title: Discovering new bioactive neuropeptides in the striatum secretome using in vivo microdialysis and versatile proteomics.
abstract: 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.
authors.affiliation: 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. benoitbernay@yahoo.fr
authors.forename: Benoît
authors.initials: B
authors.lastname: Bernay
authors.forename: Marie-Claude
authors.initials: MC
authors.lastname: Gaillard
authors.forename: Vilém
authors.initials: V
authors.lastname: Guryca
authors.forename: Anouk
authors.initials: A
authors.lastname: Emadali
authors.forename: Lauriane
authors.initials: L
authors.lastname: Kuhn
authors.forename: Anne
authors.initials: A
authors.lastname: Bertrand
authors.forename: Isabelle
authors.initials: I
authors.lastname: Detraz
authors.forename: Carole
authors.initials: C
authors.lastname: Carcenac
authors.forename: Marc
authors.initials: M
authors.lastname: Savasta
authors.forename: Emmanuel
authors.initials: E
authors.lastname: Brouillet
authors.forename: Jérôme
authors.initials: J
authors.lastname: Garin
authors.forename: Jean-Marc
authors.initials: JM
authors.lastname: Elalouf
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D008297:Male; D013058:Mass Spectrometry; D008564:Membrane Potentials; D017551:Microdialysis; D008969:Molecular Sequence Data; D008970:Molecular Weight; D017072:Neostriatum / Q000378:metabolism*; D009479:Neuropeptides / Q000032:analysis* / Q000737:chemistry; D020543:Proteome / Q000032:analysis / Q000737:chemistry; D040901:Proteomics; D051381:Rats; D017208:Rats, Wistar; D055571:Secretory Pathway
chemical_list: D000596:Amino Acids; D009479:Neuropeptides; D020543:Proteome