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.
Highlights
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
Microdialysis Fluid Collection and Analysis—In this study, we combined proteomics and peptidomics analyses of striatal microdialysates to analyze both proteins and endogenous peptides processed from peptide precursors
We present a thorough characterization of the rat striatal microdialysate using a parallel peptidome and proteome profiling drawn with different MS instruments and microdialysis probes
Summary
Two-month-old male Wistar rats were studied with procedures strictly in accordance with the recommendations of the European Economic Community (86/609/EEC) for the care and use of laboratory animals. Long Peptide Investigation—This analysis was performed on samples collected in KCl condition using probes with a 20-kDa cutoff point. Six microdialysate collections from three animals were pooled, concentrated in a SpeedVac, and directly injected into an LTQ mass spectrometer. Mascot data were transferred to an in-house developed validation software for data filtering according to a significance threshold of Ͻ0.05 and the elimination of protein redundancy on the basis of proteins being evidenced by the same set or a subset of peptides. Validated hits for each analysis were transferred to a relational database This database made it possible to carry out specific queries concerning, for example, the number of spectra and/or unique peptides assigned to a given protein, best Mascot score, and best coverage. Results were deemed significant for a p value Ͻ0.05
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