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.