The expression of Neuropeptide Y in the articular cartilage of mouse distal femur

Abstracts

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SESSION 10

POSTER-SESSION I

RELATED PEPTIDES: RF-AMIDE PEPTIDES

NEUROPEPTIDE Y1 AND Y5 RECEPTORS ACTIVATION STIMULATE AUTOPHAGIC FLUX IN MOUSE HYPOTHALAMIC NEURONS

INTRODUCTION TO NEUROPEPTIDE FF AND RELATED RF-AMIDE RECEPTORS Frédéric Simonin; Biotechnology and Cellular Signalling, CNRS UMR7242, Strasbourg University, Illkirch, France The neuropeptide FF (NPFF, FLFQPQRF-NH2) that was discovered 30 years ago belongs to the family of RF-amide neuropeptides, characterized by a conserved ArgPhe-NH2 COOH-terminal sequence. In mammals, peptides from this family target five different G proteincoupled receptors including NPFF1 and NPFF2 receptor subtypes. In this lecture, I will summarize the current knowledge on NPFF and other RF-amide related receptors from historical aspects to therapeutic opportunities. Taking advantage of most recent findings in the field, special focus will be given on molecular and pharmacological properties on RF-amide peptides and their receptors as well as their involvement in the modulation of different physiological functions including feeding, reproduction and pain. doi:10.1016/j.npep.2015.11.033

HYPOTHALAMIC NEUROPEPTIDE FF RECEPTOR-2 SIGNALING IS CRITICAL FOR THE REGULATION OF ENERGY HOMEOSTASIS Lei Zhang, I-Chieh J. Lee, Herbert Herzog; Neuroscience Division, Garvan Institute of Medical Research, Sydney NSW 2010, Australia The neuropeptide FF receptor 2 (NPFFR2) is highly expressed in the hypothalamus where it is activated by a set of RFamide peptides. However, its physiological function(s) particularly in energy homeostasis regulation is unclear. Here we show that male NPFFR2−/− mice on chow have reduced weight gain which is likely due to a reduced food intake. On the other hand, female NPFFR2−/− mice exhibited reduced adiposity and increased energy expenditure and physical activity. Although showing gender dimorphism in terms of fat deposition, phenotypes of both male and female NPFFR2−/− mice on chow are consistent with a lean phenotype. Interestingly, when fed on HFD, both male and female NPFFR2−/− show greater weight and fat gain compared to WT mice. This exacerbated diet-induced obesity in NPFFR2−/− mice is associated with reduced energy expenditure. When energy expenditure from HFD and chow studies are compared, there are significant interactions between genotype and diet effects, i.e. HFD increases energy expenditure in both NPFFR2−/− and WT mice, however this increase is significantly less in NPFFR2−/− than that in WT mice, suggesting that a lack of NPFFR2 signalling leads to an impaired diet-induced adaptive thermogenesis. In support of that, HFD significantly increases UCP-1 and PGC-1α levels in the brown adipose tissue of WT mice but not in that of NPFFR2−/− mice. In addition, tyrosine hydroxylase (TH) expression in the paraventricular nucleus of the hypothalamus (PVN), a key molecule involved in mediating brown adipose tissue thermogenesis, shows a significant increase in WT in response to HFD but not in NPFFR2−/− mice. Interestingly, while HFD significantly decreases NPY expression in the Arc in WT this counter-regulation is not seen in NPFFR2−/− mice. Taken together, these data demonstrate that NPFFR2 signalling plays important roles in the regulation of energy homeostasis with specific effects on diet-adaptive thermogenesis. doi:10.1016/j.npep.2015.11.034

Mariana Botelhoa,c,1, Célia A. Aveleiraa,1, Sara Carmo-Silvaa,c, Jorge Pascoal a, Marisa Ferreira-Marquesa, Clévio Nóbregaa, Luísa Cortesa, Jorge Valeroa, Lígia Sousa-Ferreiraa , Ana R. Álvaroa, Magda Santanaa , Sebastian Kügler b, Luís Pereira de Almeidaa,c, Cláudia Cavadasa,c,; aCNC — Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal; bCenter of Molecular Physiology of the Brain, Department of Neurology, University Medicine Göttingen, Germany; cFaculty of Pharmacy, University of Coimbra, Coimbra, Portugal 1 These authors contributed equally to this work. Caloric restriction (CR) is a robust anti-aging intervention to extend lifespan, and this effect is tied to increased autophagy. CR increases Neuropeptide Y (NPY) in the hypothalamus. NPY role on autophagy remains unknown. Therefore, the aim of this study was to investigate the role of NPY on autophagy regulation in hypothalamic neurons. Cell line of mouse hypothalamic neuron were exposed to NPY (100 nM) and autophagy flux was evaluated by measuring the autophagy biomarkers (LC3B-II and SQSTM1), by Western blotting and using live cell spinning-disk confocal microscopy with the tandem mCherry-GFP-LC3 cell-based assay. To study the involvement of NPY receptors on NPY-effect, the cells were exposed to different NPY receptors selective agonists and antagonists, and inhibitors of different intracellular pathways. Moreover, NPY–AAV was injected in mouse hypothalamic arcuate nucleus and autophagy markers were evaluated by western blot and immunohistochemistry. The results show that NPY stimulates autophagy in hypothalamic neurons through Y1 and Y5 receptors activation. Moreover, the activation of NPY Y1 receptor increases autophagic flux through PI3K and PKA signaling pathways, and NPY Y5 receptor activation enhances autophagic flux through PI3K, MEK/ERK and PKA. Since both hypothalamic autophagy and NPY levels decrease with age, modulation of NPY may act as a protective mechanism against impaired hypothalamic dysfunction associated with age and to delay aging. This work was supported by FEDER through the Programa Operacional Factores de Competitividade – COMPETE and National Funds through FCT – UID/NEU/04539/2013, PTDC/SAU-FCF/099082/2008. FCT fellowships SFRH/ BPD/73942/2010, SFRH/BD/73004/2010 and SFRH/BPD/78424/2011; QREN project “Aging, Stress and Chronic Disease: from mechanisms to therapeutics” (CENTRO-07-ST24FEDER-002006). doi:10.1016/j.npep.2015.11.035

POSTER SESSION I THE EXPRESSION OF NEUROPEPTIDE Y IN THE ARTICULAR CARTILAGE OF MOUSE DISTAL FEMUR Yuanyuan Ma; Guanghua School of Stomatology, Hospital of Stomatology, Sun Yat-sen University, Guangzhou 510055, China; Guangdong Provincial Key Laboratory of Stomatology, Guangzhou, China Neuropeptide Y (NPY) is a potential modulator of bone remodeling. We have identified the presence of NPY in the osteocytes and osteoblasts in vitro. Recently we have also found the expression of NPY in the articular cartilage of distal femur of 3-week-old C57BL/6J mice by immunohistochemistry method. In the longitudinal sections, the apparent expression of NPY is present in the matrix of epiphyseal cartilage, as well as in the immature and mature chondrocytes there. In the epiphyseal plate, the expression of NPY shows the spatial

14

Abstracts

distribution regularity. NPY is detectable in a high level in zones of reserve and proliferating cartilage. Particularly, NPY is abundant in the cytoplasm of the mature chondrocytes which range themselves in rows in the cartilage lacunae in zone of proliferating cartilage. However, there is little NPY expression in the big cartilage lacunae in zone of calcifying cartilage. It is interesting that the transitional trabecular bone, in zone of ossification, shows highly positive staining of NPY and the cells attaching to the surface of trabecular bone do as well. By the transverse sections, there are several concentric structures inside the outermost articular cartilage. In the matrix of these concentric structures, there are higher NPY expressions in both outer and inner layers than that in the middle layer. In the cartilage lacunae, we can find many NPYimmunoreactive chondrocytes. Besides, NPY is prominently present in the articular cartilage and it is also identified in the trabecular bone between the cartilage and the concentric structures. All the results reveal the spatial distribution of NPY in the articular cartilage. It is worth for further research to explore the regulating effects of NPY in the cartilage and even in the process of the endochondral ossification via the chondrocytes. doi:10.1016/j.npep.2015.11.036

POSTER SESSION I ALTERING IN THE STRUCTURE OF NEUROPEPTIDE Y DURING THE BINDING TO THE Y1 RECEPTOR INVESTIGATED BY NMR Mathias Bossea,, Anette Kaiserb, Tristan Zellmannb, Kerstin Burkertb, Rene Meierb, Peter Schmidta, Annette G. Beck-Sickingerb, Daniel Hustera; aInstitute of Medical Physics and Biophysics, University of Leipzig, Härtelstr. 16-18, D-04107 Leipzig, Germany; bInstitute of Biochemistry, University of Leipzig, Brüderstr. 34, D-04103 Leipzig, Germany G protein-coupled receptors (GPCRs) are involved in most physiological processes and have become of high interest for the research community as well as for the pharmaceutical industry. Structural studies are crucial to understand the molecular details of these processes. Unfortunately, molecular characterisation of these large transmembrane proteins is difficult. Solution nuclear magnetic resonance (NMR) spectroscopy offers the opportunity to study structural and dynamical aspects in the interaction of ligand and receptor. Using different NMR experiments we try to understand how the neuropeptide Y (NPY) bound to one of its GPCR, the neuropeptide Y receptor type 1 (Y1R). Therefore, several differently 15N/13C-labelled NPY variants were synthesized by solid phase peptide synthesis and studied bound to the receptor by NMR. The Y1R was produced recombinantly in Escherichia coli as inclusion bodies, solubilised in SDS, refolded and incorporated in DMPC/DHPC bicelles in a high micromolar concentration. We determined several changes in the NPY backbone bound to the receptor in comparison to the not bound state via recording different 1H/15N HSQC spectra by solution NMR in the presence and in the absence of the receptor. Finally, our 13C/13C correlation spectra recorded by solid state NMR indicate a change in the secondary structure of NPY bound to the receptor. Taken together, the binding of NPY to the Y1R is connected with a considerable altering in the structure of the ligand, like published for the Y2R(1) with small but crucial differences.

POSTER SESSION I P1.4 DIFFERENCES IN PHARMACOLOGY OF MONOMERIC AND DIMERIC NPY Y1 RECEPTOR FLUORESCENT ANTAGONISTS REVEALED BY HIGH CONTENT IMAGING Rachel Richardsona,, Simon Mountfordc, Oscar Liuc, Marleen Groenena, Charlie Laughtonb, Steve Briddona, Philip Thompsonc, Nick Hollidaya; a Institute of Cell Signalling, School of Life Sciences, Medical Centre, Nottingham NG7 2UH, UK; bPharmacy, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, UK; cMedicinal Chemistry, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville 3052, Australia Fluorescent ligands enable real time monitoring of ligand–receptor interactions in living cells (Vernall et al., 2014). In developing this approach for NPY receptors, we have studied monomeric and dimeric fluorescent peptides based on BVD15, a C terminal NPY analogue (Mountford et al., 2015, 2014). Here we compare Y1 pharmacology of BIDA84 ([Lys2-sulphatedCy5, Arg4]BVD15), and BIDB13, a monorhodamine derivative of the BVD15 dimer 1229U913. BIDA84 and BIDB13 bound Y1-GFP receptors in HEK293 membranes ([125I]PYY pKi 9.4 and 8.5 respectively), and were antagonists of NPY stimulated Y1 receptor ß arrestin2 recruitment (Kilpatrick et al., 2010). BIDA84 was surmountable (pKb 7.5 ± 0.2, n = 3), but the effect of BIDB13 was on-surmountable. In whole cell fluorescent ligand binding assays, BIDA84 labelling co-localised with Y1-GFP and was displaced by NPY (pIC50 7.89 ± 0.12, n = 3), BIBO3304 (8.70 ± 0.04, 4) or 1229U91 (8.75 ± 0.07, 4). IC50 values for competing ligands varied with BIDA84 concentration as predicted from competitive reversible interactions at equilibrium. BIDB13 also labelled Y1-GFP receptors selectively, but its displacement by other ligands was prevented as its concentration increased (BIBO3304 pIC50 8.27 ± 0.19 at 1 nM BIDB13, 7.48 ± 0.18 at 10 nM, b6.0 at 100 nM; n = 3–4). Molecular dynamic simulations revealed structural selection imposed on BIDA84 and BIDB13 through fluorophore addition and dimer formation. BIDA84 is therefore a high affinity fluorescent antagonist suitable for deriving quantitative Y1 receptor pharmacology. Studies of BIDB13 reveal that dimerization of the BVD15 pharmacophore generates slowly reversible receptor binding properties. RR supported by the Nottingham–Monash PhD programme. References Kilpatrick, L.E., et al., 2010. Br. J. Pharmacol. 160, 892–906. Mountford, S.J., et al., 2014. Org. Biomol. Chem. 12, 3271–3281. Mountford, S.J. et al., 2015. — this meeting. Vernall, A.J., et al., 2014. Br. J. Pharmacol. 171, 1073–1084. doi:10.1016/j.npep.2015.11.038

POSTER SESSION I STRUCTURE AND DYNAMIC SIGNALING OF THE Y2 RECEPTOR

Reference Kaiser, A., Müller, P., Zellmann, T., Scheidt, H.A., Thomas, L., Bosse, M., Meier, R., Meiler, J., Huster, D., Beck-Sickinger, A.G., Schmidt, P., 2015. Angew. Chem. Int. Ed. 54, 7446–7449. doi:10.1016/j.npep.2015.11.037

Brian Bendera,, Peter Schmidtb, Anette Kaiserc, Annette Beck-Sickingerc, Daniel Husterb, Jens Meilera; aDepartments of Pharmacology, Chemistry, and Biomedical Informatics, Center for Structural Biology and Institute of Chemical Biology, Vanderbilt University, Nashville, TN 37232, USA; b Institute of Medical Physics and Biophysics, University of Leipzig, Härtelstrasse 16-18, 04107 Leipzig, Germany; cInstitute of Biochemistry, University of Leipzig, Brüderstrasse 34, 04103 Leipzig, Germany