Figure legends
Figure 1: Activation of the κ-OR induces the autophagic
machinery in neuronal cells. κ-Neuro2A cells were (A) treated
with various concentrations of U50,488H (10, 20, 50 μM) for 6 h,(B) pre-treated with 20 nM BafA1 for 16 h, prior to U50,488H
(20 μΜ) exposure for 6 h. (C) κ-Νeuro-2A cells were pre-treated
with 50 μM naloxone for 45 min, prior to U50,488H (20, 50 μM) exposure
for 6 h. All cell lysates (50 μg) were subjected to 17 % SDS-PAGE for
western immunoblotting to detect the autophagosome formation, (top
panels, immunoblots of LC3-II and β-actin; bottom panels, quantification
of LC3-II (measured with an anti-LC3B antibody and normalized to β-actin
levels). (D) Primary neuronal cultures were pre-treated or not
with naloxone (50 μΜ) for 45 min and exposed to U50,488H (50 μΜ) for 6h.
Confocal images of 10 DIV embryonic cortical neurons, immmunostained
with an anti-LC3B antibody (1:200) to label autophagosomes, MAP2 (1:500)
to label dendrites, and the nuclear dye, Hoechst (1:1000). (E)Embryonic primary neuronal cultures were administered for 6 h with the
indicated concentrations of U50,488H (lanes 2-4). Lane 5, represents
lysates from neuronal cultures treated with 50 μΜ naloxone prior to
U50,488H administration. Top panel represents immunoblots of LC3-II and
β-actin. Quantification of LC3-II was normalized to β-actin levels
(bottom panel). All experiments were performed independently at least 3
times. Error bars represent mean values \(\pm\)SEM. Statistical analysis
was performed using Statistical analysis was performed using one or
two-way ANOVA. *p < 0.05 and **p<0.01 as compared in
the absence of agonist, #p<0.05 as compared
with samples in the presence of U50,488H.
Figure 2. Dynorphin induces κ-ΟR-mediated autophagy in neuronal
cells. (A, B) κ- Νeuro-2A cells were treated with various
concentrations (0.1, 1 and 20 μΜ) of dynorphin1-13 for 3
and 6 h. The levels of LC3-II (A) and Beclin1 (B)after dynorphin1-13 treatment for 6 h were detected by
western blotting. Data represent mean\(\pm\text{SEM}\) of three
independent experiments; Statistical analysis was performed using
one-way ANOVA. *p<0.05 as compared to untreated samples.
(C) κ-Neuro-2A cells were pre-treated for 1 h, with or without
the κ-ΟR antagonist nor- (5 μM) following administration with 1 μΜ
dynorphin for 6 h. The LC3-II levels were detected by western blotting.
β-actin served as loading control. Error bars represent mean\(\mathbf{\pm}\) of three independent experiments, *p < 0.05
as compared with untreated samples. #p< 0.05 as compared with
samples in the presence of nor-. Statistical analysis was performed
using two-way ANOVA. (D) Representative confocal images of
embryonic DIV 10 neurons were
treated or not with 1 μΜ dynorphin for 6 h and immmunostained with
antibodies against LC3B (1:200) and Tuj1 (1:1000) to label
autophagosomes and mature dendrites respectively and the nuclear dye
TO-PRO3 (1:500). Statistical analysis was performed using one-way ANOVA.
**p < 0.01 as compared with untreated cells.
Figure 3: U50,488H treatment of neuronal cultures upregulates
autophagosome biogenesis. (A) Primary neuronal cultures were treated
with 20 μΜ U50,488H for 1, 6, and 24 h and the levels of pre-autophagic
markers FIP200 (1:1000) and Ulk1(1:1000) were identified by western
blotting using the corresponding antibodies. (B) Western blot
analysis against Beclin1 antibody and normalized with β-actin of
Neuro-2A cell lysates after 20 μΜ U50,488H treatment at the indicated
time intervals. (C) κ-Neuro2A cells were treated with 20 and 50
μΜ U50,488H for 6 h and the levels of ATG5 (1:1000), Beclin1 (1:1000)
and p62 (1:1000) were detected by western blotting against the
corresponding antibodies. (D) κ-Neuro2A cells were treated with
20 μΜ U50,488H for 6 h and the mRNA levels of Becn1 and Atg5 were
examined by real-time PCR. Error bars represent average ± SΕΜ.Statistical analysis was performed using one way ANOVA. All experiments
were performed at least three times. *p < 0.05 as compared
with untreated samples.
Figure 4: κ-OR activation induces autophagy in a
Gi/o protein, ERK 1,2 kinase and p-CREB dependent
manner. κ-Neuro2A cells were pre-treated with PTX (100 ng/ml) for 16 h,
followed exposure to U50,488H (20, 50 μΜ) for 6 h. LC3-II levels (A)
were assessed in protein lysates by western immunoblotting. (B)Beclin1 levels were detected after PTX pretreatment following
administration with 20 μΜ U50,488H for 6 h. (C) κ-Neuro2A cells
were pre-treated with PTX for 16 h, prior to U50,488H (20 μΜ) for 15 min
and 6 h exposure and the levels of p-ERK1,2 were quantified. Tubulin was
used as loading control. (D) κ-Neuro2A cells were pre-treated
with the ERK1,2 inhibitor PD98059 (20 μΜ) for 2 h, followed by U50,488H
(20 μΜ) administration for 6 h. Total ERK1,2 was used for
quantification. (E) The levels of p-JNK were estimated in
κ-Neuro2A cells in the presence or not of 20 μΜ of the JNK inhibitor
SP600125 for 30 min prior to 6 h U50,488H (20 μΜ) administration. All
experiments were performed three times. Error bars represent mean values\(\pm\)SEM. Statistical analyses were performed using two-way ANOVA. *p
< 0.05 as compared with untreated cells,#p<0.05 as compared with samples with
U50,488H alone. (F) κ-Neuro-2A cells were pre-treated with or
without PD98059 (20 μΜ) for 2 h, followed by U50,488H (20 μΜ) exposure
for 6 h. The phosphorylated levels of CREB were analyzed by western
blotting using an anti-p-CREB (Ser133) (1:500) serum and quantified
using a CREB antibody (1:1000). Statistical analysis was performed using
two-way ANOVA. *p < 0.05 as compared with untreated samples
and #p<0.05 as compared with samples in the
presence of U50,488H. (G) Chromatin immunoprecipitation was
performed in κ-Neuro-2A cells as described in Materials and methods
using an anti-CREB (10 μg) (lanes 3, 4) or normal rabbit serum (NRS)
(lanes 1, 2) and immunoprecipitated Becn1 was quantified by PCR on an
agarose gel.
Figure 5: κ-ΟR activation induces autophagy in mouse hippocampus
and alters synaptosomal protein levels. Male mice were injected i.p.
with saline (vehicle) or 5 mg/kg U50,488H for six consecutive days and
the hippocampus (A), cortex (B) and striatum(C) were isolated and lysed as described in Materials and
methods. Autophagosome accumulation was measured by western blotting
using LC3B and Beclin1 antibodies. Brain lysates from hippocampus(D), cortex (E), and striatum (F) of
saline-and U50,488H-injected mice were isolated and the protein levels
of spinophilin (1:000) and PSD-95 (1:1000), were measured by
immunoblotting using the corresponding antibodies. β-tubulin (1:000) was
used as loading control. (G) Hippocampal synaptosomes of
saline- or U50,488H-injected mice were isolated and the levels of
spinophilin, PSD-95, and SNAP-25 were detected using the corresponding
antibodies and quantified by Image J software using β-actin. All data
are presented as average ± SEM from three independent experiments
(n=4/group, *p < 0.05 as compared with saline group using
one-way ANOVA with post-hoc test).
Figure 6. κ-ΟR-mediated autophagy leads to degradation
of synaptosomal proteins. (A) Hippocampal lysates ( 800 μg)
were immunoprecipitated with 2 μg of an LC3B antibody and immunoblotted
with spinophilin, PSD95 and SNAP25. NRS immunoprecipitated samples were
used as negative control. (B) κ-Neuro-2A cells were pre-treated
with 20 nM BafA1 for 24 h, following exposure with 20 μΜ U50,488H for 16
h. Cell lysates were subjected to 10% SDS-PAGE and the protein levels
of spinophilin, PSD95 and SNAP25 were detected using the corresponding
antibodies. Quantification of the synaptic proteins was normalized using
β-tubulin. Error bars represent mean values ± SEM of three independent
experiments. Statistical analysis was performed using one-way ANOVA, *p
< 0.05 as compared with the values in the absence of agonist.(C) Primary hippocampal neuronal cultures were treated for 24 h
with 20 μΜ U50,488H and labeled with a βIII-tubulin (Tuj1) antibody
(1:1000). Nuclei were stained with TO-PRO-3 (blue) (1:500). Graph
represents mean ± S.E.M of the number of branches calculated from 100
Tuj1-positive neurons of control and U50,488H-treated neuronal cultures;
scale bar: 40 μm. Statistical analysis was performed using one-way
ANOVA, ***p<0.001 as compared with the untreated samples.
Figure 7: Nor-BNI blocks FST-induced autophagy promoting
synaptic alterations in mouse hippocampus. (A) Experimental timeline of
the nor-BNI administration protocol and the 2-day repeated FST in
C57BL/6J mice. Four groups of animals [saline and nor-BNI
(non-stressed groups), and saline and nor-BNI exposed to FST (stressed
groups)], (n=6/group) were injected i.p. with saline or the specific
κ-OR antagonist nor-BNI (10 mg/kg) on day 1. One hour after injection,
mice were subjected to 15 min FST. On the second day, mice were
subjected to 4x 6 min FST trials and immediately sacrificed and
hippocampi and cortices were isolated. (B) For behavioral
analysis the immobility time of mice was quantified using a video
tracking software as described in Materials and Methods. Statistical
analysis was performed by one-way ANOVA variance, *p<0.05
relative to FST saline group. For autophagy induction, LC3-II and Beclin
1 levels were measured in hippocampus (C) and cortex(D) with Western immunoblotting. (E) The levels of
spinophilin, PSD-95 and SNAP-25 in the hippocampus were detected from
the four groups to evaluate differences between the stressed and
non-stressed animals injected with saline or nor-BNI. β-tubulin was used
as the loading control. (F) Spinophilin, PSD-95 and SNAP25
levels were detected from isolated cortical lysates (50 μg) from the
four mice groups. All data are presented as average ± SEM (n=6/group *p
< 0.05 as compared with the saline non-stressed group and#p < 0.05 compared with the saline stressed
ones using two-way ANOVA with post-hoc test).
Figure 8: Schematic representation of a putative
signaling pathway via which κ-OR activation triggers autophagy resulting
in synaptic alterations . Agonist activation of κ-ΟR in neuronal cells
leads to ERK1, 2 phosphorylation mediated by Gi/ο proteins. Αctivated
ERK1,2 subsequently phosphorylates CREB which in turn translocates to
the nucleus to activate Becn1 gene expression. Upregulation of
Beclin1 and Atg5 promotes the initiation of autophagy resulting in
alterations of hippocampal synaptic proteins enriched in dendritic
spines.