Skip to main content
NCBI home page
British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2020 Jan 30;177(7):1609–1621. doi: 10.1111/bph.14922

Morphine‐induced RACK1‐dependent autophagy in immortalized neuronal cell lines

Li‐Tao Liu 1, Ying‐Qi Song 1, Xue‐Shen Chen 1, Yin Liu 1,2, Jie‐Jun Zhu 1, Li‐Ming Zhou 1, Shi‐Jun Xu 3, Li‐Hong Wan 1,✉
PMCID: PMC7060369  PMID: 31747048

Abstract

Background and Purpose

Autophagy is a critical cellular catabolic process in cell homoeostasis and brain function. Recent studies indicate that receptor for activated C kinase 1 (RACK1) is involved in autophagosome formation in Drosophila and mice, and that it plays an essential role in morphine‐associated memory. However, the exact mechanism of the role of RACK1 in morphine‐induced autophagy is not fully understood.

Experimental Approach

SH‐SY5Y cells were cultured and morphine, rapamycin, 3‐methyladenine and RACK1 siRNA were used to evaluate the regulation of RACK1 protein in autophagy. Western blotting and immunofluorescence were used to assess protein expression.

Key Results

Activation of autophagy (i.e. autophagosome accumulation and an increase in the LC3‐II/LC3‐I ratio) induced by morphine contributes to the maintenance of conditioned place preference (CPP) memory in mice. Moreover, morphine treatment significantly increased Beclin‐1 expression and decreased the p‐mTOR/mTOR and SQSTM1/p62 levels, whereas knockdown of RACK1 prevented morphine‐induced autophagy in vitro. Furthermore, we found that in the mouse hippocampus, knockdown of RACK1 also markedly suppressed morphine‐induced autophagy (decreased LC3‐II/LC3‐I ratio and increased p‐mTOR/mTOR ratio). Importantly, morphine‐induced autophagy in a RACK1‐dependent manner. Conversely, morphine‐induced RACK1 upregulation in vitro is partially inhibited by autophagy feedback.

Conclusions and Implications

Our findings revealed a critical role for RACK1‐dependent autophagy in morphine‐promoted maintenance of CPP memory in mice and supported the notion that control of RACK1‐dependent autophagic pathways may become an important target for novel therapeutics for morphine‐associated memory.

Abbreviations

3‐MA

3‐methyladenine

ATGs

autophagy‐related proteins

CPP

conditioned place preference

MAP1LC3

microtubule‐associated protein 1 light chain 3 beta

RACK1

receptor for activated C kinase 1

What is already known

  • RACK1 is involved in autophagosome formation and plays an essential role in morphine‐associated memory.

What this study adds

  • Morphine activated autophagy in RACK1‐dependent manner.

  • Morphine‐induced RACK1 up‐regulation in vitro is partly inhibited by autophagy feedback

What is the clinical significance

  • Control of RACK1‐dependent autophagic pathways may provide a novel therapeutics of morphine‐associated memory.

1. INTRODUCTION

Macroautophagy (hereafter referred as autophagy) is an evolutionarily conserved, lysosomal degradation process that degrades long‐lived proteins and damaged cellular organelles via delivery of cytoplasmic cargo to the lysosomes (Kim & Lee, 2014; Lin & Kuang, 2014). It is generally accepted that under physiological conditions, autophagy is essential for regulating cell longevity as a pro‐survival programme triggered in response to unfavourable cellular environments (Levine & Kroemer, 2008; Kenific & Debnath, 2014) and for maintaining neuron homeostasis and normal brain function (Hayashi et al., 2014; Zhao,Sun et al., 2015).

Autophagy is a multistep process that includes initiation, phagophore nucleation, autophagosome elongation and maturation and autophagolysosome fusion, plus a distinct series of “autophagy‐related” proteins (ATGs), which make up several distinct complexes, governing each of these steps (Yang & Klionsky, 2010; Czaja et al., 2013). The serine/threonine kinase https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2271 in cooperation with ATG13 plays a pivotal role in the autophagy initiation process. After its activation by https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=1540‐mediated phosphorylation and/or the absence of https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2109‐mediated suppression (Kim, Kundu, Viollet, & Guan, 2011), ULK1 phosphorylates a set of downstream targets, such as Beclin‐1, to trigger the full autophagy cascade in mammals (Itakura & Mizushima, 2010; Russell et al., 2013). Microtubule‐associated protein 1 light chain 3 beta (MAP1LC3) is a widely accepted marker of autophagy (Al‐Younes et al., 2011). The elongation of the autophagosomes relies on the presentation of LC3‐II (the cleaved and lipidated form of MAP1LC3) on the surfaces of isolated membranes and autophagosomes (Rodríguez et al., 2016). Furthermore, an increase in the number of LC3‐II puncta indicates either the initiation or the blockade of autophagic maturation, since LC3‐II is consumed in autolysosomes (Kabeya et al., 2000; Kimura, Noda, & Yoshimori, 2007; Klionsky et al., 2008). In the final step of autophagy, polyubiquitin‐binding protein SQSTM1/p62 binds directly to Atg8/LC3 to selectively target ubiquitinated protein aggregates to lysosomal degradation (Pankiv et al., 2007). Therefore, both LC3‐II and SQSTM1/p62 are powerful markers to study the dynamics of the autophagic process.

There is growing evidence indicating that chronic https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=1627 treatment not only activates autophagy in hippocampal neurons but also potentiates LPS‐induced autophagy initiation (Wan, Ma, Anand, Ramakrishnan, & Roy, 2015; Zhao et al., 2010). Moreover, a significant decrease in mitochondrial DNA copy number was found in the hippocampus during morphine addiction (Feng et al., 2013). In particular, enhanced autophagy activation in hippocampal cells via morphine administration at a dose of 15 mg·kg−1 for 7 days has been linked to the alleviation of spatial memory impairment due to its prevention of neuronal death in the hippocampus (Pan et al., 2017). All of these findings suggest that autophagy is an important cytoprotective mechanism that contributes to learning and memory function, especially morphine‐related memory.

However, there is considerable debate on whether enhanced autophagy promotes synaptic remodelling and memory. For example, a recent in vivo study carried out by Nikoletopoulou, Sidiropoulou, Kallergi, Dalezios, and Tavernarakis (2017) revealed that increased autophagy mediates the synaptic defects caused by https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=4872 (BDNF) deficiency. By contrast, growing evidence suggests that knockout of autophagy genes can greatly impair learning and memory in mice (Zhao et al., 2015) and activation of autophagy notably rescued synaptic deficits in fragile X mice (Yan, Porch, Court‐Vazquez, Bennett, & Zukin, 2018) and morphine‐induced hippocampal synaptic impairment (Cai et al., 2016). These findings are not consistent with Pan's results that autophagic mechanisms contribute to morphine related learning and memory functions (Pan et al., 2017), which could be due to differences in the doses of morphine, the experiment schedule and the research focus.

Interestingly, our previous study demonstrated that receptor for activated C kinase 1 (RACK1), a scaffolding/anchoring protein with 7 WD repeats, plays a critical role in morphine‐induced conditioned place preference (CPP) memory by promoting synaptic plasticity (Liu, Zhu, Zhou, & Wan, 2016). Current evidence suggests that endogenous RACK1 partially co‐localized with early autophagic structures is involved in autophagosome formation in Drosophila (Erdi et al., 2012). Recently, Zhao et al. demonstrated that RACK1 directly bind to Vps15, Atg14L and Beclin‐1 to participate in the formation of autophagy initiation complexes upon its phosphorylation by AMPK at Thr50 (Zhao et al., 2015). Our previous study showed that knockdown of RACK1 in mice resulted in notable inhibition of the acquisition and maintenance of CPP memory (Liu et al., 2016), providing a model for understanding the potential in vivo mechanisms. To further study the relationship between RACK1, autophagy and morphine‐induced CPP memory, we chose mouse hippocampus tissue because it displayed obvious acquisition and maintenance of CPP memory (Liu et al., 2016). Furthermore, we systemically investigated whether morphine‐induced autophagy in immortalized neuronal cell lines is RACK1 dependent.

2. METHODS

2.1. Materials

Morphine hydrochloride was purchased from Northeast Pharmaceutical Group Shenyang No. 1 Pharmaceutical Co., Ltd. (1 ml: 10 mg, Batch No. 120305, Shenyang, China) and was dissolved in DMSO (HyClone, MZK1250). The dissolved morphine hydrochloride was then added to complete culture medium at a final concentration of 2 mM. 3‐methyladenine (3‐MA, Merck, D00146677) was dissolved in sterile PBS (HyClone, NMZK1248) and diluted to 2.5 and 5 mM before use. https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=6031 (Merck, D00135191) was dissolved in sterile PBS at final concentrations of 10 and 100 nM.

2.2. Cell culture, cell viability assay and apoptosis analysis

See Data S1.

2.3. Transient knockdown of Gnb2l1 (the gene encoding RACK1) in cells

For knockdown in SH‐SY5Y cells (https://web.expasy.org/cellosaurus/CVCL_0019), chemically synthesized, double‐stranded RACK1 siRNA (h) or negative control siRNA (NC) was purchased from Santa Cruz (sc‐36354). siRNA transfection was carried out using the JetPEI in vitro DNA transfection reagent (Polyplus Transfection, 15031C1M) according to the manufacturer's instructions. Cells were plated at 4 × 105 cells per well in six‐well plates and cultured overnight in DMEM at 37°C in 5% CO2 (v/v) in a humidified incubator with siRNA (3 μg per well) treatment for 24 hr.

2.4. Cell experiments

The effects of morphine on autophagy in cells were evaluated. At 90% confluence, cells were treated with morphine hydrochloride at 1 and 2 mM for 24 hr.

Next, we investigated the role of autophagy in morphine‐treated cells. SH‐SY5Y cells were pre‐treated with 2.5 and 5 mM of 3‐MA (Merck, D00146677) or 10 and 100 nM of rapamycin (Merck, D00135191) for 3 hr prior to treatment with 1 mM of morphine.

Finally, the role of RACK1 in morphine‐induced autophagy was investigated. Cells were randomly divided into the following treatment groups: control, negative control siRNA (siNC), RACK1 siRNA (siRACK1), morphine (1 mM), negative control siRNA (siNC) + morphine (1 mM) and RACK1 siRNA (siRACK1) + morphine (1 mM).

2.5. Animal experiments

All hippocampus samples were obtained during a previous study of the mechanism of acquisition and maintenance of morphine‐induced CPP in mice (Liu et al., 2016). Briefly, mice were trained for morphine‐induced CPP or underwent a maintenance experiment for 7 days. Next, all of the mice were anaesthetized with pentobarbital and killed via cervical dislocation. An approval by the Institutional Animal Ethics committee with the current date (permit number: 2003‐149) and with information on the strain used in the study was obtained despite the fact that this study used previously approved samples. The Sichuan University Animal Ethics Committee approved the use of C57BL/6 for the animal experiments conducted in this study (permit number: 2003‐149). Animal studies are reported in compliance with the ARRIVE guidelines (Kilkenny, Browne, Cuthill, Emerson, & Altman, 2010) and with the recommendations made by the British Journal of Pharmacology.

2.6. Autophagy assay

Autophagy was measured using an autophagy assay kit (Sigma‐Aldrich, MAK138). Briefly, cells were cultured in 10% DMEM at 37°C in 5% CO2 (v/v) in a humidified incubator overnight and treated with different concentrations (1 and 2 mM) of morphine hydrochloride for 24 hr. Untreated cells were used as the control. Next, the medium was removed from the cells and 100 μl of the autophagosome detection reagent working solution (made by diluting 10 μl of the 500× autophagosome detection reagent solution in 5 ml of the stain buffer) was added to each well. Subsequently, the cells were incubated at 37°C with 5% CO2 for 1 hr and then washed thrice with 100 μl of wash buffer. The fluorescence intensity (λ ex = 360/λ em = 520 nm) was then measured using a fluorescence microscope (Zeiss Axio Scope A1, USA). All experiments were conducted in triplicate and repeated three times.

2.7. Protein preparation and Western blot analysis

The proteins of cells and the hippocampus lysates were extracted using the E.Z.N.A.® Total DNA/RNA/Protein Kit (Omega, USA). Protein concentrations were measured using the BCA Protein Assay Kit (Pierce Biotechnology, Rockford, IL).

Equal amounts of protein (40 μg) were loaded onto 12% SDS‐PAGE gels, separated via electrophoresis, and then transferred to PVDF membranes (Millipore, Billerica, MA). The membranes were then incubated for 2 hr at room temperature separately with the following antibodies: mouse monoclonal anti‐RACK1 antibody (1:1,000; Santa Cruz Biotechnology, sc‐17754), rabbit polyclonal anti‐LC3 antibody (1:1,000; Sigma, L7543), rabbit monoclonal anti‐Beclin‐1 antibody (1:1,000; Cell Signaling Technology, D40C5), rabbit polyclonal anti‐mTOR antibody (1:1,500; Santa Cruz Biotechnology, sc‐8319), rabbit polyclonal anti‐p‐mTOR antibody (1:1,500; Santa Cruz Biotechnology, sc‐101738), rabbit polyclonal anti‐SQSTM1/p62 antibody (1:500; Abcam, ab91526) or mouse monoclonal anti‐β‐actin antibody (TA‐09, ZSGB‐BIO, Beijing, China) as an internal control. The membranes were washed thrice with Tris‐buffered saline with Tween‐20 (TBST) and then incubated with HRP‐conjugated goat anti‐rabbit (ZDR‐5306, ZSGB‐BIO, Beijing, China) or goat anti‐mouse (ZB‐2305, ZSGB‐BIO, Beijing, China) IgG (1:3,000 dilution) antibodies for 1 hr at room temperature. All proteins were detected using Western Lightning™ Chemiluminescence Reagent Plus, and the results were quantitated via scanning densitometry using the Kodak IS4400CF image analysis system and the corresponding software (Eastman Kodak, Rochester, NY).

2.8. Immunofluorescence staining of LC3

Cultured SH‐SY5Y cells were treated with 1 or 2 mM of morphine for 24 hr and then fixed in 4% paraformaldehyde for 10 min, washed thrice with TBST for 3 min at room temperature, and then blocked with Immunol Staining Blocking Solution (Beyotime) for 60 min at 4°C. The samples were then incubated with primary antibodies for 1 hr at 4°C. Anti‐LC3 antibody (1:200; Sigma, L7543) was diluted using Immunol Staining Primary Antibody Dilution Solution (Beyotime). Next, the cells were washed thrice with TBST for 3 min at room temperature and then incubated with secondary antibodies in the dark for 60 min at room temperature. The secondary antibodies were Alexa Fluor® 594‐labeled goat anti‐rabbit IgG (Beyotime) for LC3 (1:1,000). Finally, the nuclei were stained with DAPI (Beyotime, C1002) for 5 min, and digital images at 1,000× magnification were acquired using a fluorescence microscope (Zeiss Axio Scope A1, USA). Fields were chosen randomly from various regions to ensure objectivity of sampling, and a minimum of 10 cells per sample were counted. The number of LC3 puncta per cell (red fluorescence) and the mean OD (green fluorescence) were analysed using Image‐Pro Plus software 6.0 https://scicrunch.org/resources/Any/search?q=SCR_007369&l=SCR_007369; Silver Spring, USA). All experiments were conducted in triplicate and repeated three times. The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology. (Alexander et al., 2018)

2.9. Statistical analysis

The data and statistical analysis comply with the recommendations of the British Journal of Pharmacology on experimental design and analysis in pharmacology (Curtis et al., 2018). All data and images were analysed blindly. All data are presented as the mean ± SEM and statistically evaluated via two‐way ANOVA (GraphPad Prism 8 https://scicrunch.org/resources/Any/search?q=SCR_002798&l=SCR_002798), San Diego, CA). Statistical significance was considered as P < .05. Any significant main effects were subjected to the Bonferroni post hoc test and Tukey's post hoc test for group differences if F achieved P < .05. Linear regression was applied to assess the correlation of two variables via the χ 2 test, and the significance of differences was evaluated via ANOVA using SPSS software v.13.0 (https://scicrunch.org/resources/Any/search?q=SCR_002865&l=SCR_002865).

2.10. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2019/20 (Alexander et al., 2019).

3. RESULTS

3.1. Morphine‐induced hippocampal autophagy in the acquisition and maintenance of reward memory in vivo was associated with RACK1 up‐regulation

Under physiological conditions, autophagy is critical for maintaining neuron homeostasis, for regulating learning and memory function (Zhao et al., 2015) and for promoting synapse development (Shen & Ganetzky, 2009). Our previous study showed that hippocampal RACK1 promotes the maintenance of morphine reward memory in mice by enhancing hippocampal synaptogenesis (Liu et al., 2016). To assess whether autophagy is involved in the acquisition and maintenance of CPP memory in vivo, we performed Western blot analysis to detect ATGs, including LC3‐II/LC3‐I, Beclin‐1, mTOR, p‐mTOR and SQSTM1/p62, in hippocampal samples from acquisition and maintenance of CPP memory mice (the samples acquired from our previous study in 2016). It is worth noting that under these conditions (acquisition and maintenance of CPP memory), the LC3‐II/LC3‐I ratio was significantly increased. However, the Beclin‐1 level was only significantly increased after the acquisition of CPP memory. By contrast, the p‐mTOR/mTOR and SQSTM1/p62 levels were significantly decreased after the acquisition and maintenance of CPP memory (Figure 1a,b). The results of our previous study showed that the RACK1 mRNA and protein levels in the hippocampus remained high for at least 7 days after the acquisition of morphine CPP (Liu et al., 2016). We further analysed the correlation between the expression of RACK1 protein and the LC3‐II/LC3‐I ratio in the hippocampus. Interestingly, the correlation analysis revealed a significant positive correlation between these two parameters in the acquisition of morphine CPP (Figure 1c; r 2 = .573, P < .05) and the maintenance of CPP memory (Figure 1d; r 2 = .466, P < .05), with higher RACK1 protein expression predicting a higher degree of autophagy. Consistent with these observations, the correlation analysis revealed a significant positive correlation between synaptophysin (SYP) and LC3‐II/LC3‐I ratio in the hippocampus in the acquisition of morphine CPP (Figure 1c; r 2 = .659, P < .05) and the maintenance of CPP memory (Figure 1d; r 2 = .48, P < .05), with higher degrees of autophagy predicting higher SYP protein expression. All of these results suggest that autophagy is the link between RACK1 and synaptic plasticity. Based on the predication that impaired autophagy was beneficial to suppress the acquisition and maintenance of CPP memory in RACK1‐knockdown mice, we assessed the effect of shRACK1 on the altered levels of ATGs involved in the acquisition and maintenance of CPP memory. Consistent with our previous results that shRACK1 markedly suppressed the acquisition and maintenance of CPP memory in mice, shRACK1 treatment also significantly decreased the LC3‐II/LC3‐I ratio and increased the p‐mTOR/mTOR level in acquisition and maintenance of CPP memory mice compared with the results in the morphine + shNC group (Figure 1a,b). However, the Beclin‐1 and SQSTM1/p62 levels were only affected by shRACK1 in acquisition of CPP memory mice compared with the results in the morphine + shNC group (Figure 1a,b), suggesting that RACK1 may play an important role in the acquisition and maintenance of CPP memory in morphine‐treated mice partially by modifying the LC3‐II/LC3‐I and p‐mTOR/mTOR levels.

Figure 1.

Figure 1

Open in a new tab

Morphine‐induced hippocampal autophagy in the acquisition and maintenance of reward memory in vivo. (a) Representative immunoblot showing the molecular weights of the proteins used to quantify levels of LC3‐I/LC3‐II (18/16 kD), Beclin‐1 (60 kD), mTOR (211 kD), p‐mTOR (222 kD) and SQSTM1/p62 (60 kD) in hippocampus of acquisition and maintenance of morphine‐induced conditioned place preference in mice. Signals were normalized to that of β‐actin (43 kDa). (b) Comparison of the levels of the autophagy‐related proteins between the experimental groups. (c) Positive correlation between the RACK1 protein level and LC3‐II/LC3‐I in the acquisition phase (r 2 = .573, P < .05; left panel) and the maintenance phase (r 2 = .466, P < .05; right panel). (d) Positive correlation between the SYP protein levels and LC3‐II/LC3‐I in the acquisition phase (r 2 = .659, P < .05; left panel) and the maintenance phase (r 2 = .48, P < .05; right panel). Data are shown as the mean ± SEM. * P < .05 compared with the saline + shNC group (n = 5); # P < .05 compared with the morphine + shNC group (n = 5)

3.2. Morphine‐induced autophagy in vitro

To investigate whether morphine‐induced neuronal autophagy, SH‐SY5Y cells (a human neuronal cell line) and PC12 cells (a rat undifferentiated neuronal cell line) were incubated in 0.016–2 mM of morphine containing media for 48 hr and cellular viability was assessed via the CCK8 assay. Morphine did not show any cytotoxic activity on human SH‐SY5Y cells or rat PC12 cells (Figure S1a,b). Thus, 1 and 2 mM of morphine treatment for 24 hr were selected to observe the morphological changes in the subsequent experiments. Consistent with the results of the CCK8 assay, there were no morphological changes after treatment with 1 and 2 mM of morphine for 24 hr (Figure S1c). Considering that both apoptosis and autophagy usually occur prior to cell death, to determine the potential effects of morphine on apoptosis and autophagy, we first measured apoptosis via flow cytometry and Hoechst 33258 staining in human SH‐SY5Y cells following 24 hr of exposure to 1 and 2 mM of morphine (Figure S1c) and quantitatively analysed the apoptotic rate according to the FCM results. Our data indicated that morphine treatment did not result in a higher percentage of apoptotic cells relative to the apoptosis rate in the control cells. The apoptotic rate showed no significant changes in response to morphine exposure (Figure S1d), suggesting that morphine did not induce neuronal apoptosis in human SH‐SY5Y cells even at the high doses of 1 and 2 mM.

Next, we treated SH‐SY5Y cells with 1 and 2 mM of morphine for 24 hr and evaluated autophagy with an autophagy assay kit. As expected, we found that morphine notably induced autophagy (Figure 2a). Evidence of morphine‐induced autophagy was also collected by measuring autophagosome formation using an immunofluorescence antibody against LC3. During the autophagic process, LC3 is a specific marker for autophagosome formation (Al‐Younes et al., 2011). Immunofluorescence analysis of LC3 in SH‐SY5Y cell indicated that morphine significantly induced the conversion of LC3‐I, the cytoplasmic form of LC3, into LC3‐II, the autophagosomal membrane‐bound form. Quantitative analysis demonstrated that there were increased numbers of LC3 puncta in the treated cells in a dose‐dependent manner (Figure 2b). To further examine the conversion of LC3‐I to LC3‐II, cells were treated as described above and then subjected to Western blot analysis. Our data revealed that there was a dose‐dependent increase in the conversion of LC3‐I to LC3‐II in 1 and 2 mM of morphine treated SH‐SY5Y cells (Figure 2c). Similar results were found in PC12 (Figure S2a) and HT‐22 cells (Figure S3a) but not in BV‐2 cells (Figure S3b). Thus, our results support the idea that morphine induces autophagosome accumulation in neurons. We also used Western blot to examine the expression levels of several proteins known to participate in the activation of autophagy, including Beclin‐1 (a key mediator in the formation of the autophagosome), mTOR (a negative regulator of autophagy), p‐mTOR and SQSTM1/p62 (the link between LC3 and ubiquitinated substrates). We found that morphine significantly increased the Beclin‐1 level in SH‐SY5Y cells, while the p‐mTOR/mTOR and SQSTM1/p62 levels were significantly decreased under the same conditions (Figure 2d,e). Notably, degradation of SQSTM1/p62 is negatively correlated with the level of LC3‐II (Bjørkøy, Lamark, & Johansen, 2006). Significant decreases in the p‐mTOR/mTOR and SQSTM1/p62 levels further suggested that autophagy was activated by morphine, which was also supported by increased Beclin‐1 and LC3‐II/LC3‐I levels in SH‐SY5Y cells. Since increased LC3‐II levels can result from the initiation of autophagy or the blockage of autophagic maturation, https://www.guidetopharmacology.org/GRAC/LigandDisplayForward?ligandId=5535 (10 μM), which inhibits lysosome function, was used to determine whether the morphine‐induced increase in LC3‐II level was a result of increased autophagosome formation or a defect in the fusion process. As shown in Figure 3a, chloroquine (10 μM) significantly increased morphine‐induced LC3‐II expression in SH‐SY5Y cells. Similar results were found in PC12 cells (Figure S2b) and HT‐22 cells (Figure S3c). Taken together, these findings provide strong evidence that morphine induces autophagy in neurons.

Figure 2.

Figure 2

Open in a new tab

Morphine induces autophagy in vitro. (a) Autophagy was detected via an autophagy assay kit. (b) Immunofluorescence analysis of LC3 in SH‐SY5Y cells after treatment with 1 or 2 mM of morphine. The data are reported as the mean value of the results of five independent experiments. (c) Representative immunoblot showing the molecular weights of the proteins used to quantify the levels of LC3‐I/LC3‐II (18/16 kD) in 1 or 2 mM of morphine treated SH‐SY5Y cells (upper panel). Signals were normalized to that of β‐actin (43 kD). Comparison of the LC3‐II/LC3‐I intensities between the experimental groups (lower panel). (d, e) Expression levels of autophagy‐related proteins (Beclin‐1 [60 kD], mTOR [211 kD], p‐mTOR [222 kD], and SQSTM1/p62 [60 kD]) in cells after treatment with 1 or 2 mM of morphine. Data are shown as the mean ± SEM from five separate experiments. * P < .05 compared with control. # P < .05 compared with 1 mM of morphine

Figure 3.

Figure 3

Open in a new tab

Chloroquine (10 μM) enhanced morphine‐induced autophagy and RACK1 up‐regulation in vitro. (a) Representative Western blot images of RACK1 and LC3‐II/LC3‐I in SH‐SY5Y cells after treatment with chloroquine (10 μM) in normal medium or in the presence of 1 or 2 mM of morphine (upper panel). Data are shown as the mean ± SEM from five separate experiments. * P < .05 compared with the control (without morphine). # P < .05 compared with morphine alone. (b) Western blot analyses were performed to detect the expression levels of RACK1 (36 kD) in cells after treatment with morphine (1 and 2 mM; upper panel). The relative integrated density values of RACK1 are shown (lower panel). Data are shown as the mean ± SEM from five separate experiments. * P < .05 compared with the control. # P < .05 compared with 1 mM of morphine. (c) Positive correlation between RACK1 protein levels and the LC3‐II/LC3‐I ratio (r 2 = .9117, P < .05)

3.3. Morphine exposure enhanced RACK1 expression in vitro

RACK1, a https://www.guidetopharmacology.org/GRAC/ObjectDisplayForward?objectId=2831 repeat protein, plays a crucial role in promoting autophagy (Erdi et al., 2012; Zhao et al., 2015). Our previous work demonstrated that chronic morphine exposure significantly increased the RACK1 level in the mouse hippocampus (Liu et al., 2016; Wan et al., 2009). The effect of morphine‐induced RACK1 expression in cells was investigated. As expected, 1 and 2 mM of morphine treatment significantly enhanced the RACK1 expression level in a dose‐dependent manner (Figure 3b). There was a significant positive correlation between the RACK1 and LC3‐II/LC3‐I ratio in morphine treated cells (r 2 = .9117, P < .05), with higher RACK1 protein expression predicting a higher LC3‐II/LC3‐I ratio in the cells (Figure 3c). The results of the correlation analysis implied that RACK1 may be involved in morphine‐induced autophagy induction. We next used chloroquine (10 μM) to inhibit lysosome function and found that chloroquine (10 μM) significantly increased morphine (1 and 2 mM)‐induced RACK1 expression in SH‐SY5Y cells (Figure 3a). This result further solidifies the relationship between RACK1 and autophagy.

3.4. Morphine‐induced autophagy is mediated by RACK1

To determine whether morphine‐induced autophagy is mediated by RACK1, we transiently transfected cells with RACK1 siRNA and examined the effect on morphine‐induced autophagy. In the absence of morphine exposure, RACK1 siRNA (siRACK1) significantly decreased the RACK1 protein and LC3‐II/LC3‐I ratio in the cells (Figure 4a). In the presence of morphine exposure, RACK1 siRNA (siRACK1) significantly inhibited morphine‐induced autophagosome accumulation (Figure 4b–d) and rescued the increased RACK1 protein level and LC3‐II/LC3‐I ratio (Figure 4e,f). To further dissect the mechanisms underlying the inhibitory effect of RACK1 siRNA on morphine‐induced autophagy, the levels of other ATGs, including Beclin‐1, mTOR, p‐mTOR, and SQSTM1/p62, were examined via Western blotting. Interestingly, we found that morphine‐induced Beclin‐1 activation was decreased and that the inhibitory effects of morphine p‐mTOR/mTOR and SQSTM1/p62 were strengthened as a result of the siRACK1 treatment (Figure 4e,f). To confirm the link between siRACK1 on morphine, we compared the LC3‐II/LC3‐I ratio in the quantitation shown in Figure 4a (control + siRACK1) with that shown in Figure 4f (morphine + siRACK1) and the data showed that morphine treatment still enhanced LC3‐II/LC3‐I ratio in the presence of siRACK1 (Figure 4g). This result may be due to the interference efficiency of the siRACK1 (50%) we used in this study (Figure 4a).

Figure 4.

Figure 4

Open in a new tab

RACK1 mediated morphine‐induced autophagy in vitro. (a) Representative Western blot images of RACK1 and LC3‐II/LC3‐I in SH‐SY5Ycells and LC3‐II/LC3‐I in cells after transient transfection with siRACK1 or siNC (upper panel). The relative integrated density values of RACK1 and LC3‐II/LC3‐I are shown (lower panel). Data were shown as the mean ± SEM from five separate experiments. * P < .05 compared with siNC. (b) Immunofluorescence analysis of LC3 in cells after transient transfection with siRACK1 or siNC in normal medium or in the presence of 1 mM of morphine. Digital images (1,000× magnification) were acquired using a fluorescence microscope (Zeiss Axio Scope A1, USA). (c) Autophagy in cells after transient transfection with siRACK1 or siNC in normal medium or in the presence of 1 mM of morphine was measured using an autophagy assay kit. The data report the mean value of five independent experiments. (d) The number of LC3 puncta per cell (red fluorescence in b) was quantized using Image‐Pro Plus software 6. The data report the mean value of five independent experiments. (e) Representative immunoblot showing the molecular weights of the proteins used to quantify the levels of RACK1 (36 kD), LC3B‐I/LC3B‐II (18/16 kD), Beclin‐1 (60 kD), mTOR (211 kD), p‐mTOR (222 kD), and SQSTM1/p62 (60 kD) in SH‐SY5Y cells. The signals were normalized to that β‐actin (43 kD). (f) Comparison of the intensity of RACK1 and those of the autophagy‐related proteins among the experimental groups. Data are shown as the mean ± SEM from five separate experiments. * P < .05 compared with the control. # P < .05 compared with 1 mM of morphine + siNC. (g) Comparison of the LC3‐II/LC3‐I ratio in the quantitation of panel a (control + siRACK1) with that of panel e (morphine + siRACK1). Data are shown as the mean ± SEM from five separate experiments. * P < .05 compared with the control. # P < .05 compared with 1 mM of morphine + siNC

3.5. Morphine‐induced RACK1 up‐regulation in cells is partially inhibited by autophagy feedback

To determine whether morphine‐induced RACK1 up‐regulation is modulated by autophagy, rapamycin and 3‐methyladenine were used to alter the autophagy level in the cells. Interestingly, rapamycin treatment markedly suppressed the RACK1 protein level, but 3‐methyladenine treatment significantly induced RACK1 expression (Figure 5a,b), which is opposite to the observations of the LC3‐II/LC3‐I ratio, suggesting that the RACK1 level is modulated by autophagy. Moreover, in the absence or presence of 3‐methyladenine, morphine exposure enhanced the RACK1 level in the cells (Figure 5b). Importantly, in the presence of rapamycin, morphine exposure did not significantly increase RACK1 expression compared with the effect of morphine alone (Figure 5a). By contrast, in the presence of 3‐methyladenine, morphine exposure significantly increased RACK1 expression compared with the effect of morphine alone (Figure 5b). These data were inconsistent with our previous results, that RACK1 level is positively correlated with LC3‐II/LC3‐I ratio, indicating that the activated autophagy in morphine exposed cells is required to partially balance the RACK1 expression level. Thus there is a feedback loop between autophagy and RACK1. However, the feedback inhibition of RACK1 by autophagy is insufficient to resist the up‐regulation induced by morphine. Therefore, the RACK1 level still showed up‐regulation after morphine exposure.

Figure 5.

Figure 5

Open in a new tab

Autophagy partially inhibited morphine‐induced RACK1 up‐regulation via a feedback look in vitro. (a) Representative Western blot images of RACK1 in SH‐SY5Y cells after treatment with rapamycin (10 and 100 nM) in normal medium or in the presence of 1 mM of morphine (upper panel). The relative integrated density values of RACK1 are shown (lower panel). Data are the mean ± SEM from five separate experiments. * P < .05 compared with the control. # P < .05 compared with the no morphine treatment group. (b) Representative Western blot images of RACK1 in SH‐SY5Y cells after treatment with 3‐methyladenine (3‐MA; 2.5 and 5 mM) in normal medium or in the presence of 1 mM of morphine (upper panel). The relative integrated density values of RACK1 are shown (lower panel). Data are the mean ± SEM from five separate experiments. * P < .05 compared with the control (no morphine and no 3‐methyladenine treatment). # P < .05 compared with the no morphine treatment group. & P < .05 compared with morphine alone

4. DISCUSSION

Previous work in our laboratory showed that hippocampal RACK1 plays a crucial role in maintaining morphine–CPP memory in mice by modulating synaptic plasticity (Liu et al., 2016). Here, we show that parallel to the maintenance of reward memory, morphine enhanced autophagy in vivo and in vitro. Moreover, the activation of RACK1‐dependent autophagic processes plays an important role in morphine treated mice and cells. Furthermore, we show that activation of autophagic processes acts as a rescue mechanism to counter morphine‐induced RACK1 up‐regulation in vitro.

Autophagy is required for normal cell homeostasis and plays an essential role in the maintenance of brain function (Hayashi et al., 2014; Zhao et al., 2015). Recent evidence indicates that the alleviation of learning and memory impairment by morphine is strongly associated with the activation of autophagic processes (Pan et al., 2017). However, morphine‐induced reward memory involves permanent functional changes in neural circuits, which is different from normal memory. Our previous data show that morphine can induce the maintenance of reward memory by promoting synaptic plasticity (Liu et al., 2016). To determine whether morphine‐induced maintenance of CPP memory is also mediated by autophagy, the LC3‐II/LC3‐I ratio in hippocampus samples of acquisition and maintenance of CPP memory mice (acquired from our previous study) were evaluated via Western blotting. Intriguingly, our results demonstrated that morphine potently induced autophagy by increasing the LC3‐II/LC3‐I ratio in the hippocampus of acquisition and maintenance of CPP memory mice. Similarly, 1 and 2 mM of morphine markedly activated autophagy (i.e., increased autophagosome formation and LC3‐II/LC3‐I ratio) in the SH‐SY5Y human neuronal cell line without any cytotoxic activity. This finding differs from previous results that a chronic high dose of morphine‐induced apoptosis in SH‐SY5Y cells (Lin et al., 2009). The difference in the results of these studies might be due to different lengths of time of the morphine effect on SHSY5Y cells.

Autophagy is a dynamic process that includes initiation, phagophore nucleation, autophagosome elongation and maturation, and autophagolysosome fusion (Czaja et al., 2013; Yang & Klionsky, 2010). Beclin‐1, as well as LC3, is a key mediator involved in the initial step of autophagosome formation (Liang et al., 1999; Pattingre, Espert, Biard‐Piechaczyk, & Codogno, 2008). During the later stage of autophagy, LC3‐II may be degraded by SQSTM1/p62, which indicates the completion of autophagy (Kabeya et al., 2000). Furthermore, autophagosome formation increases autophagic flux, which causes increased SQSTM1/p62 protein degradation (Cheng et al., 2014). As expected, morphine treatment notably activated autophagic flux as indicated by increased formation of LC3‐II as well as elevation of the Beclin‐1 level and reduction of the SQSTM1/p621 level in vitro and in vivo. Additionally, it is well recognized that the mammalian target of rapamycin (mTOR) is a large, ubiquitous, and evolutionarily conserved protein kinase that plays a central role in the inhibition of autophagy (Wong, 2013). Phosphorylated mammalian target of rapamycin (p‐mTOR) is the activated form of mTOR. mTOR comprises two functionally distinct complexes, mTORC1 and mTORC2 (Dunlop & Tee, 2014). Activated mTORC1 is strongly linked to synaptic plasticity (Stoica et al., 2011) and acquisition of morphine CPP memory (Cui et al., 2010). We found that the mTOR level did not change in these experimental paradigms, however morphine treatment significantly decreased the expression of p‐mTOR/mTOR in vitro and in vivo. Thus, it appears that mTOR and p‐mTOR are involved in the cellular response to morphine.

Protein RACK1 (official gene name Gnb2l1) is a 36‐kDa scaffolding/anchoring protein that contains seven WD‐domain motifs (McGough et al., 2004). Previous studies have revealed a central role for RACK1 in the acquisition and maintenance of morphine CPP memory (Liu et al., 2016; Wan et al., 2009; Wan et al., 2011). Recently, emerging evidence has suggested that the scaffold protein RACK1 is involved in autophagosome formation in Drosophila and mice (Erdi et al., 2012; Zhao et al., 2015). Thus, it would be interesting to determine whether RACK1 contributes to the maintenance of morphine CPP memory by influencing autophagy. Additionally, repeated morphine use has been shown to contribute to increased synaptic plasticity in the hippocampus (Alvandi, Bourmpoula, Homberg, & Fathollahi, 2017; Han et al., 2015).

We first evaluated the correlation between the RACK1 and LC3‐II/LC3‐I ratio in the hippocampus, as well as the correlation between SYP and LC3‐II/LC3‐I ratio. Significant positive correlations were found between the LC3‐II/LC3‐I ratio and those of RACK1 and SYP, implying that autophagy is the key link between RACK1 and synaptic plasticity. By contrast, the morphine‐induced increase in LC3‐II/LC3‐I was suppressed by treatment with shRACK1. To follow up on these findings, cells were treated with morphine and transiently transfected with siRACK1 to decrease the RACK1 mRNA level in cells. Interestingly, under these conditions, morphine significantly induced RACK1 expression, parallel autophagy activation. Consistent with the in vivo results, our present investigation revealed that the RACK1 protein level is positively correlated with the LC3‐II/LC3‐I ratio in morphine treated cells. Moreover, as expected, we observed that siRACK1 significantly inhibited the morphine‐induced increase in the LC3‐II/LC3‐I ratio. In the autophagosome, LC3‐II (the cleaved and lipidated form of MAP1LC3) is present both on the exterior and in the interior of the vesicles and is degraded after its fusion with lysosomes. Thus, increased LC3‐II expression, a powerful marker of autophagy activation, indicated that either autophagy was initiated or lysosomal degradation was blocked. In agreement with these observations, in the presence of morphine exposure, siRACK1 significantly inhibited morphine‐induced autophagosome accumulation (i.e. increased autophagosome formation, LC3‐II conversion and punctate LC3 expression). One likely interpretation is that morphine‐induced autophagy is mediated by RACK1, since knockdown of RACK1 with siRACK1 led to impaired basal autophagy.

To verify whether RACK1 could regulate complete autophagy, we tested the changes in the level Beclin‐1, the LC3‐II/LC3‐I ratio and the SQSTM1/p62 abundance. As expected, morphine treatment notably activated autophagic flux as indicated by increased formation of MAP1LC3‐II, an increased Beclin‐1 level and a decrease in SQSTM1/p621 abundance. Moreover, chloroquine (10 μM) significantly increased morphine‐induced LC3‐II expression. Conversely, knockdown of RACK1 notably suppressed autophagic flux as indicated by decreased formation of MAP1LC3‐II, a decreased Beclin‐1 level, and an increase in SQSTM1/p621 abundance. Our data suggest that morphine stimulates autophagy by altering the expression levels of series of ATGs. However, in this study, we found that RACK1 knockdown notably suppressed morphine‐induced Beclin‐1 expression, which is inconsistent with our previous data from an Aβ25–35‐induced spatial memory impairment model that showed that shRACK1 significantly enhanced the expression of Beclin‐1 (Zhu et al., 2016). We attribute the discrepancy to model differences, as we know that RACK1 has multiple effects on apoptosis ( Lin et al., 2015). Next, the expression levels of ATGs in hippocampus samples from our previous study were evaluated via Western blot analysis. shRACK1 treatment significantly affected the Beclin‐1 and SQSTM1/p62 levels in acquisition of CPP memory mice. The observations that shRACK1 treatment significantly decreased the LC3‐II/LC3‐I ratio and increased the p‐mTOR/mTOR level in acquisition and maintenance of CPP memory mice are consistent with previous results that showed that shRACK1 markedly suppresses the acquisition and maintenance of CPP memory in mice. In addition, our findings suggest that alterations in RACK1 lead to increased levels of p‐mTOR/mTOR in cells and the hippocampus of acquisition and maintenance of CPP memory mice. Therefore, our data demonstrated that mTOR might be involved in morphine‐induced autophagy via a RACK1‐dependent mechanism.

A double‐faceted role of autophagy activation in synaptic remodelling and memory has been recently reported (Zhao et al., 2015; Nikoletopoulou et al., 2017). In our subsequent experiments, we evaluated the changes in the RACK1 level in response to different autophagy‐modifying treatments (rapamycin and 3‐methyladenine treatment). As we hypothesized, rapamycin (an autophagy activator) significantly increased the LC3‐II/LC3‐I ratio, which reflected autophagy activation. By contrast, 3‐methyladenine (an autophagy inhibitor) markedly decreased the LC3‐II/LCB‐I level, which reflected autophagy inhibition. Interestingly, our finding showed that rapamycin suppressed the RACK1 protein level while 3‐methyladenine induced RACK1 expression, which was opposite to the LC3‐II/LC3‐I ratio in the cells, suggesting that RACK1 might partially feedback to inhibited by autophagy. Importantly, in the presence of 3‐methyladenine, morphine exposure significantly increased RACK1 expression compared with the effect of morphine alone. Taken together, the results of our study provide evidence that the activation of the autophagic process plays a crucial role in partially counteracting morphine‐induced RACK1 up‐regulation in neurons.

Based on these data, we suggest that RACK1 is the key regulator of morphine‐induced maintenance of CPP memory in mice via activation of autophagic process. Moreover, the exact mechanism of the activation of autophagic processes in vitro involves a partial inhibitory feedback effect on morphine‐induced RACK1 up‐regulation (Figure 6). Future studies designed to identify the detailed molecular signalling mechanisms underlying the regulation of the levels of ATGs by RACK1 will be important to aid in the development of improved therapeutic interventions to treat the persistence of morphine associated memory.

Figure 6.

Figure 6

Open in a new tab

Schematic model of the proposed cellular mechanism. Up‐regulated RACK1 is the key regulator of morphine‐induced maintenance of conditioned place preference memory in mice via activation of autophagy. Moreover, the exact mechanism underlying the autophagy activation is a partial inhibitory feedback effect of morphine‐induced RACK1 up‐regulation in vitro

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

AUTHOR CONTRIBUTIONS

L.‐H.W. conceived and designed the experiments. L.‐T.L., Y.‐Q.S., X.‐S.C., and J.‐J.Z. performed most of the biological experiments. S.‐J.X. donated the BV‐2 cells. L.‐M.Z., L.‐T.L., and L.‐H.W. analysed the data. Y.L. wrote the main manuscript text. All authors reviewed the manuscript.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14207, https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14208, and https://bpspubs.onlinelibrary.wiley.com/doi/full/10.1111/bph.14206 and as recommended by funding agencies, publishers, and other organizations engaged with supporting research.

Supporting information

Figure S1. Morphine did not show any cytotoxic activity in vitro.

Figure S2. Chloroquine (10 μM) enhanced morphine‐induced autophagy and RACK1 up‐regulation in PC12 cells.

Figure S3. Chloroquine (10 μM) enhanced morphine‐induced autophagy and RACK1 up‐regulation in HT‐22 cells.

Click here for additional data file. (494.5KB, doc)

Data S1. Supporting Information

Click here for additional data file. (43KB, doc)

ACKNOWLEDGEMENTS

This study was supported by the National Natural Science Foundation of China (81100989 to L.W.). The authors would like to thank Shikun Miao for the mouse breeding and husbandry.

Liu L‐T, Song Y‐Q, Chen X‐S, et al. Morphine‐induced RACK1‐dependent autophagy in immortalized neuronal cell lines. Br J Pharmacol. 2020;177:1609–1621. 10.1111/bph.14922

Li‐Tao Liu, Ying‐Qi Song, and Xue‐Shen Chen contributed equally to this work.

REFERENCES

  1. Alexander, S. P. H. , Roberts, R. E. , Broughton, B. R. S. , Sobey, S. G. , & George, C. H. (2018). Stanford SC…Ahluwalia, A. Goals and practicalities of immunoblotting and immunohistochemistry: A guide for submission to the British Journal of Pharmacology, 175, 407–411. 10.1111/bph.14112 [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Alexander S.P.H., Keely, E.A. , Mathie, A. , Peter, J.A. , Veale, E.L. , Armstrong, J.H. , … GTP collaborators . (2019). The concise guide to pharmacology 2019/2020: Introduction and other protein target. British Journal of Pharmacology, 176, S1–S20. doi:10.1111/bph.14747 [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Alvandi, M. S. , Bourmpoula, M. , Homberg, J. R. , & Fathollahi, Y. (2017). Association of contextual cues with morphine reward increases neural and synaptic plasticity in the ventral hippocampus of rats. Addict. Biol., 22, 1883–1894. 10.1111/adb.12547 [DOI] [PubMed] [Google Scholar]
  4. Al‐Younes, H. M. , Al‐Zeer, M. A. , Khalil, H. , Gussmann, J. , Karlas, A. , Machuy, N. , … Meyer, T. F. (2011). Autophagy‐independent function of MAP‐LC3B during intracellular propagation of Chlamydia trachomatis . Autophagy, 7(8), 814–828. 10.4161/auto.7.8.15597 [DOI] [PubMed] [Google Scholar]
  5. Bjørkøy, G. , Lamark, T. , & Johansen, T. (2006). p62/SQSTM1: A missing link between protein aggregates and the autophagy machinery. Autophagy, 2, 138–139. 10.4161/auto.2.2.2405 [DOI] [PubMed] [Google Scholar]
  6. Cai, Y. , Yang, L. , Hu, G. , Chen, X. , Niu, F. , Yuan, L. , … Buch, S. (2016). Regulation of morphine‐induced synaptic alterations: Role of oxidative stress, ER stress, and autophagy. J. Cell Biol., 215(2), 245–258. 10.1083/jcb.201605065 [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Cheng, S. M. , Chang, Y. C. , Liu, C. Y. , Lee, J. Y. , Chan, H. H. , Kuo, C. W. , et al. (2014). YM155 down‐regulates survivin and XIAP, modulates autophagy and induces autophagy‐dependent DNA damage in breast cancer cells. Br. J. Pharmacol., 172, 214–234. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Cui, Y. , Zhang, X. Q. , Cui, Y. , Xin, W. J. , Jing, J. , & Liu, X. G. (2010). Activation of phosphatidylinositol 3‐kinase/Akt‐mammalian target of rapamycin signaling pathway in the hippocampus is essential for the acquisition of morphine‐induced place preference in rats. Neuroscience, 171, 134–143. 10.1016/j.neuroscience.2010.08.064 [DOI] [PubMed] [Google Scholar]
  9. Czaja, M. J. , Ding, W. X. , Donohue, T. M. Jr. , Friedman, S. L. , Kim, J. S. , Komatsu, M. , … Perlmutter, D. H. (2013). Functions of autophagy in normal and diseased liver. Autophagy, 9, 1131–1158. 10.4161/auto.25063 [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Curtis, M. J. , Alexander, S. , Cirino, G. , Docherty, J. R. , George, G. H. , Giembycz, M. A. , et al. (2018). Experimental design and analysis and their reporting II: updated and simplified guidance for authors and peer reviewers. British Journal of Pharmacology, 175(7), 987–993. 10.1111/bph.14153 [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Dunlop, E. A. , & Tee, A. R. (2014). mTOR and autophagy: A dynamic relationship governed by nutrients and energy. Semin. Cell Dev. Biol., 36, 121–129. 10.1016/j.semcdb.2014.08.006 [DOI] [PubMed] [Google Scholar]
  12. Erdi, B. , Nagy, P. , Zvara, A. , Varga, A. , Pircs, K. , Ménesi, D. , … Juhász, G. (2012). Loss of the starvation‐induced gene Rack1 leads to glycogen deficiency and impaired autophagic responses in Drosophila . Autophagy, 8, 1124–1135. 10.4161/auto.20069 [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Feng, Y. M. , Jia, Y. F. , Su, L. Y. , Wang, D. , Lv, L. , Xu, L. , & Yao, Y. G. (2013). Decreased mitochondrial DNA copy number in the hippocampus and peripheral blood during opiate addiction is mediated by autophagy and can be salvaged by melatonin. Autophagy, 9, 1395–1406. 10.4161/auto.25468 [DOI] [PubMed] [Google Scholar]
  14. Han, H. , Dong, Z. , Jia, Y. , Mao, R. , Zhou, Q. , Yang, Y. , … Cao, J. (2015). Opioid addiction and withdrawal differentially drive long‐term depression of inhibitory synaptic transmission in the hippocampus. Sci. Rep., 5, –9666. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Harding, S. D. , Sharman, J. L. , Faccenda, E. , Southan, C. , Pawson, A. J. , Ireland, S. , … NC‐IUPHAR (2018). The IUPHAR/BPS Guide to PHARMACOLOGY in 2018: Updates and expansion to encompass the new guide to IMMUNOPHARMACOLOGY. Nucleic Acids Res., 46, D1091–D1106. 10.1093/nar/gkx1121 [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Hayashi, Y. , Koga, Y. , Zhang, X. , Peters, C. , Yanagawa, Y. , Wu, Z. , … Nakanishi, H. (2014). Autophagy in superficial spinal dorsal horn accelerates the cathepsin B‐dependent morphine antinociceptive tolerance. Neuroscience, 275, 384–394. 10.1016/j.neuroscience.2014.06.037 [DOI] [PubMed] [Google Scholar]
  17. Itakura, E. , & Mizushima, N. (2010). Characterization of autophagosome formation site by a hierarchical analysis of mammalian Atg proteins. Autophagy, 6, 764–776. 10.4161/auto.6.6.12709 [DOI] [PMC free article] [PubMed] [Google Scholar]
  18. Kabeya, Y. , Mizushima, N. , Ueno, T. , Yamamoto, A. , Kirisako, T. , Noda, T. , … Yoshimori, T. (2000). LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing. EMBO J., 19, 5720–5728. 10.1093/emboj/19.21.5720 [DOI] [PMC free article] [PubMed] [Google Scholar]
  19. Kenific, C. M. , & Debnath, J. (2014). Cellular and metabolic functions for autophagy in cancer cells. Trends Cell Biol., 25, 37–45. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Kilkenny, C. , Browne, W. , Cuthill, I. C. , Emerson, M. , & Altman, D. G. (2010). Animal research: Reporting in vivo experiments: The ARRIVE guidelines. British Journal of Pharmacology, 160, 1577–1579. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Kim, J. , Kundu, M. , Viollet, B. , & Guan, K. L. (2011). AMPK and mTOR regulate autophagy through direct phosphorylation of Ulk1. Nat. Cell Biol., 13, 132–141. 10.1038/ncb2152 [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Kim, K. H. , & Lee, M. S. (2014). Autophagy—A key player in cellular and body metabolism. Nat. Rev. Endocrinol., 10, 322–337. 10.1038/nrendo.2014.35 [DOI] [PubMed] [Google Scholar]
  23. Kimura, S. , Noda, T. , & Yoshimori, T. (2007). Dissection of the autophagosome maturation process by a novel reporter protein, tandem fluorescent‐tagged LC3B. Autophagy, 3, 452–460. 10.4161/auto.4451 [DOI] [PubMed] [Google Scholar]
  24. Klionsky, D. J. , Abeliovich, H. , Agostinis, P. , Abraham, R. T. , Acevedo‐Arozena, A. , Adeli, K. , et al. (2008). Guidelines for the use and interpretation of assays for monitoring autophagy in higher eukaryotes. Autophagy, 4, 151–175. 10.4161/auto.5338 [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Levine, B. , & Kroemer, G. (2008). Autophagy in the pathogenesis of disease. Cell, 132, 27–42. 10.1016/j.cell.2007.12.018 [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Liang, X. H. , Jackson, S. , Seaman, M. , Brown, K. , Kempkes, B. , Hibshoosh, H. , & Levine, B. (1999). Induction of autophagy and inhibition of tumorigenesis by beclin1. Nature, 402, 672–676. 10.1038/45257 [DOI] [PubMed] [Google Scholar]
  27. Lin, W. , Zhang, Z. , Xu, Z. , Wang, B. , Li, X. , Cao, H. , et al. (2015). The association of receptor of activated protein kinase C1 (RACK1) with infectious bursal disease virus viral protein VP5 and voltage‐dependent anion channel 2 (VDAC2) inhibits apoptosis and enhances viral replication. J. Biol. Chem., 290, 8500–8510. [DOI] [PMC free article] [PubMed] [Google Scholar]
  28. Lin, W. J. , & Kuang, H. Y. (2014). Oxidative stress induces autophagy in response to multiple noxious stimuli in retinal ganglion cells. Autophagy, 10, 1692–1701. 10.4161/auto.36076 [DOI] [PMC free article] [PubMed] [Google Scholar]
  29. Lin, X. , Wang, Y. J. , Li, Q. , Hou, Y. Y. , Hong, M. H. , Cao, Y. L. , … Liu, J. G. (2009). Chronic high‐dose morphine treatment promotes SH‐SY5Y cell apoptosis via c‐Jun N‐terminal kinase‐mediated activation of mitochondria‐dependent pathway. FEBS, J276, 2022–2036. [DOI] [PubMed] [Google Scholar]
  30. Liu, L. , Zhu, J. , Zhou, L. , & Wan, L. (2016). RACK1 promotes maintenance of morphine‐associated memory via activation of an ERK–CREB dependent pathway in hippocampus. Sci. Rep., 6, 20183 10.1038/srep20183 [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. McGough, N. N. , He, D. Y. , Logrip, M. L. , Jeanblanc, J. , Phamluong, K. , Luong, K. , … Ron, D. (2004). RACK1 and brain‐derived neurotrophic factor: A homeostatic pathway that regulates alcohol addiction. J. Neurosci., 24, 10542–10552. 10.1523/JNEUROSCI.3714-04.2004 [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Nikoletopoulou, V. , Sidiropoulou, K. , Kallergi, E. , Dalezios, Y. , & Tavernarakis, N. (2017). Modulation of autophagy by BDNF underlies synaptic plasticity. Cell Metab., 26, 230–242.e5. 10.1016/j.cmet.2017.06.005 [DOI] [PubMed] [Google Scholar]
  33. Pan, J. , He, L. , Li, X. , Li, M. , Zhang, X. , Venesky, J. , … Peng, Y. (2017). Activating autophagy in hippocampal cells alleviates the morphine‐induced memory impairment. Mol. Neurobiol., 54, 1710–1724. 10.1007/s12035-016-9735-3 [DOI] [PubMed] [Google Scholar]
  34. Pankiv, S. , Clausen, T. H. , Lamark, T. , Brech, A. , Bruun, J. A. , Outzen, H. , et al. (2007). p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquitinated protein aggregates by autophagy. J. Biol. Chem., 282, 24131–24145. [DOI] [PubMed] [Google Scholar]
  35. Pattingre, S. , Espert, L. , Biard‐Piechaczyk, M. , & Codogno, P. (2008). Regulation of macroautophagy by mTOR and Beclin 1 complexes. Biochimie, 90, 313–323. 10.1016/j.biochi.2007.08.014 [DOI] [PubMed] [Google Scholar]
  36. Rodríguez, A. E. , López‐Crisosto, C. , Peña‐Oyarzún, D. , Salas, D. , Parra, V. , Quiroga, C. , … Lavandero, S. (2016). BAG3 regulates total MAP1LC3B protein levels through a translational but not transcriptional mechanism. Autophagy, 12, 287–296. 10.1080/15548627.2015.1124225 [DOI] [PMC free article] [PubMed] [Google Scholar]
  37. Russell, R. C. , Tian, Y. , Yuan, H. , Park, H. W. , Chang, Y. Y. , Kim, J. , … Guan, K. L. (2013). ULK1 induces autophagy by phosphorylating Beclin‐1 and activating VPS34 lipid kinase. Nat. Cell Biol., 15, 741–750. 10.1038/ncb2757 [DOI] [PMC free article] [PubMed] [Google Scholar]
  38. Shen, W. , & Ganetzky, B. (2009). Autophagy promotes synapse development in Drosophila . J. Cell Biol., 187, 71–79. 10.1083/jcb.200907109 [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Stoica, L. , Zhu, P. J. , Huang, W. , Zhou, H. , Kozma, S. C. , & Costa‐Mattioli, M. (2011). Selective pharmacogenetic inhibition of mammalian target of rapamycin complex I (mTORC1) blocks long‐term synaptic plasticity and memory storage. Proc. Natl. Acad. Sci. U. S. A., 108, 3791–3796. 10.1073/pnas.1014715108 [DOI] [PMC free article] [PubMed] [Google Scholar]
  40. Wan, J. , Ma, J. , Anand, V. , Ramakrishnan, S. , & Roy, S. (2015). Morphine potentiates LPS‐induced autophagy initiation but inhibits autophagosomal maturation through distinct TLR4‐dependent and independent pathways. Acta Physiol (Oxf.), 214, 189–199. 10.1111/apha.12506 [DOI] [PMC free article] [PubMed] [Google Scholar]
  41. Wan, L. , Su, L. , Xie, Y. , Liu, Y. , Wang, Y. , & Wang, Z. (2009). Protein receptor for activated C kinase 1 is involved in morphine reward in mice. Neuroscience, 161, 734–742. 10.1016/j.neuroscience.2009.03.064 [DOI] [PubMed] [Google Scholar]
  42. Wan, L. , Xie, Y. , Su, L. , Liu, Y. , Wang, Y. , & Wang, Z. (2011). RACK1 affects morphine reward via BDNF. Brain Res., 1416, 26–34. 10.1016/j.brainres.2011.07.045 [DOI] [PubMed] [Google Scholar]
  43. Wong, M. (2013). Mammalian target of rapamycin (mTOR) pathways in neurological diseases. Biom. J., 36, 40–50. 10.4103/2319-4170.110365 [DOI] [PMC free article] [PubMed] [Google Scholar]
  44. Yan, J. , Porch, M. W. , Court‐Vazquez, B. , Bennett, M. V. L. , & Zukin, R. S. (2018). Activation of autophagy rescues synaptic and cognitive deficits in fragile X mice. Proc. Natl. Acad. Sci. U. S. A., A115, E9707–E9716. [DOI] [PMC free article] [PubMed] [Google Scholar]
  45. Yang, Z. , & Klionsky, D. J. (2010). Mammalian autophagy: Core molecular machinery and signaling regulation. Curr. Opin. Cell Biol., 22, 124–131. 10.1016/j.ceb.2009.11.014 [DOI] [PMC free article] [PubMed] [Google Scholar]
  46. Zhao, L. , Zhu, Y. , Wang, D. , Chen, M. , Gao, P. , Xiao, W. , … Chen, Q. (2010). Morphine induces BECLIN 1‐ and ATG5‐dependent autophagy in human neuroblastoma SH‐SY5Y cells and in the rat hippocampus. Autophagy, 6, 386–394. 10.4161/auto.6.3.11289 [DOI] [PubMed] [Google Scholar]
  47. Zhao, Y. G. , Sun, L. , Miao, G. , Ji, C. , Zhao, H. , Sun, H. , … Zhang, H. (2015). The autophagy gene Wdr45/Wipi4 regulates learning and memory function and axonal homeostasis. Autophagy, 11, 881–890. 10.1080/15548627.2015.1047127 [DOI] [PMC free article] [PubMed] [Google Scholar]
  48. Zhu, J. , Chen, X. , Song, Y. , Zhang, Y. , Zhou, L. , & Wan, L. (2016). Deficit of RACK1 contributes to the spatial memory impairment via upregulating BECLIN1 to induce autophagy. Life Sci., 151, 115–121. 10.1016/j.lfs.2016.02.014 [DOI] [PubMed] [Google Scholar]

Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1. Morphine did not show any cytotoxic activity in vitro.

Figure S2. Chloroquine (10 μM) enhanced morphine‐induced autophagy and RACK1 up‐regulation in PC12 cells.

Figure S3. Chloroquine (10 μM) enhanced morphine‐induced autophagy and RACK1 up‐regulation in HT‐22 cells.

Click here for additional data file. (494.5KB, doc)

Data S1. Supporting Information

Click here for additional data file. (43KB, doc)

Articles from British Journal of Pharmacology are provided here courtesy of The British Pharmacological Society

RESOURCES