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 In mammalian brain, the striatum plays a critical role for planning and executing voluntary movements and is also involved in cognitive processes (1). The striatum makes use of a complex network architecture connecting specialized anatomical structures to achieve these highly integrated tasks. It receives projections from primary sensory and motor cortices as well as motor thalamic nuclei and sends projections to downstream basal ganglia structures, thereby influencing the control of cortical and brainstem motor systems (2). In this context, communication within and between brain structures appears as a key element for brain functioning. For cell-to-cell communication, secreted proteins play a pivotal regulatory role. To enter the secretory pathway, it has been long assumed that an N-terminal signal peptide sequence is strictly required. However, recent studies have shown that endoplasmic reticulum-and Golgi-independent or non-classical mechanisms may be responsible for protein secretion (3). The extracellular medium is thus more complex than previously suspected, and its characterization has gained a special interest (4, 5). In silico analyses suggest that mature proteins secreted via classical and non-classical mechanisms share common physicochemical properties (6). In this respect, proteomics is a powerful approach for systematically analyzing proteins present in the extracellular medium (7-9). For neurochemical monitoring of the secretome within the brain, only a few tools provide an appropriate insight into its spatial and temporal dynamics. Microdialysis, in particular, has been shown to be a powerful tool for exploring the extracellular content o...

VGF: An inducible gene product, precursor of a diverse array of neuro-endocrine peptides and tissue-specific disease biomarkers

“…Interestingly, in vivo neuronal depolarization of the rat striatum released a small number of VGF peptides, partly related to fragments found in the cerebro-spinal fluid ( Fig. 3: Bernay et al, 2009). In optical pathways, VGF mRNA and protein were strongly expressed in late gestation and early postnatal rat, especially in the dorsal lateral geniculate nucleus (Lombardo et al, 1995).…”

“…A range of VGF fragments were shown, some of which were parallel to VGF peptides released in the striatum secretome upon stimulation (see Fig. 3, ''in vivo release'': Bernay et al, 2009). …”

“…1) yields diverse VGF peptides, including some of low MW which are preferentially released by various stimuli both in vitro (Possenti et al, 1989(Possenti et al, , 1999Trani et al, 1995Trani et al, , 2002 and in vivo (Bernay et al, 2009). Peptides endowed with so far demonstrated biological activity are summarized in Fig.…”

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