Μηχανισμοί διαβίβασης σήματος στη νόσο του Πάρκινσον

Over the last years it has become increasingly clear that the protein levels of α-synuclein (α-syn) appear to be critical in the pathogenesis of Parkinson’s disease (PD). Recent evidence also suggests that specific extracellular α-syn strains are implicated in the progression of PD pathology. It is, therefore, plausible that deregulation in the normal processing of secreted α-syn may be a causative risk factor for PD. In this capacity, elucidation of the underlying mechanisms that regulate the protein levels of extracellular α-syn becomes essential. To date, the degradation mechanisms involved have received very little attention. Here, we sought to investigate factors and mechanisms that regulate the extracellular levels of α-syn exploiting a series of enzymatic, biochemical and proteomic approaches. As a source of secreted α-syn we used conditioned media from inducible SH-SY5Y cells overexpressing and secreting this protein under biologically relevant conditions. Using kallikrein-related peptidase 6 (KLK6), an extracellular enzyme known to cleave recombinant α-syn, we demonstrate, for the first time, that unlike recombinant forms, naturally secreted α-syn forms (wild-type & A53T) are resistant to direct KLK6 proteolysis. This differential susceptibility appears to be partially due to the non-covalent association of secreted α-syn with lipids. We further provide evidence that secreted α-syn can be cleaved by KLK6 indirectly through activation of a secreted metalloprotease, suggestive of the involvement of a proteolytic cascade in the catabolism of secreted α-syn. Our results clearly suggest that physiologic modifications affect the biochemical behavior of secreted α-syn and provide novel insights into mechanisms and potential targets for therapeutic interventions.Κατά τα τελευταία χρόνια έχει καταστεί ολοένα και πιο σαφές ότι τα πρωτεϊνικά επίπεδα της α-συνουκλεΐνης παίζουν σημαντικό ρόλο στην παθογένεια της νόσου του Πάρκινσον. Πρόσφατα στοιχεία δείχνουν επίσης ότι συγκεκριμένες μορφές της εξωκυττάριας α-συνουκλεΐνης εμπλέκονται στην εξέλιξη της παθολογίας της νόσου. Ως εκ τούτου, είναι εύλογο ότι η απορρύθμιση των φυσιολογικών επιπέδων της εξωκυττάριας α-συνουκλεΐνης μπορεί να αποτελεί έναν αιτιολογικό παράγοντα κινδύνου στη νόσο του Πάρκινσον. Συνεπώς, η διαλεύκανση των υποκείμενων μηχανισμών που ρυθμίζουν τα επίπεδα της εξωκυττάριας α-συνουκλεΐνης γίνεται απαραίτητη. Μέχρι σήμερα, οι αποικοδομητικοί μηχανισμοί που εμπλέκονται έχουν λάβει πολύ λίγη προσοχή. Στη παρούσα μελέτη, επιδιώξαμε να διερευνήσουμε παράγοντες και μηχανισμούς που ρυθμίζουν τα επίπεδα της εξωκυττάριας α-συνουκλεΐνης αξιοποιώντας μια σειρά ενζυμικών, βιοχημικών και πρωτεομικών προσεγγίσεων. Ως πηγή εξωκυττάριας α-συνουκλεΐνης χρησιμοποιήσαμε το θρεπτικό μέσο επαγόμενων SH-SY5Y κυττάρων που υπερεκφράζουν και εκκρίνουν την πρωτεΐνη αυτή σε φυσιολογικές συγκεντρώσεις. Χρησιμοποιώντας την καλλικρεΐνη 6, ένα εξωκυττάριο ένζυμο που είναι γνωστό ότι πρωτεολύει την ανασυνδυασμένη α-συνουκλεΐνη, δείξαμε για πρώτη φορά, ότι σε αντίθεση με τις ανασυνδυασμένες μορφές, οι φυσικά εκκρινόμενες μορφές της α-συνουκλεΐνης (αγρίου τύπου & A53T) είναι ανθεκτικές σε απευθείας πρωτεόλυση από το ένζυμο αυτό. Αυτή η διαφορική επιδεκτικότητα φαίνεται να οφείλεται, εν μέρει, στη μη ομοιοπολική σύνδεση της εκκρινόμενης α-συνουκλεΐνης με λιπίδια. Στην συνέχεια, δείξαμε ότι η εκκρινόμενη α-συνουκλεΐνη μπορεί να πρωτεολυθεί από την καλλικρεΐνη 6 εμμέσως και συγκεκριμένα μέσω της ενεργοποίησης μιας εκκρινόμενης μεταλλοπρωτεάσης, υποδηλώνοντας τη συμμετοχή ενός πρωτεολυτικού μονοπατιού στον καταβολισμό της εκκρινόμενης α-συνουκλεΐνης. Τα αποτελέσματά αυτά, υποδηλώνουν ξεκάθαρα ότι οι φυσιολογικές τροποποιήσεις της εκκρινόμενης α-συνουκλεΐνης επηρεάζουν την βιοχημική συμπεριφορά της και παρέχουν νέες γνώσεις για μηχανισμούς και πιθανούς στόχους για θεραπευτικές παρεμβάσεις

Resistance of naturally secreted a-synuclein to proteolysis

Recent evidence suggests that specific extracellular ?-synuclein (?-syn) strains are implicated in the progression of Parkinson's disease (PD) pathology. It is plausible that deregulation in the normal processing of secreted ?-syn may be a causative risk factor for PD. To date, the degradation mechanisms involved have received very little attention. Here, we sought to investigate factors that regulate extracellular ?-syn levels. We show, for the first time, that cell-secreted ?-syn forms are resistant to direct proteolysis by kallikrein-related peptidase 6 (KLK6), an extracellular enzyme known to cleave recombinant ?-syn. This differential susceptibility appears to be partially due to the association of secreted ?-syn with lipids. We further provide evidence that secreted ?-syn can be cleaved by KLK6 indirectly through activation of a secreted metalloprotease, suggestive of the involvement of a proteolytic cascade in the catabolism of secreted ?-syn. Our results clearly suggest that physiological modifications affect the biochemical behavior of secreted ?-syn and provide novel insights into mechanisms and potential targets for therapeutic intervention

Resistance of naturally secreted alpha-synuclein to proteolysis

Recent evidence suggests that specific extracellular alpha-synuclein (alpha-syn) strains are implicated in the progression of Parkinson’s disease (PD) pathology. It is plausible that deregulation in the normal processing of secreted alpha-syn may be a causative risk factor for PD. To date, the degradation mechanisms involved have received very little attention. Here, we sought to investigate factors that regulate extracellular alpha-syn levels. We show, for the first time, that cell-secreted alpha-syn forms are resistant to direct proteolysis by kallikrein-related peptidase 6 (KLK6), an extracellular enzyme known to cleave recombinant alpha-syn. This differential susceptibility appears to be partially due to the association of secreted alpha-syn with lipids. We further provide evidence that secreted alpha-syn can be cleaved by KLK6 indirectly through activation of a secreted metalloprotease, suggestive of the involvement of a proteolytic cascade in the catabolism of secreted alpha-syn. Our results clearly suggest that physiological modifications affect the biochemical behavior of secreted alpha-syn and provide novel insights into mechanisms and potential targets for therapeutic interventions

CRH Promotes the Neurogenic Activity of Neural Stem Cells in the Adult Hippocampus

Local cues in the adult neurogenic niches dynamically regulate homeostasis in neural stem cells, whereas their identity and associated molecular mechanisms remain poorly understood. Here, we show that corticotropin-releasing hormone (CRH), the major mediator of mammalian stress response and a key neuromodulator in the adult brain, is necessary for hippocampal neural stem cell (hiNSC) activity under physiological conditions. In particular, we demonstrate functionality of the CRH/CRH receptor (CRHR) system in mouse hiNSCs and conserved expression in humans. Most important, we show that genetic deficiency of CRH impairs hippocampal neurogenesis, affects spatial memory, and compromises hiNSCs' responsiveness to environmental stimuli. These deficits have been partially restored by virus-mediated CRH expression. Additionally, we provide evidence that local disruption of the CRH/CRHR system reduces neurogenesis, while exposure of adult hiNSCs to CRH promotes neurogenic activity via BMP4 suppression. Our findings suggest a critical role of CRH in adult neurogenesis, independently of its stress-related systemic function

Single-cell transcriptomic profiling of the aging mouse brain

The mammalian brain is complex, with multiple cell types performing a variety of diverse functions, but exactly how each cell type is affected in aging remains largely unknown. Here we performed a single-cell transcriptomic analysis of young and old mouse brains. We provide comprehensive datasets of aging-related genes, pathways and ligand–receptor interactions in nearly all brain cell types. Our analysis identified gene signatures that vary in a coordinated manner across cell types and gene sets that are regulated in a cell-type specific manner, even at times in opposite directions. These data reveal that aging, rather than inducing a universal program, drives a distinct transcriptional course in each cell population, and they highlight key molecular processes, including ribosome biogenesis, underlying brain aging. Overall, these large-scale datasets (accessible online at https://portals.broadinstitute.org/single_cell/study/aging-mouse-brain) provide a resource for the neuroscience community that will facilitate additional discoveries directed towards understanding and modifying the aging process