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Molecular & Cellular Proteomics 8:946-958, 2009.
© 2009 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Discovering New Bioactive Neuropeptides in the Striatum Secretome Using in Vivo Microdialysis and Versatile Proteomics ^S

Benoît Bernay^a,b,c,d, Marie-Claude Gaillard^a, Vilém Gurya^b,e,f, Anouk Emadali^b,e,f, Lauriane Kuhn^b,e,f, Anne Bertrand^f,g, Isabelle Detraz^f,g, Carole Carcenac^f,g, Marc Savasta^f,g,h, Emmanuel Brouillet^c,i, Jérôme Garin^b,e,f and Jean-Marc Elalouf^a,j

From the ^a Laboratoire de PhysioGénomique, Service de Biologie Intégrative et Génétique Moléculaire (SBIGeM), Institut de Biologie et de Technologies de Saclay (iBiTec-S), Commissariat à l'Energie Atomique (CEA), F-91191 Gif-sur-Yvette, France, ^b Laboratoire d'Etude de la Dynamique des Protéomes (LEDyP), Institut de Recherches en Technoloqies et Sciences pour le Vivant (RTSV), CEA, F-38054 Grenoble, France, ^c Molecular Imaging Research Center (MIRCen), Institut d'Imagerie Biomédicale (I2BM), 18 route du Panorama, BP6, F-92265 Fontenay-aux-Roses, F-91401 Orsay, France, ^e INSERM U880, F-38054 Grenoble, France, ^f Université Joseph Fourier, F-38054 Grenoble, France, ^g Equipe Dynamique des Réseaux Neuronaux du Mouvement, INSERM U836, Institut des Neurosciences, F-38043 Grenoble, Cedex 09, France, ^h Centre Hospitalier Universitaire de Grenoble, F-38043 Grenoble, Cedex 09, France, and ⁱ Unité de Recherche Associée CEA-CNRS 2210, 18 route du Panorama, BP6, F-92265 Fontenay-aux-Roses, F-91401 Orsay, France

The striatum, a major component of the brain basal nuclei, is central for planning and executing voluntary movements and undergoes lesions in neurodegenerative disorders such as Huntington disease. To perform highly integrated tasks, the striatum relies on a complex network of communication within and between brain regions with a key role devoted to secreted molecules. To characterize the rat striatum secretome, we combined in vivo microdialysis together with proteomics analysis of trypsin digests and peptidomics studies of native fragments. This versatile approach, carried out using different microdialysis probes and mass spectrometer devices, allowed evidencing with high confidence the expression of 88 proteins and 100 processed peptides. Their secretory pathways were predicted by in silico analysis. Whereas high molecular weight proteins were mainly secreted by the classical mode (94%), low molecular weight proteins equally used classical and non-classical modes (53 and 47%, respectively). In addition, our results suggested alternative secretion mechanisms not predicted by bioinformatics tools. Based on spectrum counting, we performed a relative quantification of secreted proteins and peptides in both basal and neuronal depolarization conditions. This allowed detecting a series of neuropeptide precursors and a 6-fold increase for neurosecretory protein VGF and proenkephalin (PENK) levels. A focused investigation and a long peptide experiment led to the identification of new secreted non-opioid PENK peptides, referred to as PENK 114–133, PENK 239–260, and PENK 143–185. Moreover we showed that injecting synthetic PENK 114–133 and PENK 239–260 into the striatum robustly increased glutamate release in this region. Thus, the combination of microdialysis and versatile proteomics methods shed new light on the secreted protein repertoire and evidenced novel neuropeptide transmitters.

^j To whom correspondence may be addressed: Laboratoire de PhysioGénomique, Bâtiment 144, SBIGeM, iBiTec-S, CEA, F-91191 Gif-sur-Yvette, France. Tel.: 33-169088022; Fax: 33-169084712; E-mail: jean-marc.elalouf{at}cea.fr

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