Department of Biology
Library of the School of Science

2022-11-29

2022

Pallaki Paschalina

Εμμανουήλ Φραγκούλης, Ομότιμος Καθηγητής Τμήματος Βιολογίας, ΕΚΠΑ
Ζαφειρούλα Γεωργούση, Ερευνήτρια Α’, ΕΚΕΦΕ «Δημόκριτος»
Διαμάντης Σίδερης, Αναπληρωτής Καθηγητής Τμήματος Βιολογίας, ΕΚΠΑ
Σπύρος Ευθυμιόπουλος, Καθηγητής Τμήματος Βιολογίας, ΕΚΠΑ
Διδώ Βασιλακοπούλου, Αναπληρώτρια Καθηγήτρια Τμήματος Βιολογίας, ΕΚΠΑ
Ιωάννης Σωτηρόπουλος, Ερευνητής Γ’, ΕΚΕΦΕ «Δημόκριτος»
Μαρία Γαϊτάνου, Ερευνήτρια Γ’, Ελληνικό Ινστιτούτο Παστέρ

Εναλλακτικά μονοπάτια της σηματοδότησης των οπιοειδών υποδοχέων που εμπλέκονται στην έκφραση γονιδίων κατά τη νευρογένεση

Greek

Alternative pathways of opioid receptors signaling involved in gene expression during neurogenesis

Opioid receptors (ORs) dynamically interact with a great variety of proteins consisting multi-functional complexes that govern various signaling pathways. These interactions alter trafficking, signaling and the fine tuning of these receptors. The current thesis focuses on the role of δ-opioid receptor (δ-OR) in neurogenesis, since δ-OR is highly expressed in brain regions (cortex, amyglada, and hippocampus) that control cognition, memory and feelings. In addition, it was found that the C-terminal region of δ-OR consists an interacting platform for a variety of proteins, apart from Gα and Gβγ subunits. Two of these interacting proteins are the Signal Transducer and Activator of Transcription 5B (STAT5B) and the Regulator of G-protein Signaling 4 (RGS4), (Leontiadis et al. 2009; Georgoussi et al 2006; 2012; Georganta et al. 2010). The Regulators of G protein signaling (RGS) negatively regulate G protein coupled receptor (GPCR) signaling and despite their initial role as GTPase activating proteins, they also exert non-canonical functions such as transcriptional regulation by interacting with various protein partners. RGS4, a member of the B/R4 family, is a multitask protein highly expressed in developing neurons and strongly associated with various neurological disorders such as Schizophrenia, stress disorders and tolerance with an unknown yet molecular mechanism. Ample evidence from our lab has previously shown that RGS4 negatively regulates opioid receptor signaling by conferring selectivity for G-protein coupling, inhibiting δ-ΟR-mediated ERK1,2 phosphorylation and accelerating DSLET-mediated receptor internalization (Georgoussi et al. 2006; Leontiadis et al. 2009).Moreover, other findings demonstrated that δ-OR forms a multiprotein signaling complex, consisting of Gi/Go proteins and the transcription factor STAT5B, that leads to neurite outgrowth and neuronal differentiation upon δ-ΟR activation (Georganta et. al 2010;2013; Georgoussi 2012). Given that RGS4 is a multitask protein expressed in the developing neurons and implicated in neurodegenerative diseases through yet unclarified mechanisms we thus intended to determine whether RGS4 could participate in signaling pathways to regulate neurotropic events upon δ-OR activation.Knowing that RGS4 and STAT5B interact at the conserved YXXL motif of the δ-CT we wondered whether the RGS4 can interact directly with STAT5B and how this interplay between RGS4 and STAT5B could regulate cellular mechanisms driving neuronal responses. The results of the present thesis demonstrate for the first time the direct interaction between RGS4 and the transcription factor STAT5B irrespective of the activation state of the receptor or state of the G-protein. The interaction between RGS4-STAT5B was also verified in the endogenous milieu of mice brains, whereas in vitro pull down experiments confirmed the interaction and further specify that the DNA-binding domain of STAT5B is responsible for the RGS4 pairing. Further functional studies reveal that RGS4 blocks STAT5B activation mediated upon δ-OR and erythropoietin receptor (EPO-R) activation. RGS4 exerts these effects by interfering in STAT5B phosphorylation, dimerization and transcriptional activation triggered by two different stimuli, DSLET and erythropoietin. In our effort to translate the biological effect of the interplay between RGS4 and STAT5B and knowing the pivotal role of STAT5B and of RGS proteins in neurogenesis and neuroprotection we study further if RGS4 is involved in neurite outgrowth and differentiation through STAT5B mediated responses. In order to answer this question, we use a neuronal cell line stably expressing δ-OR (δ-Neuro2A) as well as an in vivo loss-of-function animal model (RGS4-/-mice). Our data showed that in presence of RGS4 the DSLET-dependent neurite outgrowth of δ-Neuro2A is blocked, whereas primary cortical cultures of the RGS4-/-mice exhibit enhanced neuronal sprouting compared to wild types ones, after δ-opioid administration. Our studies also revealed a new regulatory role of RGS4 in neuronal cell proliferation since our observations showed that Neuro2A cells stably expressing the RGS4 (Neuro2A-RGS4) exhibited a significantly decreased rate of survival and proliferation as assessed by the Trypan blue and BrdU incorporation methods. Additionally, it was found that RGS4 in involved in proliferation by altering the transcriptional activity of STAT5B inducible anti-apoptotic genes such as Bcl-2 and Bcl-xl in neuronal stem cells derived from RGS4-/- mice. Further studies demonstrated that RGS4 is also implicated in neuronal homeostasis by regulating autophagy in cortical neurons possibly via a pathway involving the AKT and JNK kinases.Collectively, these findings demonstrate a non-canonical regulatory role of RGS4 in neurogenesis and neuronal homeostasis and provide novel insights into the multiple roles that RGS4 exerts in neuronal development and synaptic signaling upon opioid administration or not.

Science

Opioid receptors; neuronal cells; G-proteins; gene transcription; cell proliferation; neuronal differentiation; primary neuronal cultures

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https://pergamos.lib.uoa.gr/uoa/dl/object/3254645