New approaches to the regulation and function of κ-opioid receptor

Doctoral Dissertation uoadl:3260278 52 Read counter

Unit:
Department of Biology
Library of the School of Science
Deposit date:
2023-02-01
Year:
2023
Author:
Karousiotis Christos
Dissertation committee:
Kωνσταντινος Βοργιάς, Καθηγητής Τμήματος Βιολογίας, ΕΚΠΑ
Ζαφειρούλα Γεωργούση, Ερευνήτρια Α’, ΕΚΕΦΕ «Δημόκριτος»
Ουρανία Τσιτσιλώνη, Καθηγήτρια Τμήματος Βιολογίας, ΕΚΠΑ
Σπύρος Ευθυμιόπουλος, Καθηγητής Τμήματος Βιολογίας, ΕΚΠΑ
Διδώ Βασιλακοπούλου, Αναπληρώτρια Καθηγήτρια Τμήματος Βιολογίας, ΕΚΠΑ
Δημήτρης Κλέτσας, Διευθυντής ερευνών ΙΒΕ, ΕΚΕΦΕ «Δημόκριτος»
Ιωάννης Σωτηρόπουλος, Ερευνητής Γ’, ΕΚΕΦΕ «Δημόκριτος»
Original Title:
’ΝΕΕΣ ΠΡΟΣΕΓΓΙΣΕΙΣ ΣΤΗ ΡΥΘΜΙΣΗ ΚΑΙ ΛΕΙΤΟΥΡΓΙΑ ΤΟΥ κ-ΟΠΙΟΕΙΔΟΥΣ ΥΠΟΔΟΧΕΑ
Languages:
Greek
Translated title:
New approaches to the regulation and function of κ-opioid receptor
Summary:
The molecular mechanisms that govern anxiety, depression and associated stress-related neural responses and pathologies are poorly understood. Opioid receptors (μ-ΟR, δ-ΟR and κ-OR) regulate motivational processes and are important players in psychiatric disorders. Accumulating evidence suggests that κ-ΟR antagonists and δ-ΟR agonists exert promising antidepressant potential. Consistent with these findings the κ-ΟR and its endogenous neuropeptide ligand dynorphin A were found to play a key role in modulating stress and mood and specific κ-ΟR antagonists are currently in phase III clinical trials. Opioid receptors and their complex signalling are known to regulate neuronal plasticity and neurotransmission; however, the underlying mechanisms remain as yet, unclarified. A growing body of evidence indicates that systemic administration of κ-ΟR ligands produces mood altering effects in classical models of depression. Notably, κ-ΟR agonists and antagonists showed depressant and antidepressant effects respectively, in the forced swim test and learned helplessness in rats. Stress blocks LTP through release of endogenous opioids, a finding supporting
previous observations that stress is accompanied by release of the endogenous κ-OR agonist dynorphin and subsequent activation of the κ-ΟR with an as yet unknown mechanism. Emerging evidence suggests that G protein-coupled receptors (GPCRs) are direct sensors involved in regulation of the autophagic machinery while autophagy is recently shown to regulate synaptic morphogenesis and function. Αutophagy is a regulated process for degradation of cytosolic components and organelles through delivery to lysosomes. Its primary role is to ensure cell survival under stress conditions. In neurons, basal levels of autophagy help to control the cellular quality of proteins and protect cells from protein aggregation. Recent results suggest that autophagy regulates the development and function of axons, dendrites and synapses whereas, insufficient or excessive neuronal autophagy contributes to pathological changes in these structures. Autophagy alters the kinetics and neurotransmitter release and the density of synaptic vesicles. The modulation of vesicle numbers or evoked neurotransmitter release by presynaptic autophagy may contribute to synaptic plasticity mechanisms such as synaptic potentiation and depression. Autophagy has also been implicated in the degradation of postsynaptic receptors such as GABAA and AMPA receptors. Moreover, it was previously demonstrated that activation of the δ-ΟR with selective agonists is implicated in the regulation of neuronal differentiation, neurite outgrowth and survival through the formation of a dynamic protein complex “signalosome” composed of Gi/o, RGS proteins transcription factors and the neuronal protein spinophilin. Using in vitro and in vivo studies, this study aims to clarify the potential role of autophagy and κ-opioid receptor (κ-OR) signaling on synaptic structure and integrity. We hereby demonstrate that the selective κ-opioid receptor (κ-OR) agonist, U50,488H and the endogenous neuropeptide dynorphin induce autophagy in a time-and dose-dependent manner in neuronal cells by upregulating microtubule-associated protein light chain 3-II (LC3-II), Beclin 1, Atg5 and reducing p62 protein levels. Moreover, pre-treatment of neuronal cells with pertussis toxin blocked the above κ-OR-mediated cellular changes, indicating the involvement of Gi/o proteins in the κ-OR effects. Our molecular analysis also revealed a κ-OR-driven upregulation of becn1 gene through ERK1,2-dependent activation of the transcription factor CREB in neuronal cells. Our in vivo studies demonstrate that mice treated with U50,488H displayed profound increases of specific autophagic markers in the hippocampus with a concomitant decrease of several pre- and post-synaptic proteins such as spinophilin, PSD-95 and SNAP25, which interact with LC3, suggesting that these proteins are plausibly engulfed in the κ-OR-induced autophagic cargo. Αlso, using acute stress (force swim), a stimulus known to trigger synaptic loss and also increase levels of the natural ligand of κ-OR, dynorphin, we demonstrated that the administration of the κ-ΟR selective antagonist, nor-binaltorhimine (norBNI) before stress blocked the stress-evoked reduction of synaptic protein and the accompanied induction of autophagy in the hippocampus. Finally, pre-treatment of neuronal cells with the κ-ΟR antagonist CERC-501, which is at clinical phase III as anxiolytic drug, inhibits κ-OR mediated autophagy in vitro. These findings provide novel insights about the essential role of autophagic machinery into the mechanisms through which κ-OR and its signaling regulated brain plasticity. Elucidating these molecular mechanisms will revolutionize disease diagnosis, allow the identification of new therapeutic targets and lead to the discovery of novel therapeutic agents and effective treatments.
Main subject category:
Science
Keywords:
opioids, receptor, autophagy, stress, synaptic proteins, G proteins, κ-opioid receptor
Index:
Yes
Number of index pages:
3
Contains images:
Yes
Number of references:
290
Number of pages:
224
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