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P.1.009 Cell synchronisation as a tool to optimise expression of metabotropic glutamate receptors in inducible mammalian expression system
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Molecular neuropsychopharmacology
V1B receptor and looked for its ability to induce functional synergism. Results: Only for V1B /CRF1 receptors, there was a clear association between receptor heterodimerisation (BRETmax reach from x1 = Fluorescence/Luminescence = 0.005 to x2 = 0.02) and crosstalk-independent potentiation in both IPs (27% under 10 nM AVP and 10 nM CRF) and cAMP (29% under 1 nM CRF and 10 nM AVP) signalling pathways. By contrast, for co-transfection of V1B and CRF2 receptors, no molecular association could be observed (no saturation curve) and hormone-stimulated second messenger potentiation only reflected signalling crosstalk. Evidence for the existence of V1A /CRF1 heterodimers was found (x1 = 0.015; x2 = 0.03) but they conformationally differed to V1B /CRF1 heterodimers, and potentiation of signalling independent of second messenger crosstalk could only be observed for formation of IPs (13% under 10 nM AVP and 10 nM CRF). To further correlate heterodimerisation and crosstalkindependent potentiation, we generated a chimerical CRF1 -CRF2 receptor. BRET experiments show that this mutant has lost capacity to heterodimerise with V1B receptors. Moreover, despite it still activated cAMP pathway in a dose-dependent manner, this CRF1 mutant was no longer able to potentiate vasopressin-stimulated IPs accumulation via a mechanism independent of second messengers crosstalk. Conclusion: These results suggest that AVP-CRF synergism at least partially reflects the existence of V1B /CRF1 heterodimers, and that their formation modifies signalling at these sites. Studies underway are in line with the existence of molecular physical association of V1B /CRF1 heterodimers in native tissues. Further, the present work is consistent with a role for V1B /CRF1 heterodimers controlling ACTH secretion − and the activity of corticolimbic structures controlling mood − in the pathogenesis of stress-related psychiatric conditions like depression, anxiety disorders and schizophrenia. Reference(s) [1] Murat, B., Devost, D., Andr´es, M., Mion, J., Boulay, V., Corbani, M., Zingg, H.H., Guillon, G., 2012 V1b and CRHR1 receptor heterodimerization mediates synergistic biological actions of vasopressin and CRH. Mol Endocrinology 26(3), 502−20. [2] Young, S.F., Griffante, C., Aguilera, G., 2007 Dimerization between vasopressin V1b and corticotropin releasing hormone type 1 receptors. Cell Mol Neurobiol 27, 439–461. Disclosure statement: Work supported by the ANR, SERVIER (France) and the FRM.
P.1.009 Cell synchronisation as a tool to optimise expression of metabotropic glutamate receptors in inducible mammalian expression system B. Chruscicka1 ° , P. Branski1 , G. Burnat1 , A. Pilc1 . Polish Academy of Sciences, Neurobiology, Krakow, Poland 1
Background: The metabotropic glutamate receptors (mGluRs) are class C G protein-coupled receptors that play an important neuromodulatory role throughout the brain, as such they are involved in a number of psychiatric and neurological disorders like anxiety, depression and schizophrenia [1]. Expression of metabotropic glutamate receptors in heterologous mammalian cells is a method for their functional characterisation, and tool to study the agonist, antagonist and allosteric effects which are very attractive targets for therapeutic intervention. However, the constitutive expression of mGluRs seems to have a toxic effect on the HEK293 cells. Thus, an inducible expression system (in which the mGluRs expression is activated in the presence of tetracycline) is used [2]. Unfortunately an expression of receptors was confirmed in approximately 30% of cells after tetracycline induction. Aim: Our goal was to increase the number of HEK293 cells with an induced expression of mGlu2 receptor using synchronisation of cell cycle. Methods: cDNA fragment encoding mGlu2 receptor was cloned into a pcDNA5/FRT/TO vector under the control of a tetracycline regulated promoter. HEK293 cells containing inducible mammalian expression system (FlpIn T-REx host cells line from Invitrogen) were treated with GeneJuice transfection reagent, and then selected by hygromycin. Synchrony of stably transfected cells was performed by means of three different synchronisation protocols. To obtain G2 /M phase block, cells were supplemented with nocodazole for 18 h. To arrest cells at the beginning of S phase a double thymidine block was used (16 h and 18 h). G0 /G1 population of cells was obtained by serum starvation for 48 h [3]. The efficacy of cell synchronisation was verified by flow cytometric analysis of DNA content after propidium iodide-staining. For evaluation a surface expression of mGlu2 receptor flow cytometer immunofluorescence staining was performed. Results: We received 45.2±0.9% cells at the beginning of S phase after double thymidine block, 60.6±0.9% cells in M phase after nocodazole treatment and 77.9±8.0% cells in G0 /G1 phase after serum starvation. Untreated cells are characterised by 52.7±1.8% cells in G0 /G1 , 31.3±0.9% in S and 15.0±1.0% in G2 /M phase of cycle. Cell synchronisation seems to have statistically
Molecular neuropsychopharmacology significant effect on mGlu2 receptor expression level [One-way ANOVA, F(3.41) = 22.8, p < 0.0001]. Moreover Dunnett’s Multiple Comparison Test analysis shows that level of mGlu2 receptor surface expression in cells after double thymidine block was significantly higher compared to receptor expression level in asynchronously growing cells (p < 0.01). In contrast, the expression level of mGlu2 receptor was significantly decreased in M phase blocked cells (p < 0.01) and cells after serum starvation (p < 0.01). Conclusions: The results indicate that modification of the inducible expression system by cell synchronisation seems to have significant effect on surface mGluR2 expression level. Reference(s) [1] Palucha, A., Pilc, A., 2007. Metabotropic glutamate receptor ligands as possible anxiolytic and antidepressant drugs. Pharmacology & Therapeutics 115, 116– 147. [2] Van Craenenbroeck, K., Vanhoenacker, P., Leysen, J.E., Haegeman, G., 2001. Evaluation of the tetracycline- and ecdysone-inducible systems for expression of neurotransmitter receptors in mammalian cells. European Journal of Neuroscience 14, 968–976. [3] Yu, J.N., Ma, S.F., Miao, D.Q., Tan, X.W., Liu, X.Y., Lu, J.H., Tan, J.H., 2006. Effects of cell cycle status on the efficiency of liposome-mediated gene transfection in mouse fetal fibroblasts. Journal of Reproduction and Development 52, 373–382.
P.1.010 The identification of protein tyrosine phosphatase, non-receptor type 1 in hippocampal modulation of food anticipatory behaviour E. Kostrzewa1 ° , L.A.W. Verhagen1 , C. Gelegen1 , H.A. van Lith2 , D.A. Collier3 , M. Mitsogiannis1 , E. de Vries1 , M. van Gestel1 , R.A. Adan1 , M.J.H. Kas1 . 1 Rudolf Magnus Institute of Neuroscience, Neuroscience and Pharmacology, Utrecht, The Netherlands; 2 Utrecht University, Faculty of Veterinary Medicine, Utrecht, The Netherlands; 3 King’s College, Institute of Psychiatry, London, United Kingdom Background: Activity based anorexia (ABA) is a rodent model of hyperactivity evoked by food restriction. The paradoxical hyperactivity during food restriction is reminiscent of that observed in patients suffering from eating disorders. One of the parameters of this model, food anticipatory activity (FAA) enables us to study neurobiological mechanisms that synchronise the timing
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of food availability and changes in arousal in order to be prepared for upcoming relevant environmental events [1]. Methods: To identify chromosomes contributing to FAA, 21 mouse strains from a chromosome substitution panel (host strain expressing FAA (C57BL/6J) and a donor strain lacking FAA (A/J)) were tested using the ABA model (n = 8−10 mice per strain). Subsequently, we generated an F2 population from chromosome substitution strains 2 that lacks FAA. F2 mice (n = 125) were genotyped and phenotyped to fine map a locus on mouse chromosome 2 associated with FAA. In addition, genome-wide micro-array gene expression analysis was performed to identify differentially expressed genes in the hippocampus of F2 mice selected for their high or low FAA-levels. To functionally validate the candidate gene we generated shRNA viral vector (pAAV-shbase containing GFP cassette, titer 7.8×1012 genomic copies/ml) and injected C57BL/6J mice in the dentate gyrus (DG) three weeks prior to the ABA model. We verified virus spread using ISH for GFP and assessed the potential neuronal damage by performing IHC for NeuN and ISH for miRNA124. Level of candidate gene knockdown was quantified by radioactive ISH. Animals’ body weight changes, food intake and running wheel activity (RWA) during ABA were recorded. Results: We genetically mapped a Quantitative Trait Locus on mouse chromosome 2 associated with FAA. The combined genetic fine mapping and genome-wide gene expression data revealed Ptpn1 (protein tyrosine phosphatase, non-receptor type 1) as a candidate gene for FAA. To validate this candidate gene, we performed stereotactic injections of a virus containing anti-Ptpn1 shRNA. The virus infected neurons in DG and partly in CA fields without detectable neuronal damage. The expression of Ptpn1 in DG was significantly reduced in the antiPtpn1 group in comparison to the control virus injected group (Student t-test: t(8) = 2.50, p < 0.05). There were no differences in body weight, food intake nor baseline RWA (Student t-test: t(8) = −0.493, p = 0.635) between antiPtpn1 and control virus treated mice. Similarly, total RWA during the restriction phase did not differ significantly between the groups (Student t-test: t(8) = 0.126, p = 0.903 respectively). Normalised FAA, (total FAA as a percentage of total RWA), was found to be significantly decreased in the Ptpn1 knockdown group compared to the control group (Student t-test: t(8) = 2.459, p < 0.05). A positive association was found between the level of Ptpn1 mRNA expression in the DG and the magnitude of FAA (r = 0.642, p = 0.045). Conclusions: This data confirm that FAA levels are associated with Ptpn1 gene expression in the DG. As Ptpn1 is a key regulator of insulin and leptin receptor sensitivity, these findings provide a possible mechanism through
Molecular neuropsychopharmacology
V1B receptor and looked for its ability to induce functional synergism. Results: Only for V1B /CRF1 receptors, there was a clear association between receptor heterodimerisation (BRETmax reach from x1 = Fluorescence/Luminescence = 0.005 to x2 = 0.02) and crosstalk-independent potentiation in both IPs (27% under 10 nM AVP and 10 nM CRF) and cAMP (29% under 1 nM CRF and 10 nM AVP) signalling pathways. By contrast, for co-transfection of V1B and CRF2 receptors, no molecular association could be observed (no saturation curve) and hormone-stimulated second messenger potentiation only reflected signalling crosstalk. Evidence for the existence of V1A /CRF1 heterodimers was found (x1 = 0.015; x2 = 0.03) but they conformationally differed to V1B /CRF1 heterodimers, and potentiation of signalling independent of second messenger crosstalk could only be observed for formation of IPs (13% under 10 nM AVP and 10 nM CRF). To further correlate heterodimerisation and crosstalkindependent potentiation, we generated a chimerical CRF1 -CRF2 receptor. BRET experiments show that this mutant has lost capacity to heterodimerise with V1B receptors. Moreover, despite it still activated cAMP pathway in a dose-dependent manner, this CRF1 mutant was no longer able to potentiate vasopressin-stimulated IPs accumulation via a mechanism independent of second messengers crosstalk. Conclusion: These results suggest that AVP-CRF synergism at least partially reflects the existence of V1B /CRF1 heterodimers, and that their formation modifies signalling at these sites. Studies underway are in line with the existence of molecular physical association of V1B /CRF1 heterodimers in native tissues. Further, the present work is consistent with a role for V1B /CRF1 heterodimers controlling ACTH secretion − and the activity of corticolimbic structures controlling mood − in the pathogenesis of stress-related psychiatric conditions like depression, anxiety disorders and schizophrenia. Reference(s) [1] Murat, B., Devost, D., Andr´es, M., Mion, J., Boulay, V., Corbani, M., Zingg, H.H., Guillon, G., 2012 V1b and CRHR1 receptor heterodimerization mediates synergistic biological actions of vasopressin and CRH. Mol Endocrinology 26(3), 502−20. [2] Young, S.F., Griffante, C., Aguilera, G., 2007 Dimerization between vasopressin V1b and corticotropin releasing hormone type 1 receptors. Cell Mol Neurobiol 27, 439–461. Disclosure statement: Work supported by the ANR, SERVIER (France) and the FRM.
P.1.009 Cell synchronisation as a tool to optimise expression of metabotropic glutamate receptors in inducible mammalian expression system B. Chruscicka1 ° , P. Branski1 , G. Burnat1 , A. Pilc1 . Polish Academy of Sciences, Neurobiology, Krakow, Poland 1
Background: The metabotropic glutamate receptors (mGluRs) are class C G protein-coupled receptors that play an important neuromodulatory role throughout the brain, as such they are involved in a number of psychiatric and neurological disorders like anxiety, depression and schizophrenia [1]. Expression of metabotropic glutamate receptors in heterologous mammalian cells is a method for their functional characterisation, and tool to study the agonist, antagonist and allosteric effects which are very attractive targets for therapeutic intervention. However, the constitutive expression of mGluRs seems to have a toxic effect on the HEK293 cells. Thus, an inducible expression system (in which the mGluRs expression is activated in the presence of tetracycline) is used [2]. Unfortunately an expression of receptors was confirmed in approximately 30% of cells after tetracycline induction. Aim: Our goal was to increase the number of HEK293 cells with an induced expression of mGlu2 receptor using synchronisation of cell cycle. Methods: cDNA fragment encoding mGlu2 receptor was cloned into a pcDNA5/FRT/TO vector under the control of a tetracycline regulated promoter. HEK293 cells containing inducible mammalian expression system (FlpIn T-REx host cells line from Invitrogen) were treated with GeneJuice transfection reagent, and then selected by hygromycin. Synchrony of stably transfected cells was performed by means of three different synchronisation protocols. To obtain G2 /M phase block, cells were supplemented with nocodazole for 18 h. To arrest cells at the beginning of S phase a double thymidine block was used (16 h and 18 h). G0 /G1 population of cells was obtained by serum starvation for 48 h [3]. The efficacy of cell synchronisation was verified by flow cytometric analysis of DNA content after propidium iodide-staining. For evaluation a surface expression of mGlu2 receptor flow cytometer immunofluorescence staining was performed. Results: We received 45.2±0.9% cells at the beginning of S phase after double thymidine block, 60.6±0.9% cells in M phase after nocodazole treatment and 77.9±8.0% cells in G0 /G1 phase after serum starvation. Untreated cells are characterised by 52.7±1.8% cells in G0 /G1 , 31.3±0.9% in S and 15.0±1.0% in G2 /M phase of cycle. Cell synchronisation seems to have statistically
Molecular neuropsychopharmacology significant effect on mGlu2 receptor expression level [One-way ANOVA, F(3.41) = 22.8, p < 0.0001]. Moreover Dunnett’s Multiple Comparison Test analysis shows that level of mGlu2 receptor surface expression in cells after double thymidine block was significantly higher compared to receptor expression level in asynchronously growing cells (p < 0.01). In contrast, the expression level of mGlu2 receptor was significantly decreased in M phase blocked cells (p < 0.01) and cells after serum starvation (p < 0.01). Conclusions: The results indicate that modification of the inducible expression system by cell synchronisation seems to have significant effect on surface mGluR2 expression level. Reference(s) [1] Palucha, A., Pilc, A., 2007. Metabotropic glutamate receptor ligands as possible anxiolytic and antidepressant drugs. Pharmacology & Therapeutics 115, 116– 147. [2] Van Craenenbroeck, K., Vanhoenacker, P., Leysen, J.E., Haegeman, G., 2001. Evaluation of the tetracycline- and ecdysone-inducible systems for expression of neurotransmitter receptors in mammalian cells. European Journal of Neuroscience 14, 968–976. [3] Yu, J.N., Ma, S.F., Miao, D.Q., Tan, X.W., Liu, X.Y., Lu, J.H., Tan, J.H., 2006. Effects of cell cycle status on the efficiency of liposome-mediated gene transfection in mouse fetal fibroblasts. Journal of Reproduction and Development 52, 373–382.
P.1.010 The identification of protein tyrosine phosphatase, non-receptor type 1 in hippocampal modulation of food anticipatory behaviour E. Kostrzewa1 ° , L.A.W. Verhagen1 , C. Gelegen1 , H.A. van Lith2 , D.A. Collier3 , M. Mitsogiannis1 , E. de Vries1 , M. van Gestel1 , R.A. Adan1 , M.J.H. Kas1 . 1 Rudolf Magnus Institute of Neuroscience, Neuroscience and Pharmacology, Utrecht, The Netherlands; 2 Utrecht University, Faculty of Veterinary Medicine, Utrecht, The Netherlands; 3 King’s College, Institute of Psychiatry, London, United Kingdom Background: Activity based anorexia (ABA) is a rodent model of hyperactivity evoked by food restriction. The paradoxical hyperactivity during food restriction is reminiscent of that observed in patients suffering from eating disorders. One of the parameters of this model, food anticipatory activity (FAA) enables us to study neurobiological mechanisms that synchronise the timing
S11
of food availability and changes in arousal in order to be prepared for upcoming relevant environmental events [1]. Methods: To identify chromosomes contributing to FAA, 21 mouse strains from a chromosome substitution panel (host strain expressing FAA (C57BL/6J) and a donor strain lacking FAA (A/J)) were tested using the ABA model (n = 8−10 mice per strain). Subsequently, we generated an F2 population from chromosome substitution strains 2 that lacks FAA. F2 mice (n = 125) were genotyped and phenotyped to fine map a locus on mouse chromosome 2 associated with FAA. In addition, genome-wide micro-array gene expression analysis was performed to identify differentially expressed genes in the hippocampus of F2 mice selected for their high or low FAA-levels. To functionally validate the candidate gene we generated shRNA viral vector (pAAV-shbase containing GFP cassette, titer 7.8×1012 genomic copies/ml) and injected C57BL/6J mice in the dentate gyrus (DG) three weeks prior to the ABA model. We verified virus spread using ISH for GFP and assessed the potential neuronal damage by performing IHC for NeuN and ISH for miRNA124. Level of candidate gene knockdown was quantified by radioactive ISH. Animals’ body weight changes, food intake and running wheel activity (RWA) during ABA were recorded. Results: We genetically mapped a Quantitative Trait Locus on mouse chromosome 2 associated with FAA. The combined genetic fine mapping and genome-wide gene expression data revealed Ptpn1 (protein tyrosine phosphatase, non-receptor type 1) as a candidate gene for FAA. To validate this candidate gene, we performed stereotactic injections of a virus containing anti-Ptpn1 shRNA. The virus infected neurons in DG and partly in CA fields without detectable neuronal damage. The expression of Ptpn1 in DG was significantly reduced in the antiPtpn1 group in comparison to the control virus injected group (Student t-test: t(8) = 2.50, p < 0.05). There were no differences in body weight, food intake nor baseline RWA (Student t-test: t(8) = −0.493, p = 0.635) between antiPtpn1 and control virus treated mice. Similarly, total RWA during the restriction phase did not differ significantly between the groups (Student t-test: t(8) = 0.126, p = 0.903 respectively). Normalised FAA, (total FAA as a percentage of total RWA), was found to be significantly decreased in the Ptpn1 knockdown group compared to the control group (Student t-test: t(8) = 2.459, p < 0.05). A positive association was found between the level of Ptpn1 mRNA expression in the DG and the magnitude of FAA (r = 0.642, p = 0.045). Conclusions: This data confirm that FAA levels are associated with Ptpn1 gene expression in the DG. As Ptpn1 is a key regulator of insulin and leptin receptor sensitivity, these findings provide a possible mechanism through