Cannabidiol induces autophagy via ERK1/2 activation in neural cells

Autophagy is a lysosomal catabolic process essential to cell homeostasis and is related to the neuroprotection of the central nervous system. Cannabidiol (CBD) is a non-psychotropic phytocannabinoid present in Cannabis sativa. Many therapeutic actions have been linked to this compound, including autophagy activation. However, the precise underlying molecular mechanisms remain unclear, and the downstream functional significance of these actions has yet to be determined. Here, we investigated CBD-evoked effects on autophagy in human neuroblastoma SH-SY5Y and murine astrocyte cell lines. We found that CBD-induced autophagy was substantially reduced in the presence of CB1, CB2 and TRPV1 receptor antagonists, AM 251, AM 630 and capsazepine, respectively. This result strongly indicates that the activation of these receptors mediates the autophagic flux. Additionally, we demonstrated that CBD activates autophagy through ERK1/2 activation and AKT suppression. Interestingly, CBD-mediated autophagy activation is dependent on the autophagy initiator ULK1, but mTORC1 independent. Thus, it is plausible that a non-canonical pathway is involved. Our findings collectively provide evidence that CBD stimulates autophagy signal transduction via crosstalk between the ERK1/2 and AKT kinases, which represent putative regulators of cell proliferation and survival. Furthermore, our study sheds light on potential therapeutic cannabinoid targets that could be developed for treating neurodegenerative disorders.

Current scientific efforts have focused on describing the broad spectrum of therapeutic effects for cannabidiol (CBD), the major non-psychotomimetic phytocannabinoid derived from the plant Cannabis sativa 1 . Extensive studies have shown that CBD has several pharmacological properties, including antidepressant 2 , anti-inflammatory 3 , antiemetic 4 , neuroprotective 5,6 , analgesic 7 , antibacterial 8 , anticonvulsant 9 , anxiolytic 10 , antipsychotic 11 , antitumor 12 and immunomodulatory activity 13 . However, the underlying mechanisms involved in these properties are still under discussion.
Canonical cannabinoid receptors include CB1 and CB2. The CB1 isoform is highly expressed in the hippocampus, amygdala, basal ganglia, cortex and cerebellum. On the other hand, the CB2 isoform is mainly found in peripheral and immune tissues as well as in microglial and cerebral granule cells 14 . Previous work has shown that endo/exocannabinoids activate G i/o coupled CB1 and CB2 receptors and inhibit adenylyl cyclase 15 , consequently increasing the influx of K + into the cell, resulting in membrane hyperpolarization in the pre-synaptic neurons 16 . Additionally, it has been reported that CBD can modulate the activity of non-canonical receptors, including serotonin 1A receptor (5HT 1A ) 17 , peroxisome proliferator-activated receptor γ (PPARγ) 18 , G-protein coupled receptor 55 (GPR55 19 ), μ-and δ-opioid 20 , as well as Transient Receptor Potential Vanilloid 1 (TRPV1) cation channels 21 . It is plausible that this broad range of receptor targets could account for the plethora of CBDinduced pharmacological effects in different tissues. Concerning the TRPV1 receptor, it is a tetrameric protein and a member of the Transient Receptor Potential (TRP) superfamily of nonselective cation channels 22 . Several studies have reported that the TRPV1 receptor is activated by spice ingredients such as capsaicin, low pH, heat (> 43 °C), endovanilloids and endocannabinoids such as N-arachidonoylethanolamine (known as anandamide), and/or exocannabinoids such as CBD 21,[23][24][25] .

CBD regulates cell death in a concentration and time-dependent manner. Previous studies
have demonstrated in different cell lines that a relationship exists between autophagy and CBD-mediated cell death 31,37 . To explore this possibility, we used the Propidium Iodide (PI) cell cycle assay to monitor nuclear DNA content in SH-SY5Y cell populations in the presence and absence of CBD and monitored cell death as a function of time. In this assay, the sub-G 0 /G 1 fraction is an indicator of cell death 38 .

CBD induces autophagic flux in a concentration-dependent manner.
While several studies have shown that CBD can potentially modulate autophagy [39][40][41] , none evaluated whether this drug activates autophagy in a neuronal cell model. For this purpose, we applied different concentrations of CBD (5, 10 and 50 µM) for 2 h in the presence or absence of 10 mM ammonium chloride (NH 4 Cl), a lysosome inhibitor added 1 h before the end of treatment and assessed autophagic flux status. Then we measured LC3-II protein expression, which is indicative of autophagosome accumulation 42,43 . However, the same treatment was not able to modulate the p62 levels ( Supplementary Fig. S1E,F).
The data analysis of the LC3-II expression patterns in the presence and absence of the lysosome inhibitor revealed a significant interaction between CBD and NH 4 Cl groups [F 3,52 = 3.499; p = 0.022]. Moreover, we found that CBD increased LC3-II levels in a concentration-dependent manner, a result that was further potentiated by NH 4 Cl. These results confirm that CBD treatment modulated autophagic flux of SH-SY5Y cells. Sidak's posthoc test showed that 10 and 50 µM CBD under NH 4 Cl treatment increased LC3-II immunoreactivity relative to the control group (untreated cells) both in the presence (p < 0.05) and absence (p < 0.01) of NH 4 Cl ( Fig. 2A).
Next, we determined if the observed CBD-mediated increase in LC3-II expression on autophagic flux was time-dependent. Here SH-SY5Y cells were treated with CBD (1, 5 and 10 µM) for 1, 2 or 4 h, and in some cases, NH 4 Cl was added 1 h before the end of CBD treatment to block autophagy. Analyses of the autophagic flux status, assessed by LC3-II immunoreactivity, indicated interactions between CBD and NH 4 Cl treatment and time interval [F 6,78 = 3.96, p = 0.002]. Additionally, Sidak's post-hoc test showed higher levels of LC3-II levels in cells treated with 10 µM CBD than the respective control group (NH 4 Cl) after 1, 2 (p < 0.001) and 4 h (p = 0.002) of CBD treatment. Notably, NH 4 Cl potentiated LC3-II levels only in the cells treated with 10 µM CBD, which reached a maximum effect at 1 and 2 h after CBD treatment. Since NH 4 Cl treatment had no additional effect on LC3-II levels with 1 and 5 µM of CBD-treated cells, we can conclude that autophagic flux was not activated with those concentrations (Fig. 2B www.nature.com/scientificreports/ To further investigate the ability of CBD to induce autophagy in SH-SY5Y cells, we used a stable cell line overexpressing a fluorescent-tagged mCherry-LC3. These cells were subjected to starvation (STV) in Earle's Balanced Salt Solution (EBSS) and treated with 10 µM CBD for 2 h. The one-way ANOVA showed that CBD induced the formation of a more significant number of intracellular red puncta [F 2,22 = 16.148, p < 0.001]. Furthermore, Dunnett's post-hoc test found that red puncta were significantly more prevalent in the cells treated with CBD (p = 0.002) and STV (p < 0.001) when compared to the control group (Fig. 2D,E). These data indicate that CBD activated the autophagosome and autolysosome formation.
The role of CB1, CB2 and TRPV1-receptor activation in CBD-induced autophagy in SH-SY5Y and astrocytes cells. The observation that 10 µM CBD did not reduce cell viability but did promote autophagic flux led us to investigate whether CBD could activate autophagy via canonical cannabinoid receptors. For this purpose, we assessed the autophagic flux in two groups of SH-SY5Y or astrocyte cells. One group of each cell type was treated with 10 µM CBD for 2 h. The other groups of cells were pre-treated (30 min) with CB1, CB2 and TRPV1 antagonists, AM 251, AM 630 and CPZ (10 µM), respectively, in the presence or absence of the lysosome inhibitor NH 4 Cl during the last hour of the treatment.
The three-way ANOVA identified a significant interaction for CBD, NH 4 Cl and the respective antagonists in each condition (p < 0.05). For example, Sidak's post-hoc test showed that NH 4 Cl blocked autophagic flux, as evidenced by the augmented levels of LC3-II in the NH 4 Cl treated group relative to the control group (p < 0.05). Additionally, CBD treatment consistently increased LC3-II expression (i.e., autophagic flux) under conditions of NH 4 Cl blockade when compared to the respective control groups. Notably, AM 251, AM 630 or CPZ attenuated LC3-II expression to untreated NH 4 Cl-blocked control levels in both SH-SY5Y cells ( Fig. 3A-C) and astrocytes ( Fig. 3D-F). A summary of the statistics and the respective F and p-values associated with these experiments are provided in Supplementary Tables S1 and S2. These results provide evidence that CBD-induced autophagy is mediated by the CB1, CB2 and TRPV1 receptors.
CBD activates autophagy via crosstalk with the ERK1/2 and PI3K/AKT pathways. Having identified receptors that appear to be involved in the CBD-induced autophagy, we then focused attention on the putative signaling pathway(s). Previous studies have shown that the PI3K/AKT and MAPK/ERK pathways regulate www.nature.com/scientificreports/ cell metabolism, growth, proliferation 35,36 and participate in autophagy signal transduction 44,45 . Thus, we monitored the phosphorylated and total levels of ERK1/2 (Tyr204/Thr202) and AKT (Ser473) after treating SH-SY5Y cells with 10 µM CBD for 5, 10, 30 min, 1 and 2 h (Fig. 4A). This approach showed that CBD increased ERK1/2 phosphorylation in a time-dependent, bell-shaped fashion. A one-way ANOVA with a polynomial contrast to data regarding p-ERK/total ERK ratio detected a significant effect of the quadratic term on the time interval [F 1,24 = 4.307; p = 0.049]. Furthermore, Dunnett's post-hoc test found that 10 µM CBD significantly increased the p-ERK1/2/total ERK1/2 ratio after only 10 min of treatment (p = 0.04). It should be pointed out that this value returned to control levels after 2 h. Next, we investigated whether CBD-mediated ERK1/2 activation could induce autophagy in SH-SY5Y cells. In this experiment, untreated and CBD-treated cells were pre-treated with 20 µM of the Mitogen-Activated Protein Kinase (MAPK) kinase 1/2 inhibitor (MEK1/2), U0126, for 30 min and assessed CBD autophagic flux 46 . The two-way ANOVA showed that U0126 effectively inhibited CBD-induced ERK phosphorylation [F 1,12 = 7.120; p = 0.017] (Fig. 4B). Additionally, the three-way ANOVA showed that U0126 pre-treatment abolished the CBDinduced increase in LC3-II levels [F 1,24 = 4.763 p = 0.037] (Fig. 4C). These results confirm that ERK1/2 signaling is an upstream regulator of CBD-mediated autophagy.
Similar experiments were also utilized to evaluate the p-AKT/total AKT ratio for up to 2 h after CBD. The one-way ANOVA highlighted a significant effect for CBD treatment [F 5,24 = 5.236; p = 0.002]. The Dunnett's posthoc test showed that CBD treatment led to a sustained reduction in p-AKT (Ser473) levels during the entire experimental period (p < 0.01) (Fig. 4D).
Lastly, we evaluated the effects of CBD on AMP-activated protein kinase α (AMPKα) phosphorylation at Thr172 after 1, 2 and 4 h. While there was a tendency for the p-AMPKα/total AMPKα ratio to appear reduced  To further clarify that the ERK1/2 and AKT pathways were modulating mammalian target of rapamycin complex 1 (mTORC1) and Unc51-like kinase (ULK1) signaling via coordinated mechanisms, we evaluated downstream regulators of mTORC1, including the phosphorylation levels of p70 ribosomal protein S6 kinase β 1 (p70S6K1; Thr389) and ULK1 (Ser757) following CBD treatment for 1, 2 and 4 h. As shown in Our findings collectively reveal a possible non-canonical autophagy activation mechanism in neural cells that involves crosstalk between the upstream regulators ERK1/2, AKT kinase, and downstream ULK1 signaling, which is not dependent on mTORC1 activity. Furthermore, the present study highlights the therapeutic potential of cannabinoid treatment for neurodegenerative disorders (Fig. 6).

Discussion
Studies investigating the utility and efficacy of CBD in a variety of conditions and diseases have been gaining momentum in the last decades. Much of this increase in interest is due to the broad spectrum of potential therapeutic benefits and lack of psychotomimetic effects 1 . Recent studies have reported that cannabinoid-induced autophagy in cancerous and non-cancerous cells can elicit either protective or harmful effects 48,49 . Herein we sought to evaluate the mechanism by which CBD regulates autophagy, an essential protein-recycling pathway in neural cells.
In our study, 10 µM CBD modulated the autophagic flux without inducing cytotoxicity 41,50 . We focused on CBD nontoxic concentrations to evaluate the autophagic flux and compared unstimulated cells and CBD-stimulated cells with different concentrations in the presence, or absence, of the lysosomal inhibitor NH 4 Cl according  www.nature.com/scientificreports/ to Autophagy Guidelines 51 . In order to discriminate the increased autophagosome formation promoted by the CBD treatment, the inhibition of lysosomal activity was necessary to lead the "undigested" lipidated LC3-II to accumulation. Under NH 4 Cl blockade, LC3-II is available to be analyzed by comparing the levels in the untreated cells with respect to the one treated with lysosomal inhibitors (e.g. NH 4 Cl) to measure the rate of autophagosome degradation defined as autophagic flux 51 . Interestingly, the NH 4 Cl treatment induced a further increased LC3-II in CBD-treated cells to higher levels when compared to control cells, indicating that LC3-II formation are not due to inhibition of autophagic machinery but due to enhanced autophagy progression that can lead to faster LC3-II degradation, as observed in control cells. Therefore, based on the results obtained, we believe that the increase in autophagic flux mediated by CBD alone was not observed due to the high degradation of LC3-II without the lysosomal inhibitor, NH 4 Cl. However, using the autophagy inhibitor we are able to observe the increase in autophagosomes (LC3-II formation) mediated by CBD. This observation indicates that a specific concentration of CBD can activate autophagy and protect cells from potentially damaging conditions. Such effects are particularly relevant to neurodegenerative diseases since the autophagy-mediated degradation of misfolded proteins and protein aggregates is necessary for cellular homeostasis and survival 52,53 . As shown by Casarejos et al. (2013), Sativex, a drug that contains Δ 9 -THC and CBD, can increase autophagy, consequently reducing tau and amyloid proteins deposition in a tauopathy mouse model 54 . The membranes were incubated with specific antibodies directed towards phosphorylated kinase ERK1/2 (Tyr204/Thr202) or total ERK1/2 protein. Treatment with 10 µM CBD (10 µM) increased phosphorylated ERK1/2 (p-ERK) protein levels in SH-SY5Y cells after 10 min. (B) Cells pre-treated with 20 µM of the ERK1/2 inhibitor U0126 for 30 min and then exposed to 10 µM CBD for 10 min did not exhibit increased levels of phosphorylated ERK1/2 (p-ERK) protein in SH-SY5Y cells. (C) Cells were pre-treated with 20 µM U0126 for 30 min and then exposed to 10 µM CBD for up to 2 h, in the presence or absence of the lysosome inhibitor NH 4 Cl, at the last hour of the treatment. The 10 µM CBD treatment increased LC3-II levels under NH 4 Cl treatment, which was reverted with U0126 treatment. (D) Total cell lysates from SH-SY5Y cells treated with 10 µM CBD for 5, 10, 30 min, 1 and 2 h. Immunoblots were incubated with a specific antibody for phosphorylated AKT (Ser473) or with an antibody that recognizes total AKT and an antibody against GAPDH. CBD decreased the levels of phosphorylated AKT protein in the SH-SY5Y cells. (E) Cells were treated with 10 µM CBD for 1, 2 and 4 h, and the blots were incubated with a specific antibody for phosphorylated AMPK (Thr172) or with an antibody that recognizes total AMPK and an antibody directed against α-tubulin. No effect on AMPK phosphorylation (Thr172) was observed. Analysis was conducted with one, two, or three-way ANOVA followed by Sidak's post-hoc test; *p < 0.05, ** p < 0.01 and ***p < 0.001. Full-length blots are presented in Supplementary Figure S3 31,40,50 , similar to the results presented here. Despite these promising results, the pathophysiological circumstances and the underlying CBDmediated autophagy activation mechanisms must be thoroughly considered when deciding whether CBD should be administered or not and will require further investigation. Besides the autophagy-related effects, cannabinoids have also been shown to modulate intracellular signaling pathways involved in neuronal survival and apoptosis 55,56 . In this sense, it is essential to determine if the cannabinoid induces autophagy and elucidate the mechanism(s) of action and pathological processes to determine the therapeutic benefits they can evoke 57,58 . Indeed, it is for this reason that we also examined the activation pathways associated with the CBD receptors. Total lysates from SH-SY5Y cells were treated with 10 µM CBD for 1, 2 and 4 h. The blots were incubated with a specific antibody for phosphorylated p70S6K1 (Thr389) and ULK1 (Ser757) or with an antibody that recognizes total p70S6K1 and ULK1. No effect was observed on the mTOR targets (A) p70S6K1 (Thr389) and (B) ULK1 (Ser757) after treatment with CBD, thus indicating that autophagy activation is independent of mTORC1. Data are expressed as means ± S.E.M., and data points are displayed as dots. The analysis was conducted with a one-way ANOVA followed by Dunnett's post-hoc test. *p < 0.05, ** p < 0.01 relative to respective CTR group. (C) The autophagic flux was evaluated in SH-SY5Y cells pretreated with 1 µM of the ULK1/2 inhibitor MRT68921 for 30 min) and treated with 10 µM CBD for 2 h, in the presence or absence of the lysosome inhibitor NH 4 Cl, at the last hour of the treatment. The 10 µM CBD treatment increased LC3-II levels under NH 4 Cl blockade, which was abolished MRT6892 pre-treatment. Samples were subjected to western blotting using anti-LC3 and anti-GAPDH antibodies. Representative images of LC3-II are shown above the bar graphs. The bar graphs are reported as the means ± S.E.M of LC3-II levels after GAPDH normalization. Analysis was conducted with Three-way ANOVA followed by Sidak's post-hoc test; *p < 0.05, ** p < 0.01 and ***p < 0.001. Full-length blots are presented in Supplementary Figure S4 www.nature.com/scientificreports/ For example, using CB1 and CB2 antagonists, AM 251 and AM 630, respectively 40,59-61 , as well as the capsazepine, the vanilloid receptor antagonist, which is widely used to inhibit TRPV1 receptors 40,62 , we found that all of the employed antagonists tested attenuated CBD-induced autophagy in both SH-SY5Y cells and astrocytes. These results strongly indicate that the activation of these three CBD-sensitive receptors is involved in autophagosome formation and degradation. While identifying these receptors as active participants in the observed CBD-induced autophagy provided valuable insights into the mechanism of action, it was still necessary to assess the effects CBD elicits after binding the receptors.
The PI3K/AKT and MAPK/ERK pathways are considered crucial regulators of cell metabolism, growth, proliferation 35,36 and vital to autophagy signal transduction 44,45 . Here we showed that CBD could activate autophagy by stimulating ERK1/2 and inhibit AKT signaling via the differential phosphorylation of these proteins. It is worth mentioning that CBD did not affect AMPK phosphorylation and, consequently, its activity, which further confirms the crucial role of the crosstalk between the autophagy-related kinases, ERK1/2 and AKT. Indeed, the regulation of PI3K/AKT is negatively modulated by MAPK/ERK activation. This conclusion is in line with a previous study that showed the inhibition of ERK1/2 signaling, using U0126, increased AKT phosphorylation at Thr308 and Ser473, and the activation of the ERK/MAPK cascade attenuated growth factor-induced AKT phosphorylation 63 . Furthermore, another study reported that U0126-mediated inhibition of ERK1/2 signaling blocks the proliferation of neural progenitor cells 64 .
Since ERK1/2 activation has been associated with cannabinoid receptors 65 , it is plausible that CB1 activation by cannabinoids is involved in neuronal protection and proliferation 66 . In this context, the link between CBD-mediated autophagy and AKT is relevant to neurodegenerative diseases. For example, 10 µM CBD exerted protective effects via AKT reduction in an SH-SY5Y cell model of Parkinson's disease induced by the toxin Figure 6. Schematic representation of the signaling pathway mediated by CBD in SH-SY5Y cells. At 10 µM, CBD activates autophagy, resulting in the accumulation of LC3-II and increased presence of LC3-positive puncta. AM 251, AM 630 and CPZ antagonists abrogated the observed LC3-II accumulation, thus implicating canonical cannabinoids (CB1 and CB2) and TRPV1 receptors in CBD-mediated autophagy. Additionally, CBD activated the ERK1/2 signaling cascade by increasing phosphorylation at Tyr204/Thr202 after 10 min of CBD treatment but suppressed the PI3K/AKT pathway by reducing AKT phosphorylation at S473. On the other hand, CBD treatment did not change AMPK phosphorylation at Thr172 or the phosphorylation levels of autophagy-related proteins ULK1 (Ser757) and p70S6K (Thr389). In conclusion, our findings suggest that CBD-induced autophagy activation is dependent on ULK1 and occurs in an mTORC1 independent-manner. Furthermore, the effects on the ERK1/2 and PI3K/AKT pathways, which regulate cell survival and proliferation, indicate that CBD could elicit neuroprotective actions. The illustration was produced using smart servier medical art vectors for publications and presentations licensed under the Creative Commons (CC BY 3.0) available at smart.servier.com. www.nature.com/scientificreports/ 1-methyl-4-phenylpyridinium (MPP + ) 67 . Indeed, CB1 68 or TRPV1 receptors 69 have been shown to differentially modulate the PI3K/AKT pathways, depending on the cell type, treatment or injury 70,71 . It has been demonstrated that mTORC1 activity negatively regulates the ULK1 complex, which is required for autophagy initiation 72 . For this purpose, we investigated if the CBD-induced autophagy would be dependent on ULK1 pathway. Pre-treatment with MRT68921, a potent and selective ULK1/2 inhibitor 47 , abolished the further increase in LC3-II observed in the presence of CBD. Collectively, our findings suggest a cross talk between the upstream regulators ERK1/2 and AKT kinase through ULK1 signaling, but not dependent on mTORC1 activity. Here, we found that CBD activated the autophagic flux in an mTORC1 independent-manner since its protein targets, p70S6K1 (Thr389) and ULK1 (Ser757), were not modulated upon CBD treatment. These findings are consistent with the study of Hiebel et al. that demonstrated CB1 receptors could modulate autophagy activation in an mTORC1 and Beclin-1 independent manner 73 . In contrast, here we report that CBD-mediated autophagy depends on the proautophagic factor ULK1, suggesting a novel noncanonical autophagy signaling pathway involving direct cross talk by ERK1/2 and PI3K/AKT. These upstream autophagic-related kinases have a putative role in linking growth factor signals and mTORC1 activity 74 .

Scientific Reports
Concerning AKT, once activated, it phosphorylates and inactivates the tuberous sclerosis complex (TSC1/2 complex), a critical negative regulator of mTORC1 activity 74 . In contrast, CBD inhibits AKT but not mTORC1. Additionally, ERK1/2 inhibits TSC2 in response to growth factor 75 . Herein, we found that CBD inhibits AKT and activates ERK1/2; hence, it is likely that ERK1/2 would inhibit TSC2, resulting in unaltered mTORC1 and stimulated ULK1 activity. This possible mechanism was previously reported in a study showing that trehalose promoted cell clearance in neurodegenerative storage diseases models 76 . While the present study revealed novel therapeutic targets for the treatment of neurodegenerative processes, future studies will need to be carried out to understand the exact mechanisms related to regulating autophagosome biogenesis.

Conclusions
As depicted in Fig. 6, the present study demonstrated that CBD plays an important role in autophagy activation by regulating the phosphorylation of ERK1/2 and AKT kinases with involvement of ULK1, but in an mTORC1independent pathway. The activation of these signaling pathways involves the cannabinoid receptors, CB1 and CB2, as well as TRPV1 receptors, which are essential for promoting neuronal cell survival and proliferation. This study not only provides details about the underlying mechanisms involved in cannabinoid-mediated autophagy activation but also highlights the promising potential of cannabidiol therapy for neurodegenerative disorders.

Material and methods
Drugs. Cannabidiol  Cell culture. The human neuroblastoma (SH-SY5Y-ATCC-CRL-2266) and murine immortalized astrocyte 77 cell lines were cultured in high-glucose Dulbecco's Modified Eagle Medium, supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA, USA) and maintained at 37°C in a 5% CO 2 atmosphere 78 . These neural cell lines have been extensively used in in vitro studies involving cell death mechanisms and express functional CB1, CB2 and TRPV1 receptors 79,80 . The cells were transferred to 6-well plates (5 × 10 5 cells/well) for the flow cytometry, immunoblotting and immunofluorescence assays (Thermo Fisher Scientific). The Institutional Ethics Committee of the Federal University of São Paulo (UNIFESP) approved all of the experimental protocols and procedures (Protocol number 5810061017).
Cell death measurements. Cells were treated with seven concentrations of CBD (1, 2, 5, 10, 25, 50, 100 µM). As a positive control, 1 µM staurosporine (STS), a nonselective protein kinase inhibitor that leads cells to apoptosis 81 , was used. The cells were fixed using cold ethanol (50% in PBS) after 24 or 48 h of treatment. The fixed cells were then pelleted and stained with 25 μg/mL Propidium Iodide (PI) in PBS. Flow cytometry experiments to calculate the percentage of cells in the sub-G 0 /G 1 phase were performed with the BD FACSCalibur platform (Becton-Dickinson, Mountain View, CA, USA), and the data were acquired with the FL2 channel considering 10,000 events. The results were analyzed using the FlowJo Software (BD Biosciences, Franklin Lakes, NJ, USA).
Retroviral transduction and confocal microscopy for autophagy evaluation. As previously described 82 , to produce the virus for this protocol, 15 µg of the pLPCX-mCherry-LC3 plasmid and 5 µg of vesicular stomatitis virus G protein expression plasmid were transfected into the 293 gp/bsr cell line using the calcium phosphate protocol 83 . After 48 h, the retroviral particles present in the supernatant were recovered, supplemented with polybrene 4 µg/mL and stored at -80 °C until the transduction. The mCherry-LC3-overexpressing SH-SY5Y cells were plated on 13 mm glass coverslips at a density of 5 × 10 5 cells/mL. After 24 h, the cells were treated with 10 µM CBD for 2 h. Following this treatment, cells were fixed with 4% paraformaldehyde and observed under a Zeiss LSM 780 Confocal Microscopy (Carl Zeiss, Oberkochen, Germany). Images were analyzed with the ImageJ software (NIH, Bethesda, MD, USA).
Statistics. The Shapiro-Wilk and Levene's tests were used to evaluate data normality and homogeneity of variance assumptions, respectively, and parametric hypothesis tests were employed according to the data distribution. Briefly, one, two, or three-way Analysis of Variance (ANOVA) was applied to experimental factors. The results were used to estimate single treatment and multi-treatment interaction effects on the observed variable. Differences between groups were detected using Sidak's or Dunnett's post-hoc tests when appropriate. Data are expressed as the mean ± standard error of the mean (S.E.M.), and the significance level was set at p < 0.05. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.