships during wing development between a chromatin regulator, the Enhancer of Trithorax and Polycomb Corto, ERK and its scaffold protein dMP1, are examined here.
Results : Genetic interactions show that corto and dMP1 act together to antagonize rolled (which encodes ERK) in the future intervein cells, thus promoting intervein fate. Although Corto, ERK and dMP1 are present in both cytoplasmic and nucleus compartments, they interact exclusively in nucleus extracts. Furthermore, Corto, ERK and dMP1 co localize on several sites on polytene chromosomes, suggesting that they regulate gene expression directly on chromatin. Finally, Corto is phosphorylated. Interestingly, its phosphorylation pattern differs between cytoplasm and nucleus and changes upon ERK activation.
Picard M. Morisset S., Cloix J. F., Bizot J. C., Guerin M., Beneteau V., Guillaumet G. and Hevor T. K. (2010)
A novel pyridine derivative, 8-4-[(6-methoxy-2,3-dihydro-[1,4]dioxino[2,3-b]pyridine-3-ylmethyl)-amino]-butyl -8-aza-spiro[4.5]decane-7,9-dione hydrochloride, termed JB-788, was designed to selectively target 5-HT(1A) receptors. In the present study, the pharmacological profile of JB-788 was characterized in vitro using radioligands binding tests and in vivo using neurochemical and behavioural experiments. JB-788 bound tightly to human 5-HT(1A) receptor expressed in human embryonic kidney 293 (HEK-293) cells with a K(i) value of 0.8 nM. Its binding affinity is in the same range as that observed for the (+/-)8-OH-DPAT, a reference 5HT(1A) agonist compound. Notably, JB-788 only bound weakly to 5-HT(1B) or 5-HT(2A) receptors and moreover the drug displayed only weak or indetectable binding to muscarinic, alpha(2), beta(1) and beta(2) adrenergic receptors, or dopaminergic D(1) receptors. JB-788 was found to display substantial binding affinity for dopaminergic D(2) receptors and, to a lesser extend to alpha(1) adrenoreceptors. JB-788 dose-dependently decreased forskolin-induced cAMP accumulation in HEK cells expressing human 5-HT(1A), thus acting as a potent 5-HT(1A) receptor agonist (E(max.) 75%, EC(50) 3.5 nM). JB-788 did not exhibit any D(2) receptor agonism but progressively inhibited the effects of quinpirole, a D(2) receptor agonist, in the cAMP accumulation test with a K(i) value of 250 nM. JB-788 induced a weak change in cAMP levels in mouse brain but, like some antipsychotics, transiently increased glycogen contents in various brain regions. Behavioral effects were investigated in mice using the elevated plus-maze. JB-788 was found to increase the time duration spent by animals in anxiogenic situations. Locomotor hyperactivity induced by methamphetamine in mouse, a model of antipsychotic activity, was dose-dependently inhibited by JB-788. Altogether, these results suggest that JB-788 displays pharmacological properties, which could be of interest in the area of anxiolytic and antipsychotic drugs.
Le système histaminergique : une cible pour de nouveaux traitements des déficits cognitifs - The Histaminergic System : a Target for Innovative Treatments of Cognitive DeficitsThérapie 65 (5) 415-522 - doi : 10.2515/therapie/2010058
L’histamine exerce ses effets centraux en activant des récepteurs H1,H2 et H3. Le récepteur H3 inhibe la libération de l’histamine cérébrale. Ainsi, les agonistes inverses H3, en levant ce frein, augmentent l’activité des neurones à histamine. Le système histaminergique est un système majeur de la cognition et les agonistes inverses H3 sont pressentis comme thérapeutique potentielle des déficits cognitifs de la maladie d’Alzheimer (AD). Ils sont d’autant plus attendus que d’autres traitements de la maladie, tels que la tacrine ou la mémantine, augmentent aussi, mais par d’autres mécanismes, la neurotransmission histaminergique. Il existe une perte importante de neurones histaminergiques dans l’AD, mais la mesure des taux du métabolite de l’histamine dans le LCR de patients atteints d’AD montre que leur activité globale n’est diminuée que de 25 %. Ces données montrent qu’il devrait bien être possible d’activer les neurones histaminergiques dans l’AD.
The central effects of histamine are mediated by H1, H2 and H3 receptors. The H3 receptor inhibits histamine release in brain. Therefore, H3 receptor inverse agonists, by suppressing this brake, enhance histamine neuron activity. The histaminergic system plays a major role in cognition and H3 receptor inverse agonists are expected to be a potential therapeutics for cognitive deficits of Alzheimer’s disease (AD). They are eagerly awaited inasmuch as other treatments of the disease, such as tacrine or memantine, also enhance, through different mechanisms, histaminergic neurotransmission. An important loss of histaminergic neurons has been observed in AD. In contrast, levels of the histamine metabolite in the CSF of AD patients show that their global activity is decreased by only 25%. This indicates that activating histamine neurons in AD can be envisaged.
Effects of betahistine at histamine H3 receptors : mixed inverse agonism/agonism in vitro and partial inverse agonism in vivoJournal of Pharmacology and Experimental Therapeutics 334 (3) 945-954 - doi : 10.1124/jpet.110.168633
We previously suggested that therapeutic effects of betahistine in vestibular disorders result from its antagonist properties at histamine H(3) receptors (H(3)Rs). However, H(3)Rs exhibit constitutive activity, and most H(3)R antagonists act as inverse agonists. Here, we have investigated the effects of betahistine at recombinant H(3)R isoforms. On inhibition of cAMP formation and [(3)H]arachidonic acid release, betahistine behaved as a nanomolar inverse agonist and a micromolar agonist. Both effects were suppressed by pertussis toxin, were found at all isoforms tested, and were not detected in mock cells, confirming interactions at H(3)Rs. The inverse agonist potency of betahistine and its affinity on [(125)I]iodoproxyfan binding were similar in rat and human. We then investigated the effects of betahistine on histamine neuron activity by measuring tele-methylhistamine (t-MeHA) levels in the brains of mice. Its acute intraperitoneal administration increased t-MeHA levels with an ED(50) of 0.4 mg/kg, indicating inverse agonism. At higher doses, t-MeHA levels gradually returned to basal levels, a profile probably resulting from agonism. After acute oral administration, betahistine increased t-MeHA levels with an ED(50) of 2 mg/kg, a rightward shift probably caused by almost complete first-pass metabolism. In each case, the maximal effect of betahistine was lower than that of ciproxifan, indicating partial inverse agonism. After an oral 8-day treatment, the only effective dose of betahistine was 30 mg/kg, indicating that a tolerance had developed. These data strongly suggest that therapeutic effects of betahistine result from an enhancement of histamine neuron activity induced by inverse agonism at H(3) autoreceptors.
Rautureau, G., Jouvensal, L., Vovelle, F., Schoentgen, F., Locker, D. & Decoville, M. (2009)
An optimized extended DNA kappa B site that enhances plasmid DNA nuclear import and gene expression.J. Gene Med. 11, 401-411. Beghdadi W, Porcherie A, Schneider BS, Morisset S, Dubayle D, Peronet R, Dy M, Louis J, Arrang JM and Mecheri S. (2009)
Davenas E, Rouleau A, Morisset S, and Arrang JM. (2008)
Humbert-Claude M, Morisset S, Gbahou F and Arrang JM. (2007)
Histamine H3 and dopamine D2 receptor-mediated [35S]GTPg[S]binding in rat striatum : evidence for additive effects but lack of interactionsBiochem Pharmacol (2007), 73 (8) : 1172-1181 Arrang J, Morisset S and Gbahou F. (2007)
Salvaing, J ; Decoville, M ; Mouchel-Vielh, E ; Bussiere, M ; Daulny, A ; Boldyreva, L ; Zhimulev, I ; Locker, D ; Peronnet, F (2006)
Corto and DSP1 interact and bind to a maintenance element of the Scr Hox gene : understanding the role of Enhancers of trithorax and PolycombBmc Biology 4 Art No. 9
Background : Polycomb-group genes (PcG) encode proteins that maintain homeotic (Hox) gene repression throughout development. Conversely, trithorax-group (trxG) genes encode positive factors required for maintenance of long term Hox gene activation. Both kinds of factors bind chromatin regions called maintenance elements ( ME). Our previous work has shown that corto, which codes for a chromodomain protein, and dsp1, which codes for an HMGB protein, belong to a class of genes called the Enhancers of trithorax and Polycomb (ETP) that interact with both PcG and trxG. Moreover, dsp1 interacts with the Hox gene Scr, the DSP1 protein is present on a Scr ME in S2 cells but not in embryos.
Cloning, high yield over-expression, purification, and characterization of CG18594, a new PEBP/RKIP family member from Drosophila melanogasterProtein Expression and Purification 48 (1) 90-97
The phosphatidylethanolamine-binding protein (PEBP) family is widely distributed in various species, from bacteria to mammals. These proteins seem to modulate important cell mechanisms : they control heterotrimeric G-proteins, inhibit the MAP-kinase and NF kappa B signaling pathways, and also serine proteases (thrombin, neuropsin, and chymotrypsin). In order to establish structure-function relationships for this family of proteins, our study focuses on PEBPs expressed within a single organism : Drosophila melanogaster, which constitutes a model system that lends itself well to establishing links between genes’ expression and the corresponding proteins’ functions, and to studying physiological mechanisms such as development.
Dejardin, J ; Rappailles, A ; Cuvier, O ; Grimaud, C ; Decoville, M ; Locker, D ; Cavalli, G (2005)
Polycomb and trithorax group (PcG and trxG) proteins maintain silent and active transcriptional states, respectively, throughout development(1). In Drosophila, PcG and trxG proteins associate with DNA regions named Polycomb and trithorax response elements (PRE and TRE), but the mechanisms of recruitment are unknown. We previously characterized a minimal element from the regulatory region of the Abdominal-B gene, termed Ab-Fab. Ab-Fab contains a PRE and a TRE and is able to maintain repressed or active chromatin states during development(2).
DSP1, a Drosophila HMG protein, is involved in spatiotemporal expression of the homoeotic gene Sex combs reducedBiology of The Cell 97 (10) 779-785
Background information. The Pc-G (Polycomb group) and trx-G (trithorax group) genes play a key role in the regulation of the homoeotic genes. The homoeotic gene Scr (Sex combs reduced) contained in the Antennapedia complex specifies segmental identity of the labial and prothoracic segments in Drosophila. Regulation of Scr requires the action of different enhancer elements spread over several kilobases. We previously identified an HMGB (high mobility group)-like protein DSP1 (dorsal switch protein 1), which works like a trx-G protein for the normal Scr expression. Results. In the present study, we attempted to characterize the regulatory sequences involved in the maintenance of the Scr activation by DSP1.
Martin, D ; Daulny, A ; Decoville, M ; Locker, D (2003)
Mutagenesis analysis of the interaction between the dorsal Rel homology domain and HMG boxes of DSP1 proteinJournal of Biochemistry 134 (4) 583-589
DSP1 is an HMG-like protein of Drosophila melanogaster consisting of 386 amino acids with two HMG domains at the C-terminal end. It was shown to interact with Dorsal protein through the HMG domains and to enhance its DNA binding. Each HMG domain consists of approximately 80 amino acid residues, forming three alpha helices folded into an L-shaped structure. We have compared the interaction of various truncated and mutated forms of DSP1 with the dorsal Rel homology domain (RHD). In particular, we have mutated the conserved tryptophan residue 212 or 302 in A or B boxes or the lysine-rich region ((KKRK256)-K-253) of the A/B linker. Analysis by circular dichroism revealed that the protein tertiary structure is affected in these mutants. However, these mutations do not abolish the DSP1 binding to Dorsal, except if the two HMG boxes are altered, i.e., in a double mutant or in mutant isolated domain. Finally, studies on the enhancement of Dorsal DNA binding by DSP1 revealed that the DNA affinity is maximum in the presence of wild-type DSP1, is dramatically reduced when box A is altered, and is completely abolished when box B is altered.
DSP1 is an HIVIG-box protein which has been implicated in the regulation of homeotic genes in Drosophila melanogaster. Here we report that DSP1 is also involved in the regulation of the kni gap gene. Analysis of the phenotype of a null mutation of dsp1 (dspl(1)) reveals that the absence of maternal DSP1 results in A4 segmentation defects that are correlated with a diminution of the kni expression domain. Genetic interaction studies demonstrate that a bcd mutation enhances the A4 defect of dsp1(1). We present in vitro and in vivo evidences for a direct interaction between DSP1 and Bicoid, mediated by the BCD homeodomain and the HMG box of DSP1. Finally, we show by immunoprecipitation of cross-linked chromatin the association of DSP1 with the kni-regulating region and discuss the potential mechanism of DSP1-mediated activation of kni. (C) 2003 Wiley-Liss, Inc.
Drosophila DSP1 and rat HMGB1 have equivalent DNA binding properties and share a similar secondary foldJournal of Biochemistry 133 (4) 533-539
The protein DSP1 belongs to the group of HMG-box proteins, which share the common structural feature of the HMG-box. This approximately 80 amino acid long motif binds DNA via the minor groove. DSP1 was discovered as a,transcriptional co-repressor of Dorsal in Drosophila melanogaster and then was shown to participate to the remodeling of chromatin. By means of sequence alignment and gene organization, DSP1 was classified as the fly homologue of the vertebrate proteins HMGB1/2. DSP1 contains two HMG boxes flanked by two glutamine-rich domains at the N-terminus. In addition, the HMG domain of DSP1 displays two differences in its primary sequence as compared to the vertebrate HMGB1 : a shorter acidic tail and a linker between the two boxes longer by 6 amino acids. By comparing several functional parameters of DSP1 with those of HMGB1, the present study establishes the functional equivalence of both proteins in terms of DNA recognition. The major structural difference between the two proteins, the glutamine-rich N-terminal tail of DSP1, which does not exist in HMGB1, did not interfere with any of the studied DNA-binding properties of the proteins.
Therapeutic implications of constitutive activity of receptors : the example of the histamine H3 receptorJ Neural Transm (2003) Suppl:1-16 Ilien B., Franchet C., Bernard P., Morisset S., Weill C. Bourguignon J., Hibert M. and Galzi J.L. (2003)
Fluorescence resonance energy transfer to probe human muscarinic M1 receptor structure and drug binding propertiesJ. Neurochem. (2003) 85:768-778 Gbahou F, Rouleau A, Morisset S, Parmentier R, Crochet S, Lin JS, Ligneau X, Tardivel-Lacombe J, Stark H, Schunack W, Ganellin CR, Schwartz JC and Arrang JM. (2003)
Rouleau A., Ligneau X., Tardivel-Lacombe J., Morisset S., Gbahou F., Schwartz J.C., Arrang J.M. (2002)
Histamine H3-receptor-mediated [35S]GTP gamma[S] binding : evidence for constitutive activity of the recombinant and native rat and human H3 receptorsBr J Pharmacol. (2002) 135, 383-392 Morisset S., Pilon C., Tardivel-Lacombe J., Weinstein D., Rostene W., Betancur C., Sokoloff P., Schwartz J.C. and Arrang J.M. (2002)
Acute and chronic effects of methamphetamine on tele-methylhistamine levels in mouse brain : selective involvement of the D(2) and not D(3) receptorJ. Pharmacol. Exp. Ther. (2002) 300, 621-628 Chotard C., Ouimet T., Morisset S., Sahm U., Schwartz J.C. and Tottier S. (2002)
Effects of histamine H3 receptor agonist and antagonist on histamine co-transmitter expression in rat brainJ. Neural. Transm. (2002) 109, 293-306
Decoville, M ; Giacomello, E ; Leng, M ; Locker, D (2001)
The Drosophila dsp1 gene, which encodes an HMG-like protein, was originally identified in a screen for corepressors of Dorsal. Here we report that loss of dsp1 function causes homeotic transformations resembling those associated with loss of function in the homeotic genes Sex combs,reduced (Scr), Ultrabithorax (Ubx), and Abdominal-B. The expression pattern of Scr is altered in dsp1 mutant imaginal discs, indicating that dsp1 is required for normal expression of this gene. Genetic interaction studies reveal that a null allele of dsp1 enhances trithorax-group gene (trx-G) mutations and partially suppresses Polycomb-group gene (Pc-G) mutations. On the contrary, overexpression of dsp1 induces an enhancement of the transformation of wings into halteres and of the extra sex comb phenotype of Pc. In addition, dsp1 male mutants exhibit a mild transformation of A4 into A5. Comparison of the chromatin structure at the Mcp locus in wild-type and dsp1 mutant embryos reveals that the 300-bp DNase I hypersensitive region is absent in a dsp1 mutant context. We propose that DSP1 protein is a chromatin remodeling factor, acting as a trx-G or a Pc-G protein depending on the considered function.
The histamine H3 receptor : gene organization, multiple isoforms, constitutive activity and molecular pharmacologyDans ‘’Histamine Research in the New Millenium’’. Edité par T. Watanabe, H. Timmermann et K. Yanai. Elsevier, Amsterdam, (2001), 9-21
Decoville, M ; Giraud-Panis, MJ ; Mosrin-Huaman, C ; Leng, M ; Locker, D (2000)
Formation of the dorsoventral axis in Drosophila melanogaster is mediated through control of the expression of several genes by the morphogen Dorsal. In the ventral part of the embryo Dorsal activates twist and represses ren amongst others, Recently, several proteins have been shown to assist Dorsal in the repression of ten, one of which is DSP1, a HMG box protein that was isolated as a putative co-repressor of Dorsal. In this report we used a DSP1 null mutant to ascertain in vivo the involvement of DSP1 in Dorsal-mediated repression of ten but not in the activation of twist.