Exploring the mitochondrial degradome by the TAILS proteomics approach in a cellular model of Parkinson’s disease

Abstract Among neurodegenerative diseases, Parkinson's disease (PD) is the second most frequent worldwide. PD is primarily characterized by the specific loss of nigral dopaminergic neurons, accompanied by the onset of cardinal motor symptoms [1]. Mitochondrial dysfunction appears to have a major contribution in neuron cells death [2,3]. Dopaminergic neurons are more sensitive to mitochondrial dysfunction than other neuron types since dopamine (DA) itself can undergo spontaneous oxidation when not correctly stored in acidic vesicles, thus fostering mitochondrial damage and oxidative stress [4]. A highly conserved system of mitochondrial proteases provides a tool to ensure the maintenance of proper mitochondrial proteins activity [5]. Hence, the use of a proteomics approach to study the complete repertoire of mitochondrial proteases (i.e., mitochondrial degradome) and their protein targets, in cellular models mimicking early pathogenetic events in PD, represents a great chance for the discovery of new candidate disease biomarkers. The human neuroblastoma SH-SY5Y cell line is widely used as a cellular model to study PD pathogenesis, since a marked increase in the cytoplasmic DA concentration can be reached upon the addition of DA to the culture medium. Using this model, we have recently collected evidence supporting the hypothesis of DA-induced activation of mitochondrial proteases [6-8]. A procedure termed TAILS (Terminal Amine Isotopic Labeling of Substrates) has been recently designed for degradomics analysis [9]. In the present work, we re-adapted the TAILS protocol to our specific aim, which is a comprehensive characterization of mitochondrial proteases and their substrates in a cellular model of altered DA homeostasis in SH-SY5Y cells, which mimics the early steps in PD pathogenesis. To this aim, SH-SY5Y cells were treated with DA (0.25 mM, 24 hours), mitochondria-enriched fractions were isolated and the TAILS bench protocol was performed as described by Overall and coworkers [9]. As results, we identified so far 62 peptides whose presence and abundance is influenced by dopamine. Among these, we found an interesting proteolytic peptide overexpressed in DA-treated cells, belonging to the mitochondrial 39S ribosomal protein L49. Using the MEROPS database, we have matched the cleavage site with that of a candidate protease, named neprilysin, that has been already associated to neurodegeneration because of its ability to degrade the amyloid beta peptide.