Discussion
Previous studies have shown that
in the brain the κ-OR is involved in motivation, stress-related
responses and adult neurogenesis and that κ-ΟR agonists and antagonists
exert potent pro- and anti-depressant effects, respectively, in rodents
(6, 26).
However, the mechanistic details of the aberrant synaptic function and
resulting behavior mediated by κ-OR upon stress remain elusive. In the
present study, we demonstrate that κ-OR plays a role in stress-induced
autophagy, which leads to synaptic alterations. κ-ΟR-induced autophagy
occurs primarily in the hippocampus despite κ-ΟR’s high expression
levels in cortex and striatum. Gi/o proteins and U50,488H-induced ERK1,2
activation are responsible for κ-OR-mediated autophagy. An intriguing
finding of the present study has been that U50,488H-dependent-CREB
activation regulates the transcription of the becn1 gene,
confirming that κ-ΟR activation leads to transcriptional induction of
specific autophagic genes. We are thus proposing a putative G protein
dependent signaling pathway for the control of autophagy by κ-ΟR.
Activated κ-OR leads to activation of ERK1,2 which phosphorylates CREB,
with the later promoting alterations of autophagic genes leading to
synaptic protein changes (Fig. 8). This proposal is in agreement with
previous observations which have shown that heterotrimeric G proteins
control autophagic sequestration in HT-29 cells
(27, 28),
and that Gαi3, which is activated by κ-OR
(8), plays a crucial role in
autophagosomal membrane compartmentalization
(29) and autophagy initiation
(30). It is also compatible with previous
findings suggesting that a dynamic interplay between Gαi3, the activator
of G-protein signaling 3 and Gα-interacting vesicle-associated protein
(GIV), are signaling components that determine whether autophagy is
induced or inhibited (31), and that Gαi3
interacts with RGS4 to inhibit autophagy
(32).
A number of different GPCRs have been shown to regulate autophagy albeit
through different mechanisms (15). Thus,
dopamine D2 and D3 receptors were shown to be positive regulators of
autophagy, involving the Akt-mTOR and AMP-activated protein kinase
(AMPK) signaling pathways (33).
Similarly, activation of the β2-adrenergic receptor upregulates
autophagy and increases collagen degradation in order to maintain
cardiac extracellular matrix homeostasis
(34). Methamphetamine exposure also
induces autophagy via the κ-OR as a pro-survival response against
apoptotic endothelial cell death, an effect that is also mediated by
ERK1,2 activation and inactivation of the Akt/mTOR pathway
(16). In the mouse hippocampus, acute or
chronic morphine administration upregulates autophagic flux as
protective response towards morphine-induced neuronal death and
consequent spatial memory deficits (35).
Moreover, chronic morphine administration alters synaptic plasticity in
the hippocampus and results in spine and excitatory synapse density
reduction, via generation of reactive oxygen species leading to
Endoplasmic Reticulum (ER) stress activation
(18). Finally, it was noted that
morphine-mediated autophagy involves activation of ER stress with
subsequent downstream astrocyte activation via the μ-opioid receptor
(36), a finding confirming that opioids
are indeed potential positive regulators of autophagy.
The immediate responses to stressful stimuli include neuro-morphological
changes in multiple brain areas including the hippocampus
(37). Acute stress reduces the density of
dendritic spines, alters the location of postsynaptic elements of
excitatory synapses, and impairs long-term potentiation and memory
(14, 37).
Chronic stress has been reported to enhance autophagy in rodents
(38, 39).
Furthermore, a role for autophagy in depressive-like behaviors and
cognitive impairment has been demonstrated following prenatal stress
(40). Our current findings extend the
existing evidence by demonstrating a plausible scenario whereas the
dynorphin/κ-ΟR system initiates autophagy, which leads to stress-induced
synaptic alterations.
Neuronal autophagy plays a major role in brain function by modulating
synaptic organization and morphogenesis
(12, 13).
It contributes to synaptic plasticity by degrading specific synaptic
proteins such as PSD-95, PICK1 and SHANK3, which play important roles in
synaptic function and spine modeling. This implies a direct link between
autophagy and pruning of synaptic connections during postnatal
development (13). In agreement with these
predictions, our findings demonstrate that U50,488H-κ-ΟR activation of
primary neuronal hippocampal cultures reduces the number of neurite
branches. In addition, sub-chronic U50,488H administration in mice led
to degradation of the key scaffolding synaptic proteins, spinophilin,
PSD95 and SNAP25, particularly in the hippocampus, but not in the cortex
or striatum. We specifically chose to examine these proteins as they are
implicated in dendritic spine remodeling. Spinophilin localizes in the
postsynaptic compartment, is enriched in dendritic spines, and modulates
spine morphogenesis and maturation through the regulation of the actin
cytoskeleton (41). It also interacts
directly with opioid receptors and other GPCRs to regulate their
trafficking and signaling that leads to synaptic alterations
(41-43). On the other hand, SNAP-25 plays
a crucial role pre-synaptically by mediating synaptic vesicle fusion. Of
note is that SNAP-25 and PSD-95 are substrates of autophagic degradation
modulating dendritic spine morphology and function
(44) .
Stress blocks long term potentiation through release of endogenous
opioids including the release of the endogenous κ-opioid neuropeptide,
dynorphin. Activation of κ-ΟR in vivo promotes aversion,
dysphoria, depression, and anxiety-like behaviors
(5, 6,
45, 46).
Conversely, κ-OR antagonists prevent many effects of stress and
counteract stress-induced behavioral responses and for this reason, are
considered as novel therapeutics for stress-related disorders
(4, 47).
Forced swim stress in rats elevates dynorphin A levels in the
hippocampus [2], while chronic autophagy deficiency in dopamine
neurons results in increased size of axon profiles, increased evoked
dopamine release and rapid presynaptic recovery [12]. Another
interesting finding of the present study has been that FST in mice
promoted autophagy, as indicated by the elevated levels of autophagic
markers and this effect was prevented by administration of the κ-OR
antagonist, nor-BNI. This suggests that the endogenous dynorphin/κ-OR
system is involved in stress-induced autophagy and could be part of the
orchestration of structural changes observed in the hippocampus under
stress exposure. Interestingly, a concomitant decrease of the three
synaptic proteins, spinophilin, PSD-95 and SNAP-25, was also detected in
the hippocampus but not the cortex of stressed animals. We thus
postulate that degradation of these synaptic proteins could be
attributed to their engulfment in the autophagosome. These findings are
further supported by the observation that LC3 interacts with these three
key protein substrates that possess putative LIR motifs in their
sequence. In turn, this suggests that under acute stress, the release of
dynorphin triggers the autophagic machinery leading to synaptosomal
alterations in the hippocampus. It is interesting to note that similar
synaptosomal alterations that are crucial for dendritic spine remodeling
and are caused by autophagic degradation have also been reported
previously under conditions of nutritional stress, through a
BDNF-regulated mechanism (13). Autophagy
was also reported to play a crucial role in postnatal spine pruning in
layer V pyramidal neurons (48),
suggesting that it plays a significant role in synaptic organization and
morphogenesis.
Based on the present findings, we conclude that an interplay exists
between κ-ΟR-mediated autophagy and stress-mediated synaptosomal
alterations. Indeed, we propose the existence a signaling pathway (Fig.
8) correlating κ-OR-induced autophagy in neurons with synaptic
hippocampal alterations under stress conditions. This κ-OR-mediated
autophagy mechanism results in synaptic dysfunction in hippocampus that
may contribute to the cognitive changes observed upon stress exposure.
Given that κ-OR antagonists (47,
49) are in phase II clinical trials for
stress-related mood and anxiety disorders, it would be interesting to
explore whether these drugs effectively alleviate stress-related
pathologies via κ-OR-mediated-autophagy.