Results
Selective κ-ΟR agonists induce the autophagic flux in neuronal
cells. It is known that neuronal autophagy is involved in cell growth,
survival and synaptic plasticity (11,
13). Here, we investigated whether
specific κ-OR agonists could trigger autophagy in neuronal cells and
modulate synaptic organization. We thus treated Neuro-2A cells, stably
expressing κ-ΟR, with U50,488H, a κ-OR-specific agonist, and monitored
the levels of the lipidated LC3 (LC3-II), a reliable and specific marker
of autophagosome formation located at the membrane of the autophagosome.
As shown in Fig. 1A, increasing concentrations of U50,488H for 6 h
caused a dose-dependent increase in LC3-II accumulation. Addition of the
lysosomal inhibitor bafilomycin A1 (BafA1), which prevents the fusion of
autophagosomes with lysosomes indicated a significant increase in LC3-II
levels in cells exposed simultaneously to U50,488H and BafA1, relative
to BafA1 alone (Fig. 1B), indicating that κ-OR activation upregulates
the autophagic flux. Finally, LC3-II accumulation was markedly reversed
upon treatment with the opioid antagonist, naloxone, prior to U50,488H
exposure, further confirming the κ-ΟR-dependent autophagic activation
(Fig. 1C).
To recapitulate U50,488H-mediated autophagy in a native neuronal milieu,
we treated cortical neuronal cultures to U50,488H, that resulted in a
significant increase of LC3-positive autophagosomes that appeared as
puncta, compared with untreated controls (Fig. 1D, images a, e); an
effect that was abrogated when cells were pre-treated with naloxone
(images e, i). Consistent with these findings, immunoblot analysis of
primary neuronal cultures showed that naloxone blocked the increase in
LC3-II accumulation caused by U50, 488H exposure (Fig. 1E).
Collectively, these results demonstrate that κ-OR activation induces
autophagy in neuronal cells.
To deduce whether other κ-OR agonists exert similar effects on autophagy
initiation, we exposed Neuro-2A cells to varying concentrations of the
endogenous κ-OR neuropeptide dynorphin1-13, which also
resulted in increased LC3-II and Beclin 1 levels (Fig. 2A, B). This
effect was blocked by the selective κ-ΟR antagonist nor-BNI (Fig. 2C).
Finally, dynorphin1-13-treated primary neuronal cultures
indicated an increase of LC3-positive puncta compared to control neurons
(Fig. 2D compare images a with e). These data suggest that κ-OR-induced
autophagy is not selective to U50,488H, but can also be mediated by the
endogenous κ-ΟR neuropeptide dynorphin.
Because a key initial event of the autophagosome biogenesis is the
formation of the pre-autophagosomal structure (PAS), composed of ULK1,
which is a complex of a serine/threonine protein, with the focal
adhesion kinase (FIP200) and other proteins, we examined the timing of
U50,488H-mediated early autophagic events. Treatment of primary neuronal
cultures with U50,488H for 1, 6, and 24 h, triggered a marked increase
of FIP200 and ULK1 protein levels reaching a peak at 6 h agonist
exposure (Fig. 3A). In parallel, U50,488H treatment of Neuro-2A cells
for various time intervals indicated a time-dependent increase of
Beclin1, a key mediator of autophagosome formation. This increase peaked
at 6 h following U50,488H administration (Fig. 3B). Similarly, as shown
in Fig. 3C, exposure of Neuro-2A cells to U50,488H increased the protein
levels of ATG5 and Beclin 1, with a concomitant decrease of p62 known to
increase when autophagy is inhibited and decrease when autophagy is
induced (21). Additionally, Becn1and Atg 5 mRNA levels were also elevated after 6 h U50,488H cell
exposure (Fig. 3D). These results clearly demonstrate that κ-OR is
involved in autophagosome biogenesis in neuronal cells.
Identification of the κ-OR signaling pathway that regulates the
autophagic machinery. The κ-OR is coupled to pertussis toxin-sensitive
Gi/o proteins to regulate a variety of effectors
(1, 8). Το
define the role of G proteins, we pretreated Neuro-2A cells with
pertussis toxin (PTX), which ribosylates Gαi/o subunits. PTX blocked
U50,488H-mediated increase of LC3-II and Beclin 1 levels (Fig. 4A, B),
suggesting that Gi/o proteins are important players in κ-ΟR-mediated
autophagy. To examine whether ERK1,2 is implicated in κ-OR- induction of
autophagy the levels of ERK1,2 phosphorylation of Neuro-2A cells were
assessed in the presence or absence of PTX. U50,488H enhanced ERK1,2
phosphorylation after 15 min and 6 h post-exposure and this effect was
abolished by PTX exposure (Fig. 4C). Moreover, when the cells were
pretreated with the ERK1,2 inhibitor PD98059 prior to agonist
activation, U50,488H-mediated-ERK1,2 phosphorylation was abolished with
a concomitant decrease of the LC3-II and Beclin1 levels, relative to the
untreated cells (Fig. 4D). On the other hand, JNK activation cannot
recapitulate the effects of ERK1,2 phosphorylation on κ-OR-mediated
induction of the autophagic pathway. Indeed, no effects on LC3-II
accumulation were detected in U50,488H-treated cells relative to the
untreated ones upon pre-treatment with the JNK inhibitor SP600125 (Fig.
4E). These results suggest that ERK1,2 is implicated in κ-OR-mediated
autophagy.
κ-OR regulates Beclin1 transcription via CREB activation.Because it is known that CREB regulates various autophagic genes
(22) and that a consensus CRE binding
site (TGACGTCA) exists in the mouse Becn1 promoter, we sought to
determine if autophagic genes are regulated by p-CREB upon U50,488H
exposure. CREB was phosphorylated in response to U50,488H cell exposure,
and this effect was abolished by the ERK1,2 inhibitor PD98059 (Fig. 4F).
Moreover, chromatin immunoprecipitation (ChIP) assay in isolated
chromatin fragments of Neuro-2A cells indicated that CREB binding in theBecn1 promoter was greatly enhanced by U50,488H exposure relative
to untreated cells (Fig. 4G). Therefore, κ-ΟR-mediated increase in
Beclin1 levels appears to involve transcriptional activation of the
beclin1 gene by ERK1,2-activated CREB.
U50,488H administration induces autophagy and promotes synaptic
alterations in mouse hippocampus. We next sought to examine whether we
could recapitulate U50,488H-mediated κ-ΟR autophagy in vivo and
determine whether specific brain regions are involved. To this end, mice
were injected with saline (vehicle) or U50,488H for 7 consecutive days
and the levels of LC3-II and Beclin1 were measured in the hippocampus,
cortex and striatum. U50,488H resulted in a significant increase of
LC3-II and Beclin1 in the mouse hippocampus as compared to vehicle, but
with no significant changes in cortical and striatal lysates (Fig.
5A-C). Collectively, these results suggest that κ-OR-mediated autophagy
is detected specifically to the mouse hippocampus.
Autophagy contributes to synaptic plasticity by degrading specific
proteins that are essential for synaptic function and spine remodeling
(13). To elucidate whether
U50,488H-mediated autophagy leads to synaptic alterations, initially the
levels of proteins enriched in dendritic spines such as PSD-95 and
spinophilin, were examined in the hippocampus, cortex and striatum of
U50,488H-treated mice. As shown in Fig. 5D, spinophilin and PSD-95 in
hippocampal lysates were significantly decreased in U50,488H-treated
mice compared with saline-treated controls. However, no significant
alterations for these proteins were detected in the cortex or striatum
(Fig. 5E, F). This was further confirmed in isolated synaptosomes where
a pronounced decrease of spinophilin, PSD-95, as well as SNAP25 was
detected in U50,488H-injected mice, as compared to control ones (Fig.
5G). All these suggest that these synaptic proteins are degraded,
possibly by being engulfed in the κ-ΟR-mediated autophagic cargo.
To test this hypothesis and in view of the known interaction of LC3 with
autophagic cargos through the LC3-interacting regions (LIR) of various
proteins, spinophilin, SNAP25 and PSD-95 among them
(13, 23),
we examined whether these proteins interact with LC3.
Co-immunoprecipitation studies of hippocampal lysates using an LC3
antibody indicated that spinophilin, PSD-95 and SNAP25 do interact with
LC3 (Fig. 6A). Moreover, to further define whether these synaptic
protein alterations are indeed due to autophagy induction we measured
their levels in the presence of BafA1. Treatment of Neuro-2A cells with
U50,488H decreases the levels of spinophilin and PSD95. Inhibition of
autophagy by BAF1 treatment did not alter these protein levels,
suggesting that U50,488H-κ-ΟR activation indeed leads to degradation of
these synaptosomal proteins (Fig. 6B).
Finally, to verify whether these
κ-OR-mediated effects are due to alterations in neuronal sprouting, the
number of branches in U50,488H-treated hippocampal neuronal cultures
were measured. U50,488H significantly reduced the number of branches
relative to the controls (Fig. 6C), suggesting that κ-ΟR-induced
autophagy modulates neuronal morphogenesis, possibly by degrading key
synaptic proteins.
Activation of the endogenous κ-OR/dynorphin system upon stress
upregulates autophagy in the hippocampus and results in synaptic
alterations. It is well documented that the κ-ΟR/dynorphin system plays
an important role in anxiety and stress-related behaviors and that κ-OR
antagonists exhibit anxiolytic effects
(24, 25).
To examine whether stress-induced endogenous dynorphin release impacts
on autophagy regulation, we examined the consequences of acute stress on
autophagy in the hippocampus. To this end, mice injected with either
vehicle or the κ-OR selective antagonist, nor-BNI, which is known to
exert anxiolytic effects, were subjected to a two-day modified forced
swim test (FST) (Fig. 7A). Male C57BL/6J mice were divided into 4 groups
saline-control or saline-FST (stressed) and norBNI-not stressed or
nor-BNI-FST (stressed). nor-BNI significantly decreased immobility time
following the FST, compared to saline-treated mice, suggesting that
nor-BNI attenuates stress-related behavior (Fig. 7B). Subsequently, the
levels of the autophagic markers LC3-II and Beclin1 were measured in
isolated hippocampal lysates and found to be significantly increased in
stressed animals (FST) relative to vehicle injected-non-stressed ones
(Fig. 7C). In contrast, this
increase in autophagic markers was not detected in nor-BNI-treated mice
under control or FST conditions when compared with the saline-control
group (Fig. 7C). Moreover, as expected, no significant alterations of
LC3-II levels were detected in the cortices of the same treatment groups
(Fig. 7D), confirming that the hippocampus is the target region for
κ-ΟR-induced autophagy under acute stress.
Finally, to confirm that dynorphin/κ-OR-induced autophagy-mediated
changes in the structural reorganization of hippocampal synapses during
stress may be rescued by nor-BNI, we measured the levels of synaptic
proteins in hippocampi of stressed and naïve animals subjected to
nor-BNI, or saline treatment. Our results demonstrated that spinophilin,
PSD-95, and SNAP-25 protein levels were significantly reduced in
stressed animals relative to the control ones (Fig. 7E). In contrast,
the levels of these synaptic proteins in nor-BNI injected mice prior to
FST were at the same levels as the control nor-BNI-injected ones devote
of the stressor (Fig. 7E, lanes 5-8). Again, no significant alterations
in cortical lysates of these proteins were detected, irrespective of the
stress-related regime or nor-BNI administration status (Fig. 7F).
Collectively, these results suggest that the endogenous dynorphin
release due to the acute FST results in κ-ΟR-mediated induction of
autophagy that in turn leads to aberrant hippocampal synaptic
alterations.