Introduction
The κ-opioid receptor (κ-OR), that is distributed in the central and peripheral nervous system mediates the diverse effects of opioids ranging from pain perception, neurotransmitter release and respiratory depression to the regulation of a variety of psychiatric disorders including anxiety and addiction (1). The κ-OR and its endogenous neuropeptide, dynorphin A, were found to play a key role in modulating anxiety and stress-related behaviors. Thus, stress has been shown to increase endogenous dynorphin levels and up-regulate κ-OR signaling in the nucleus accumbens and the CA3 region of the hippocampus (2). Ablation of κ-OR from brain dopaminergic neurons produced anxiolytic effects, confirming that the regulation of dopaminergic neurotransmission by κ-OR is critical for manifestation of stress and anxiety (3).
Recent results also suggest that κ-ΟR antagonists possess promising antidepressant potential, indicating that the κ-ΟR and its endogenous neuropeptide ligand, dynorphin A, are critical mediators of stress and mood disorders with specific κ-ΟR antagonists being currently tested in phase II clinical trials (4-6). However, the signaling constituents responsible for the neurobiological responses that regulate these physiological phenomena have yet to deduced.
In the brain, the κ-OR is coupled to pertussis toxin sensitive Gi/o proteins to regulate a variety of downstream effectors including adenylyl cyclase, K+ and Ca2+channels, phospholipase C, and ERK1/2 phosphorylation (1, 7). Such diverse signaling events are mediated not only by interactions with G proteins but also by other proteins that determine the generated signal and alterations in the trafficking, targeting and fine tuning of this receptor (1, 8, 9).
Macroautophagy, herein referred as autophagy, is a highly conserved degradation process in which proteins and organelles are engulfed in autophagic vesicles and subsequently targeted for degradation in lysosomes (10). Autophagy plays an important role in many organisms upon exposure to stress but is also considered to be an important physiological mechanism in neuronal homeostasis. In neurons, autophagy occurs constitutively under physiological conditions, while impaired autophagy is implicated in many neurodevelopmental and neurodegenerative disorders (11). Recent evidence suggests that autophagy regulates the development and function of axons, dendrites and synapses, whereas aberrations in neuronal autophagy contribute to pathological changes. Autophagy alters the kinetics of neurotransmitter release and the density of synaptic vesicles and is also implicated in the degradation of postsynaptic receptors such as the GABAA and AMPA receptors (11, 12). Autophagy contributes to such alterations by degrading specific synaptic proteins involved in spine remodeling and retraction suggesting a direct link between autophagy and pruning of synaptic connections. Consequently, the targeting of neuronal autophagy may have great clinical implications in terms of treatment of various psychiatric disorders (13, 14).
Additional findings also suggest that GPCRs are direct regulators of autophagy (15). Previous studies have shown that exposure of SH-SY5Y and endothelial cells to morphine or methamphetamine respectively, induces autophagy through the involvement of opioid receptors by as yet undefined mechanisms (16, 17). Moreover, other studies have shown that morphine dysregulates synaptic balance in the hippocampus via a novel signaling pathway involving reactive oxygen species, endoplasmic reticulum stress and autophagy (18). Although opioid receptors and interacting, Gi/o and Regulators of G protein signaling (RGS) proteins, were shown to play key roles in neuronal signaling (8, 19, 20), it is unknown whether κ-OR activation by specific agonists can induce the autophagic machinery in neuronal cells and whether these effects could result in synaptic alterations implicated in stress-related behaviors.
The present study demonstrates a novel signaling pathway via which a specific representative of opioid receptors, κ-ΟR, induces autophagy resulting in synaptosomal integrity changes. In addition, we show that administration of the κ-OR specific antagonist, norBNI to mice, during acute stress exposure [daily forced swim test (FST)], prevents autophagy induction and stress-induced degradation of synaptic proteins. These results provide a novel insight to the role of this receptor in the regulation of neuronal autophagy and demonstrate that κ-ΟR-mediated autophagy is responsible for specific changes in stress-induced synaptic alterations.