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.

Fig. 1.

Experimental strategy. Illustration of the various collection and preparative steps for MS analysis of the striatal microdialysate. CSF, cerebrospinal fluid.

Fig. 2.

Venn diagrams of striatum microdialysate data. The number of proteins (A) and peptides (B) detected by Q-TOF (dark) and Orbitrap (light) analysis is shown. Data obtained with 20- and 100-kDa-cutoff probes were combined for this analysis. C, number of proteins detected in microdialysates sampled with 20-kDa (dark) or 100-kDa (light)-cutoff probes. D, number of endogenous peptides detected in peptidomics analysis by Q-TOF (dark) and Orbitrap (light) analysis of samples collected with 20-kDa-cutoff probes.

Fig. 3.

SC of microdialysate sampled with 20- or 100-kDa cutoff points. SC was obtained from proteome and peptidome data. Proteome, samples collected with either 100- or 20-kDa-cutoff membranes and undergoing trypsin digestion before MS analysis. Peptidome, samples collected with 20-kDa-cutoff membranes and submitted to MS analysis without trypsin digestion. For the 100-kDa proteome, six biological samples were analyzed. For the 20-kDa proteome and peptidome, three biological samples were studied. SC was grouped from cellular localization (A) and molecular function (B).

Fig. 4.

Molecular function of endogenous peptides. Spectrum counting was carried out on samples collected in aCSF or KCl condition using 20-kDa-cutoff probes.

Fig. 5.

Spectrum counting of neuropeptide precursors. Samples were collected in aCSF or KCl condition using 20-kDa-cutoff probes. SC was normalized by total SC and averaged from six analyses. Each error bar corresponds to the standard error of the mean. *, p < 0.05 by Mann and Whitney test.

Fig. 6.

Identification of new PENK neuropeptides. Q-TOF MS/MS spectra of MH²⁺ MDELYPVEPEEEANGGEILA (PENK 114–133) (A) and MH²⁺ FAESLPSDEEGESYSKEVPEME (PENK 239–260) (B) from microdialysate sampled in KCl condition are shown.

Fig. 7.

Long peptide analysis of a new PENK neuropeptide. MS (A) and MS/MS (B) spectra of the peptide DADEGDTLANSSDLLKELLGTGDNRAKDSHQQESTNNDEDSTS (PENK 143–185) detected in microdialysate sampled in KCl condition are shown.

Fig. 8.

Effect of peptides infusion on amino acid concentrations in the striatum. Changes, recorded at 10-min intervals, in DOPAC, dopamine, HVA, glutamate (GLU), and GABA concentrations during the delivery (—) of 10⁻⁴ m peptides PENK 239–260 or PENK 114–133 through the dialysis probe. Results are expressed as percentage of base line. Each error bar corresponds to the standard error of the mean. Asterisks indicate statistically significant changes (*, p < 0.05; **, p < 0.01; ***, p < 0.005) in analysis of variance and Fisher's post hoc least significant difference test for comparisons with the base line. Each point is the mean value obtained from three to four animals.