AUTOPHAGY 2016, VOL. 12, NO. 8, 1310–1329 http://dx.doi.org/10.1080/15548627.2016.1183844

BASIC RESEARCH PAPER

Cocaine induces astrocytosis through ER stress-mediated activation of autophagy Palsamy Periyasamy, Ming-Lei Guo, and Shilpa Buch Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USA

ABSTRACT

ARTICLE HISTORY

Cocaine is known to induce inflammation, thereby contributing in part, to the pathogenesis of neurodegeneration. A recent study from our lab has revealed a link between macroautophagy/autophagy and microglial activation. The current study was aimed at investigating whether cocaine could also mediate activation of astrocytes and, whether this process involved induction of autophagy. Our findings demonstrated that cocaine mediated the activation of astrocytes by altering the levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 in both human A172 astrocytoma cells and primary human astrocytes. Furthermore, cocaine treatment resulted in increased formation of endogenous MAP1LC3B puncta in human astrocytes. Additionally, astrocytes transfected with the GFPMAP1LC3B plasmid also demonstrated cocaine-mediated upregulation of the green fluorescent MAP1LC3B puncta. Cocaine-mediated induction of autophagy involved upstream activation of ER stress proteins such as EIF2AK3, ERN1, ATF6 since blockage of autophagy using either pharmacological or genesilencing approaches, had no effect on cocaine-mediated induction of ER stress. Using both pharmacological and gene-silencing approaches to block either ER stress or autophagy, our findings demonstrated that cocaine-induced activation of astrocytes (measured by increased levels of GFAP) involved sequential activation of ER stress and autophagy. Cocaine-mediated-increased upregulation of GFAP correlated with increased expression of proinflammatory mediators such as TNF, IL1B, and IL6. In conclusion, these findings reveal an association between ER stress-mediated autophagy and astrogliosis in cocaine-treated astrocytes. Intervention of ER stress and/or autophagy signaling would thus be promising therapeutic targets for abrogating cocaine-mediated neuroinflammation.

Received 24 July 2015 Revised 20 April 2016 Accepted 25 April 2016

Introduction Cocaine is a potent psychostimulant whose repeated abuse significantly alters the functioning of the central nervous system (CNS). Cocaine potentially modulates the immune response in the CNS leading to a neuroinflammatory state that is characterized by enhanced activation of glial cells such as the astrocytes and microglia, in the brains of addicts.1 Astrocytes are the most abundant glial cells in the brain and play critical roles in a plethora of CNS processes such as, neurogenesis through the formation, maintenance, and pruning of synapses and neuronal survival through the uptake and release of glutamate, scavenging of free radicals, and the production of cytokines and nitric oxide to maintain neuronal homeostasis.2 Paradoxically, astrocytes can also become rapidly activated following exposure to diverse toxic stimuli, leading in turn, to a pathological state termed as reactive astrocytosis that is characterized by upregulation of the intermediate filament proteins GFAP (glial fibrillary acidic protein) and VIM (vimentin), both of which are associated with cellular hypertrophy and proliferation.3 In addition, astrocytosis has been reported as a hallmark feature underlying various psychiatric and neurological

KEYWORDS

astrogliosis; autophagy; cocaine; ER stress; GFAP; proinflammatory cytokines; unfolded protein response

disorders, such as Alzheimer disease,4 amyotrophic lateral sclerosis,5 cerebral ischemia,6 epilepsy,7 HIV infection,8 multiple sclerosis,9 and Parkinson disease.10 Cocaine binds and inhibits the dopamine transporter activity resulting in elevation of synaptic dopamine concentration leading, in turn, to increased excitability of D1-type neurons in the striatum.11 Over the years, the role of cocaine in activating glia and thereby contributing to the development of addiction has gained momentum.11-13 Glia such as the astrocytes play critical roles in supporting and maintaining neuronal homeostasis, based on their ability to form the tripartite synapses with neurons to modulate synaptic transmission. In microglia, cocaine has been demonstrated to induce the activation via both the endoplasmic reticulum (ER) stress14 and autophagy pathways.15 The mechanism(s), by which cocaine induces astrocyte activation however, remains less clear. The goal of the current work was to elucidate the molecular mechanisms by which cocaine induced astrogliosis. Numerous studies suggest that ER stress stimulates an evolutionarily conserved adaptive mechanism recognized as the unfolded protein response, which combines the early inhibition of protein synthesis with a later upregulation of genes that

CONTACT Shilpa Buch [email protected] Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE 68198-5880, USA. Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/kaup. Supplemental data for this article can be accessed on the to publisher’s website. © 2016 Taylor & Francis

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stimulate protein folding or clearance. ER stress response is transmitted via activation of 3 ER transmembrane proteins: ERN1 (endoplasmic reticulum [ER] to nucleus signaling 1), EIF2AK3 (eukaryotic translation initiation factor 2 a kinase 3), and ATF6 (activating transcription factor 6) that co-operatively induce global gene expression changes to restore ER homeostasis. Prolonged ER stress, if not alleviated, leads to chronic and sustained unfolded protein response which, in turn, activates apoptosis by upregulating ATF4 (activating transcription factor 4). ATF4 then promotes both the transcription of prosurvival genes and the expression of the proapoptotic transcription factor, DDIT3 (DNA-damage inducible transcript 3) and caspases, culminating in cell death.16 Emerging data also suggest a possible interplay between ER stress and the activation of autophagy.16 Autophagy is an evolutionarily conserved, key physiological process that promotes the turnover of cellular organelles and macromolecules through lysosomal degradation, thereby regulating cellular homeostasis and differentiation. Autophagy initiation begins with the membrane distention from either the ER or Golgi complex, followed by formation of a double-membrane structure known as the autophagosome, which, in turn, sequesters cellular components and subsequently fuses with the lysosome to form an autolysosome, the site for degradation of the sequestered cargo by the action of the lysosomal hydrolytic enzymes. Autophagy-related genes such as ATG5 (autophagy-related 5), and ATG7 (autophagy- related 7) are crucial for the formation of autophagosomes. MAP1LC3B (microtubule-associated protein 1 light chain 3 b) is a mammalian ortholog of yeast Atg8. During the initiation step, ATG12 (autophagy-related 12) conjugates ATG5 and promotes the conjugation of MAP1LC3B-I to phosphatidylethanolamine leading to the conversion of the soluble form of MAP1LC3B-I to the lipidated form, MAP1LC3B-II. MAP1LC3B-II attaches to the membrane of phagophores and is commonly used as a unique marker of autophagosomes as a measure of autophagic activity. Efficient autophagic activity is also essential for cell survival; however, similar to ER stress, extended autophagic activity can also lead to cell death.16 Autophagy has been identified as a critical player in various diseases such as cancer, diabetes, cardiovascular and neurodegenerative diseases.17 Taken together, the current work is based on the hypothesis that exposure of astrocytes to cocaine leads to induction of ER stress with subsequent activation of autophagy that ultimately culminates into increased cellular activation that is accompanied by increased expression of GFAP and proinflammatory cytokines. To validate this hypothesis, we examined the expression profiles of ER stress and autophagy signaling pathway markers, intracellular autophagosome formation, measured autophagic flux through MAP1LC3B turnover and SQSTM1 (sequestosome 1) degradation, GFAP expression patterns, and the levels of proinflammatory cytokines in both human A172 astrocytoma cells and primary human astrocytes following exposure to cocaine. Our findings highlight the association between ER stress-mediated activation of autophagy and astrogliosis in cocaine-treated human astrocytes. Intervention of either ER stress and/or autophagy signaling would thus provide a promising therapeutic target for astrocytosis induced by cocaine.

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Results Cocaine induced ER stress in the human astrocytoma cell line A172 and in primary human astrocytes Herein we sought to examine whether exposure of both the human A172 astrocytic cell line as well as primary human astrocytes to cocaine could lead to the induction of the signature ER stress sensors. Since activation of ER stress mainly relies on the concentration and exposure time of the ER stressors,18 it was imperative to first ascertain these 2 critical factors in the in vitro study. Our findings demonstrated that cocaine dose-dependently (1 to 100 mM) activated the classical ER stress signaling markers in human A172 astrocytoma cells at 12 h postexposure. As shown in Figure 1A and 1B, with increasing concentrations of cocaine, there was a corresponding increase in the phosphorylation level of the ER sensors, EIF2AK3 and EIF2S1 (eukaryotic translation initiation factor 2, subunit 1 a), leading in turn, to a significant (P < 0.05) increase in the phosphorylated (p)-EIF2AK3:EIF2AK3 and p-EIF2S1: EIF2S1 ratios, in human A172 astrocytoma cells. Convincingly, the protein levels of ATF6, ERN1, HSPA5 (heat shock protein 5), and DDIT3 were significantly (P < 0.05) increased in a dose-dependent manner (Fig. 1C to 1F). We demonstrated that human A172 astrocytoma cells exposed to either 10 or 100 mM cocaine had identical effects on the induction of the ER stress pathway. Lower concentration of 10 mM cocaine was thus chosen for subsequent experiments. It has been revealed that the plasma concentrations of cocaine in humans, following intranasal cocaine administration range between 0.4 to 1.6 mM,19 while the plasma cocaine concentrations in tolerant abusers reach levels up to 13 mM.20 In addition, the cocaine concentrations in postmortem brains of chronic cocaine users following acute intoxications have been reported higher than 100 mM.21 We thus rationalized that cocaine concentrations ranging from 1 to 100 mM would be compatible to the levels observed in humans. Next, we sought to examine the optimal time of cocaine-mediated induction of ER stress signaling in human A172 astrocytoma cells treated with 10 mM cocaine. Cells were exposed to cocaine for the indicated time periods (0, 1, 3, 6, 12, and 24 h) and assessed for the protein levels of ER stress markers. As shown in Figure. 1G to 1L, cocaine time-dependently induced the levels of ER stress marker proteins in human A172 astrocytoma cells. We acknowledge that human A172 astrocytoma cells cannot be considered as a true astrocyte model since these are transformed cells. Next, we thus sought to validate the time- and dose-dependent activation of ER stress by cocaine in primary human astrocytes. Cells were treated with 1 to 100 mM cocaine for 12 h (time-dependent experiment) or 10 mM cocaine for 0 to 24 h (dose-dependent experiment) to detect the protein levels of key ER stress markers by western blotting. Similar to the findings in human A172 astrocytoma cells, primary human astrocytes treated with cocaine also demonstrated both dose-dependent (Fig. 2A to 2F), and time-dependent (Fig. 2G to 2L) activation of ER stress markers. Collectively, our findings demonstrated that cocaine induced ER stress in both

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Figure 1. Cocaine-mediated dose- and time-dependent activation of ER stress in human A172 astrocytoma cells. (A to F) Representative western blots showing the dosedependent activation of ER stress marker proteins, such as (A) p-EIF2AK3:EIF2AK3, (B) p-EIF2S1:EIF2S1, (C) ATF6, (D) ERN1, (E) HSPA5 and (F) DDIT3, in overnight serumstarved human A172 astrocytoma cells that were treated with 1, 10 and 100 mM cocaine for 12 h. (G to L) Representative western blots showing the time-dependent activation of ER stress marker proteins, such as (G) p-EIF2AK3:EIF2AK3, (H) p-EIF2S1:EIF2S1, (I) ATF6, (J) ERN1, (K) HSPA5 and (L) DDIT3, in overnight serum-starved human A172 astrocytoma cells that were treated with 10 mM cocaine for the indicated time durations. ACTB was probed as a protein loading control for all experiments. The data are presented as mean § SEM from 3 independent experiments. One-way ANOVA followed by post hoc test least significant difference was used to determine the statistical significance. , P < 0.05 vs control group.

human A172 astrocytoma cells as well as primary human astrocytes, thereby suggesting a role for ER stress activation in astrocytes exposed to cocaine. Cocaine induced autophagy in human A172 astrocytoma cells and primary human astrocytes Activation of ER stress signaling is closely linked to autophagy induction as the distended ER membrane serves at least a part, in the formation of the autophagosomal membrane.22 Since cocaine induced ER stress in human astrocytes, we next sought to determine whether cocaine treatment could also induce autophagy in these cells. Human A172 astrocytoma cells treated with 10 mM cocaine for varying time points were assessed for the protein levels of classic autophagic markers such as BECN1 (Beclin 1,

autophagy related), ATG5, MAP1LC3B-II and SQSTM1 by western blotting. Interestingly, as represented in Figure 3A to 3D, cocaine significantly (P < 0.05) induced the levels of BECN1, ATG5, and MAP1LC3B-II proteins in a timedependent manner whereas the levels of SQSTM1 protein, a marker for autophagic flux, showed a time-dependent decrease in human A172 astrocytoma cells, suggesting thereby the induction of autophagy by cocaine. We next aimed to validate the same phenomenon in primary human astrocytes exposed to cocaine. Primary human astrocytes treated with 10 mM cocaine time-dependently increased the protein levels of BECN1, ATG5, and MAP1LC3B-II (Fig. 3E to 3G) with a concomitant decrease in the levels of SQSTM1 protein (Fig. 3H). Furthermore, we found that cocaine treatment did not significantly alter the mRNA expression of SQSTM1 in both human A172 astrocytoma cells

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Figure 2. Cocaine-mediated dose- and time-dependent activation of ER stress in primary human astrocytes. (A to F) Representative western blots showing the dosedependent activation of ER stress marker proteins, such as (A) p-EIF2AK3:EIF2AK3, (B) p-EIF2S1:EIF2S1, (C) ATF6, (D) ERN1, (E) HSPA5 and (F) DDIT3, in overnight serumstarved primary human astrocytes that were treated with 1, 10 and 100 mM cocaine for 12 h. (G to L) Representative western blots showing the time-dependent activation of ER stress marker proteins, such as (G) p-EIF2AK3:EIF2AK3, (H) p-EIF2S1:EIF2S1, (I) ATF6, (J) ERN1, (K) HSPA5 and (L) DDIT3, in overnight serum-starved primary human astrocytes that were treated with 10 mM cocaine for the indicated time durations. ACTB was probed as a protein loading control for all experiments. The data are presented as mean § SEM from 3 independent experiments. One-way ANOVA followed by post hoc test least significant difference was used to determine the statistical significance. , P < 0.05 vs control group.

as well as in primary human astrocytes (Fig. S1A and S1B). Intriguingly, however, cocaine-mediated reduction of SQSTM1 protein over time was primarily due to increased protein degradation when assessed by protein clamping experiments using cycloheximide (Fig. S1C to S1F). Cocaine increased intracellular autophagosome formation in human astrocytes Having demonstrated that cocaine was capable of inducing autophagy in both human A172 astrocytoma cells and primary human astrocytes, we next sought to confirm our findings by detecting autophagosome formation, as reflected by the MAP1LC3B puncta, in astrocytes exposed to cocaine. Herein we transfected human A172 astrocytoma cells or the primary human astrocytes with an overexpressing GFP-MAP1LC3B plasmid, followed by exposure of astrocytes to 10 mM cocaine

for 12 h and subsequent detection of green fluorescent MAP1LC3B puncta by immunofluorescence imaging. Compared to the control astrocytes, cocaine significantly increased the formation of exogenous MAP1LC3B puncta in human A172 astrocytoma cells and primary human astrocytes (Fig. 4A, 4B, and 4E), thereby confirming the induction of autophagosome formation by cocaine in GFP-MAP1LC3B-overexpressing astrocytes. Next, we wanted to examine the effect of cocaine on the endogenous formation of MAP1LC3B-positive autophagosomes in human A172 astrocytoma cells as well as primary human astrocytes. Immunofluorescence analysis revealed significantly increased formation of endogenous MAP1LC3B puncta in cocaine-treated human A172 astrocytoma cells compared with the control astrocytes not treated with cocaine (Fig. 4C and 4F). Similar findings were obtained in cultured primary human astrocytes treated with 10 mM cocaine for 12 h (Fig. 4D and 4F). To validate these results and

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Figure 3. Cocaine-mediated activation of autophagy in human A172 astrocytoma cells and primary human astrocytes. (A to D) Representative western blots showing the time-dependent activation of autophagy marker proteins, such as (A) BECN1, (B) ATG5, (C) MAP1LC3B-II and (D) SQSTM1, in overnight serum-starved human A172 astrocytoma cells that were treated with 10 mM cocaine for the indicated time durations. (E to H) Representative western blots showing the time-dependent activation of autophagy marker proteins, such as (E) BECN1, (F) ATG5, (G) MAP1LC3B-II and (H) SQSTM1, in overnight serum-starved primary human astrocytes that were treated with 10 mM cocaine for the indicated time durations. ACTB was probed as a protein loading control for all experiments. The data are presented as mean § SEM from 3 independent experiments. One-way ANOVA followed by post hoc test least significant difference was used to determine the statistical significance. , P < 0.05 vs control group.

to decipher the ability of cocaine to regulate the autophagosome-lysosome fusion efficiency, human astrocytes were transfected with a plasmid encoding tandem fluorescent-tagged MAP1LC3B plasmid.23 As shown in Fig. S2A, human A172 astrocytoma cells transfected with tandem fluorescent-tagged MAP1LC3B reporter plasmid followed with exposure to 10 mM cocaine for 12 h caused a significant increase in both red and yellow punctate fluorescence indicating complete

autophagosome maturation in lysosomes as well as effective activation of autophagy (Fig. S2C and S2D). These findings were further validated in primary human astrocytes transfected with tandem fluorescent-tagged MAP1LC3B reporter plasmid followed by exposure to 10 mM cocaine (Fig. S2B to S2D). Taken together, these results provide strong evidence that cocaine exposure upregulated the autophagic flux thereby increasing autophagosome clearance in human astrocytes.

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Since autophagy can also be associated with cell viability, we next determined the viability of human A172 astrocytoma cells and primary human astrocytes exposed to 10 mM cocaine. As shown in Figure 4G and 4H, exposure of both human A172 astrocytoma cells and primary human astrocytes to 10 mM cocaine for either 12 or 24 h, had no effect on cell viability.

Figure 4. (For figure legend, see page 1316)

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Cocaine increased autophagic flux by increasing MAP1LC3B turnover and SQSTM1 degradation in human astrocytes Since cocaine increased the number of autophagosomes in human astrocytes (Fig. 4), it was essential to distinguish whether

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the accumulation of autophagosomes was due to increased upstream activation of autophagy or rather due to a blockade of autophagosome-lysosomal fusion at later stages. To determine this, we measured the autophagic flux through MAP1LC3B turnover, by measuring MAP1LC3B-II degradation in autolysosomes, in astrocytes treated with cocaine. Human astrocytes were exposed to 10 mM cocaine (for 24 h) followed by treatment of cells with an inhibitor of autophagosome-lysosome fusion, bafilomycin A1 (BAF) (that was added at a saturating concentration, 400 nM in the last 4 h of the 24 h treatment period), and assessed for accumulation of MAP1LC3B-II. As shown in Figure 4I, exposure of human A172 astrocytoma cells with 10 mM cocaine followed by treatment with BAF resulted in a significant increase in the accumulation of MAP1LC3B-II levels compared to astrocytes treated with either BAF or cocaine alone. Increased accumulation of MAP1LC3B-II protein confirmed increased autophagic flux in human A172 astrocytoma cells exposed to cocaine. Similar to findings in human A172 astrocytoma cells, cocaine treatment also increased autophagic flux in primary human astrocytes (Fig. 4J). In addition to the MAP1LC3B turnover assay, an alternative method to monitor the autophagic flux is to assess the preferential degradation of SQSTM1 by autophagy. SQSTM1 is a ubiquitin-binding protein that is selectively integrated into phagophores by directly binding to the MAP1LC3B protein which, in turn, results in its selective degradation by autophagy. Accelerated degradation or decreased levels of SQSTM1 protein are thus an indicator of the autophagic flux activation inside the cell. As shown in Figure 3D and 3H, it was evident that SQSTM1 levels time-dependently decreased in both cocainetreated human A172 astrocytoma cells as well as primary human astrocytes. These findings thus underpin the potential of cocaine to upregulate the autophagic flux in human astrocytes. To confirm whether cocaine-mediated autophagy resulted in preferential degradation of SQSTM1, human astrocytes were exposed to 10 mM cocaine for 24 h, followed by treatment of cells with 400 nM BAF in the last 4 h, as described above and assessed for SQSTM1 protein levels. As shown in Figure 4K, human A172 astrocytoma cells treated with cocaine followed by BAF, demonstrated a significant increase in SQSTM1 levels compared with astrocytes treated with cocaine alone. Interestingly, however, SQSTM1 levels in human astrocytes treated with cocaine and BAF were significantly decreased when compared with human astrocytes treated with BAF alone. These findings were also validated in primary human astrocytes

(Fig. 4L). Taken together these results confirmed that degradation of SQSTM1 protein by cocaine in human astrocytes involved autophagy-dependent clearance and ensued because of increased formation of autophagosomes. Increased autophagic flux rather than a blockade in the autophagosome degradation pathway was thus responsible for SQSTM1 degradation in astrocytes exposed to cocaine. Cocaine-mediated autophagy involved upstream activation of ER stress in human astrocytes We next determined the effect of pharmacological autophagy inhibitors, such as 3-methyladenine (3-MA), and wortmannin, on cocaine-mediated autophagy in human astrocytes. In addition, we also silenced the expression of the BECN1 gene by using BECN1 siRNA to examine its effect on cocaine-mediated autophagy in human astrocytes. Overnight serum staved human A172 astrocytoma cells were pretreated with 3-MA (5 mM) and wortmannin (100 nM) for 1 h, respectively. Then, the pretreated astrocytes were treated with 10 mM cocaine for another 12 h to study the protein levels of autophagy and ER stress signaling markers by western blotting. As shown in Figure 5A to 5D, cocaine-mediated induction of autophagy was significantly inhibited in human A172 astrocytoma cells that were pretreated with 3-MA or wortmannin through regulating the protein levels of autophagy markers, such as BECN1, ATG5, MAP1LC3B-II, and SQSTM1 similar to control astrocytes. However, the protein levels of ER stress markers, such as HSPA5 (Fig. 5E), p-EIF2S1:EIF2S1 (Fig. 5F), ATF6 (Fig. S3A) and DDIT3 (Fig. S3B) did not change significantly in cocainetreated human A172 astrocytoma cells that were pretreated with either 3-MA or wortmannin than that of cocaine-treated human A172 astrocytoma cells. These findings were further validated in primary human astrocytes pretreated with pharmacological inhibitors of autophagy, such as 3-MA, and wortmannin, followed by exposure to 10 mM cocaine (Fig. 6A to F, Fig. S4A and S4B). After utilizing pharmacological inhibitors like 3-MA or wortmannin, we next employed a genetic approach (BECN1 siRNA transfection) to validate cocaine-mediated autophagy in human A172 astrocytoma cells. Human A172 astrocytoma cells transfected with either BECN1 siRNA or scrambled siRNA were then treated with 10 mM cocaine for 12 h to determine the protein levels of autophagy and ER stress signaling markers by western blotting. As expected, cocaine exposure in BECN1

Figure 4. (see previous page) Cocaine increased autophagic flux by increasing MAP1LC3B turnover and SQSTM1 degradation in human astrocytes. (A) Representative fluorescent photomicrographs showing the GFP-MAP1LC3B puncta formation in human A172 astrocytoma cells transfected with GFP-MAP1LC3B plasmid and treated with 10 mM cocaine for 12 h. (B) Representative fluorescent photomicrographs showing the GFP-MAP1LC3B puncta formation in primary human astrocytes treated with 10 mM cocaine for 12 h. Scale bar: 20 mm. (C) Representative fluorescence photomicrographs showing the endogenous MAP1LC3B puncta formation in human A172 astrocytoma cells treated with 10 mM cocaine for 12 h. (D) Representative fluorescence photomicrographs showing the endogenous MAP1LC3B puncta formation in primary human astrocytes treated with 10 mM cocaine for 12 h. Scale bar: 20 mm. (E) Bar graph showing the quantification number of GFP-MAP1LC3B puncta in human A172 astrocytoma cells and primary human astrocytes treated with 10 mM cocaine for 12 h. (F) Bar graph showing the quantification number of endogenous MAP1LC3B puncta in human A172 astrocytoma cells and primary human astrocytes treated with 10 mM cocaine for 12 h. (G) Bar graph showing the percentage of cell viability measured by MTS assay in overnight serum-starved human A172 astrocytoma cells treated with 10 mM cocaine for 12 and 24 h. (H) Bar graph showing the percentage of cell viability measured by MTS assay in overnight serum-starved primary human astrocytes treated with 10 mM cocaine for 12 and 24 h. (I and J) Representative western blots showing the protein levels of MAP1LC3B-II in human A172 astrocytoma cells as well as primary human astrocytes treated with 10 mM cocaine (for 24 h) followed by treatment with 400 nM BAF, which was added in the last 4 h of the 24 h treatment period. (K and L) Representative western blots showing the protein levels of SQSTM1 in human A172 astrocytoma cells as well as primary human astrocytes treated with 10 mM cocaine (for 24 h) followed by treatment with 400 nM BAF, which was added in the last 4 h of the 24 h treatment period. ACTB was probed as a protein loading control for all experiments. The data are presented as mean § SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. , P < 0.05 vs control group; #, P < 0.05 vs BAF group.

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Figure 5. Cocaine-mediated autophagy involved upstream activation of ER stress in human A172 astrocytoma cells. (A to D) Representative western blots showing the protein levels of autophagy markers, such as (A) BECN1, (B) ATG5, (C) MAP1LC3B-II and (D) SQSTM1, in human A172 astrocytoma cells pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 mM cocaine for 12 h. (E and F) Representative western blots showing the protein levels of ER stress markers, such as (E) HSPA5 and (F) p-EIF2S1:EIF2S1, in human A172 astrocytoma cells pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 mM cocaine for 12 h. (G) Representative western blots showing the protein levels of an autophagy marker, such as BECN1, in human A172 astrocytoma cells transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. (H) Representative western blots showing the protein levels of ER stress marker such as HSPA5 in human A172 astrocytoma cells transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. ACTB was probed as a protein loading control for all experiments. The data are presented as mean § SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. , P < 0.05 vs control group; #, P < 0.05 vs cocaine group.

siRNA-transfected human A172 astrocytoma cells failed to induce autophagy as evidenced by the decreased protein levels of BECN1 and MAP1LC3B-II, and increased protein level of SQSTM1 compared with scrambled siRNA-transfected astrocytes (Fig. 5G, Fig. S3E and S3F). Conversely, the protein levels

of ER stress markers, such as HSPA5, ATF6, and DDIT3 remained significantly higher in BECN1 siRNA-transfected human A172 astrocytoma cells treated with cocaine compared with the scrambled siRNA-transfected human A172 astrocytoma cells exposed to cocaine (Fig. 5H, Fig. S3C and S3D).

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Figure 6. Cocaine-mediated autophagy involved upstream activation of ER stress in primary human astrocytes. (A to D) Representative western blots showing the protein levels of autophagy markers, such as (A) BECN1, (B) ATG5, (C) MAP1LC3B-II and (D) SQSTM1, in primary human astrocytes pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 mM cocaine for 12 h. (E and F) Representative western blots showing the protein levels of ER stress markers, such as (E) HSPA5 and (F) p-EIF2S1:EIF2S1, in primary human astrocytes pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 mM cocaine for 12 h. (G) Representative western blots showing the protein levels of an autophagy marker, such as BECN1, in primary human astrocytes transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. (H) Representative western blots showing the protein levels of ER stress marker such as HSPA5 in primary human astrocytes transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. ACTB was probed as a protein loading control for all experiments. The data are presented as mean § SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. , P < 0.05 vs control group; #, P < 0.05 vs cocaine group.

These findings were further validated in primary human astrocytes transfected with BECN1 siRNA followed by 10 mM cocaine exposure (Fig. 6G and 6H, Fig. S4C to S4F). Taken together, our results further confirm that ER stress activation is

an upstream event for induction of autophagy in astrocytes exposed to cocaine. Since our results indicated that ER stress activation resulted in autophagy induction, we next wanted to investigate whether

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Figure 7. Cocaine-mediated autophagy involved upstream activation of ER stress in human A172 astrocytoma cells. (A and B) Representative western blots showing the protein levels of ER stress markers, such as (A) HSPA5, and (B) p-EIF2S1:EIF2S1, in human A172 astrocytoma cells pretreated with the ER stress inhibitors, such as 10 mM salubrinal or 50 mM 4-PBA, for 1 h following treatment with 10 mM cocaine for 12 h. (C to F) Representative western blots showing the protein levels of autophagy markers, such as (C) BECN1, (D) ATG5, (E) MAP1LC3B-II and (F) SQSTM1, in human A172 astrocytoma cells pretreated with 10 mM salubrinal or 50 mM 4-PBA for 1 h following treatment with 10 mM cocaine for 12 h. (G) Representative western blots showing the protein levels of an ER stress marker, HSPA5, in human A172 astrocytoma cells transfected with EIF2AK3 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. (H) Representative western blots showing the protein levels of autophagy marker, BECN1 in human A172 astrocytoma cells transfected with EIF2AK3 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. ACTB was probed as a protein loading control for all experiments. The data are presented as mean § SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. , P < 0.05 vs control group; #, P < 0.05 vs cocaine group.

cocaine-mediated autophagy could also be inhibited by ER stress inhibitors, such as salubrinal (10 mM for 1 h) or sodium 4-phenylbutyrate (4-PBA; 50 mM for 1 h), in human astrocytes. Human A172 astrocytoma cells were pretreated either with 10 mM salubrinal or 50 mM 4-PBA for 1 h, followed by 10 mM

cocaine exposure (12 h), to determine the protein levels of ER stress markers, HSPA5, p-EIF2S1:EIF2S1, ATF6, and DDIT3, and autophagy markers, BECN1, ATG5, MAP1LC3B-II and SQSTM1. As represented in Figure 7A and 7B; Fig. S5A and S5B, the levels of HSPA5, p-EIF2S1:EIF2S1, ATF6, and DDIT3

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Figure 8. Cocaine-mediated autophagy involved upstream activation of ER stress in primary human astrocytes. (A and B) Representative western blots showing the protein levels of ER stress markers, such as (A) HSPA5, and (B) p-EIF2S1:EIF2S1, in primary human astrocytes pretreated with the ER stress inhibitors, such as 10 mM salubrinal or 50 mM 4-PBA, for 1 h following treatment with 10 mM cocaine for 12 h. (C to F) Representative western blots showing the protein levels of autophagy markers, such as (C) BECN1, (D) ATG5, (E) MAP1LC3B-II and (F) SQSTM1, in primary human astrocytes pretreated with 10 mM salubrinal or 50 mM 4-PBA for 1 h following treatment with 10 mM cocaine for 12 h. (G) Representative western blots showing the protein levels of an ER stress marker, HSPA5, in primary human astrocytes transfected with EIF2AK3 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. (H) Representative western blots showing the protein levels of autophagy marker, BECN1 in primary human astrocytes transfected with EIF2AK3 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. ACTB was probed as a protein loading control for all experiments. The data are presented as mean § SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. , P < 0.05 vs control group; #, P < 0.05 vs cocaine group.

proteins were notably inhibited in human A172 astrocytoma cells pretreated with the ER stress inhibitors, salubrinal or 4PBA, followed by cocaine treatment. Interestingly, the protein levels of classical autophagy markers such as BECN1, ATG5,

MAP1LC3B-II and SQSTM1 were inhibited by the ER stress inhibitors, salubrinal or 4-PBA, in human A172 astrocytoma cells exposed to cocaine (Fig. 7C to 7F). Next, we employed a genetic approach, i.e. EIF2AK3 siRNA transfection to

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Figure 9. Cocaine-mediated astrogliosis involved ER stress and autophagy signaling pathways. (A) Representative western blots showing the time-dependent activation of GFAP protein in overnight serum-starved human A172 astrocytoma cells that were treated with 10 mM cocaine. (B) RT-qPCR showing the relative expression of GFAP mRNA and (C) Representative western blot showing the levels of GFAP protein in human A172 astrocytoma cells pretreated with 10 mM salubrinal and 50 mM 4-PBA for 1 h following treatment with 10 mM cocaine for 12 h. (D) RT-qPCR showing the relative expression of GFAP mRNA and (E) Representative western blot showing the expression of GFAP protein in human A172 astrocytoma cells pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 mM cocaine for 12 h. (F) Representative western blot showing the levels of GFAP protein in human A172 astrocytoma cells transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. (G) Representative western blots showing the time-dependent activation of GFAP protein in overnight serum-starved primary human astrocytes that were treated with 10 mM cocaine. (H) RT-qPCR showing the relative expression of GFAP mRNA and (I) Representative western blot showing the levels of GFAP protein in primary human astrocytes pretreated with 10 mM salubrinal and 50 mM 4-PBA for 1 h following treatment with 10 mM cocaine for 12 h. (J) RT-qPCR showing the relative expression of GFAP mRNA and (K) Representative western blot showing the expression of GFAP protein in primary human astrocytes pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 mM cocaine for 12 h. (L) Representative western blot showing the levels of GFAP protein in primary human astrocytes transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. ACTB was probed as a loading control for all experiments. The data are presented as mean § SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. , P < 0.05 vs control group; #, P < 0.05 vs cocaine group.

corroborate that ER stress was an upstream event for cocainemediated activation of autophagy in astrocytes. Human A172 astrocytoma cells transfected with either EIF2AK3 siRNA or scrambled siRNA were exposed to 10 mM cocaine (12 h) and assessed for the levels of autophagy and ER stress signaling marker proteins by western blotting. As expected, EIF2AK3 siRNA-transfected astrocytes demonstrated downregulation of

both the ER stress (Fig. 7G, Fig. S5C to S5F) and autophagy (Fig. 7H, Fig. S5G and S5H) markers and, cocaine exposure failed to exert any further effect. These findings were further validated in primary human astrocytes that were either pretreated with pharmacological inhibitors of ER stress, such as salubrinal or 4-PBA (Fig. 8A and 8F, Fig. S6A and S6B), or that were transfected with EIF2AK3 siRNA followed by cocaine

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exposure (Fig. 8G and 8H, Fig. S6C to S6H). These findings further confirmed that ER stress is an upstream event for cocainemediated activation of autophagy in human astrocytes. Cocaine-mediated astrogliosis involved ER stress and autophagy signaling pathways Next, we sought to explore whether cocaine-mediated ER stress and autophagy played a role in astrogliosis. Overnight serumstarved human A172 astrocytoma cells were treated with 10 mM cocaine for the indicated times and monitored for expression of astrocyte activation marker, GFAP. As shown in Figure 9A, exposure of human A172 astrocytoma cells to cocaine resulted in a time-dependent and significant upregulation of GFAP. Furthermore, pretreatment of human A172 astrocytoma cells to ER stress inhibitors, salubrinal (10 mM for 1 h) and 4-PBA (50 mM for 1 h), significantly blocked cocaine-mediated upregulation of GFAP mRNA and protein levels compared with cocaine alone treated astrocytes (Fig. 9B and 9C). Similarly, pretreatment of human A172 astrocytoma cells with the autophagy inhibitors, 3MA (5 mM) and wortmannin (100 nM) notably inhibited the cocaine-mediated induction of GFAP mRNA and protein levels compared with the cocaine alone treated astrocytes (Fig. 9D and 9E). Similar to pharmacological blocking, gene-silencing of BECN1 in human A172 astrocytoma cells also significantly inhibited cocaine-mediated induction of GFAP protein (Fig. 9F). These findings were also validated in primary human astrocytes (Fig. 9G to 9L). Interestingly, exposure of both human A172 astrocytoma cells as well as primary human astrocytes to either the inducer of ER stress (tunicamycin) or autophagy (rapamycin) resulted in induction of GFAP protein levels (Fig. S7A and S7B). Reciprocally, genetic knockdown of EIF2AK3 failed to upregulate GFAP protein in both human A172 astrocytoma cells and primary human astrocytes following cocaine exposure (Fig. S7C and S7D), thereby underscoring the roles both ER stress and autophagy signaling pathways in cocaine mediated activation of astrocytes.

cytokines both in human A172 astrocytoma cells (Fig. 10C) and in primary human astrocytes (Fig. 10D). Similar to pharmacological blocking, gene-silencing of BECN1 and EIF2AK3 also significantly inhibited cocaine-mediated upregulation of proinflammatory cytokines in both human A172 astrocytoma cells (Fig. 10E and 10G) as well as in primary human astrocytes (Fig. 10F and 10H). Overall, these findings thus implicate the role of ER stress and autophagy in mediating astrocyte activation, as evidenced by increased expression of GFAP and proinflammatory cytokines, in response to cocaine. Chronic cocaine administration induced the ER stress and autophagy in vivo To further explore the in vivo relevance of our findings, we next sought to determine the induction of ER stress and autophagy in experimental mice treated chronically with cocaine. As shown in Figure 11, chronic intraperitoneal administration of cocaine (20 mg/kg body weight/day for 7 consecutive d) induced a significant increase in the levels of ER stress marker proteins, such as (A) HSPA5 and (B) p-EIF2S1:EIF2S1, as well as the autophagy markers such as (C) BECN1, (D) MAP1LC3B-II, and (E) SQSTM1 in the striatum compared to saline administered control mice. Furthermore, mice that were intraperitoneally administered the ER stress inhibitor, salubrinal (1 mg/kg body weight for 7 d) prior to cocaine administration failed to exhibit cocaine-mediated upregulation of either ER stress or autophagy markers in the striatum compared with saline administered control mice (Fig. 11A to 11E). As expected chronic cocaine administration also resulted in concomitant upregulation of GFAP protein (Fig. 11F), as well as the proinflammatory cytokines, such as Tnf, Il1b, and Il6 mRNAs in the striatum (Fig. 11G). Interestingly, pretreatment of mice with the ER stress inhibitor, salubrinal significantly abrogated cocainemediated upregulation of GFAP protein and the proinflammatory cytokines. These findings thus underscore the role of ER stress and autophagy in cocaine-mediated activation of astrocytes.

Discussion Cocaine-mediated autophagy induced the expression of proinflammatory cytokines in human astrocytes Having confirmed that cocaine-mediated ER stress and autophagy signaling induced the expression of GFAP in human astrocytes, we next sought to examine whether the activation of ER stress and autophagy pathways also played roles in the induction of cocaine-mediated generation of inflammatory responses in astrocytes. Overnight serum-starved human A172 astrocytoma cells as well as primary human astrocytes were pretreated with either the ER stress inhibitors (salubrinal or 4PBA) or autophagy inhibitors (3-MA or wortmannin) for 1 h followed by exposure to cocaine (10 mM) for 12 h and assessed for expression of proinflammatory cytokines. As shown in Figure 10A and 10B, inhibition of ER stress significantly attenuated cocaine-induced expression of TNF (tumor necrosis factor), IL1B (interleukin 1 b), and IL6 (interleukin 6) mRNA in human A172 astrocytoma cells as well as in primary human astrocytes. Furthermore, pretreatment of astrocytes with the autophagy inhibitors, similar to the ER inhibitor, also failed to induce cocaine-mediated upregulation of the proinflammatory

Astrocytes are the most abundant cells in the CNS and are indispensable for multifarious essential functions such as homeostasis, defense and regeneration, and the sustainment of neuronal physiology. Loss in astrocyte functioning and their reactivity to toxic stimuli, such as exposure to drugs of abuse, infection, injury or disease, accelerates the neuroinflammatory process.4,24,25 Concurrently, there is a growing body of evidence suggesting that reactive astrocytosis and microgliosis are classical features underlying neuroinflammation observed in drug abusers and in various neurodegenerative diseases.1,26-29 Astrogliosis is accompanied by the release of a wide range of neurotoxic mediators comprising of chemokines, complement factors, cytokines, growth factors and reactive oxygen species.30-33 All of these mediators are potentially noxious not only to neurons but also to the neighboring cells, thereby contributing to disruption of cellular homeostasis in the CNS. These sequelae of neuroinflammatory signaling have been implicated in the pathogenesis of various neurodegenerative diseases with diverse etiologies.28 The cellular and molecular mechanism(s) leading to astrogliosis mediated by diverse agents as well as the

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Figure 10. Cocaine-mediated autophagy induced the expression of proinflammatory cytokines in human astrocytes. (A) RT-qPCR analysis of mRNA expression profile of proinflammatory cytokines such as TNF, IL1B, and IL6 in overnight serum-starved human A172 astrocytoma cells pretreated with 10 mM salubrinal and 50 mM 4-PBA for 1 h following treatment with 10 mM cocaine for 12 h. (B) RT-qPCR analysis of mRNA expression profile of proinflammatory cytokines such as TNF, IL1B, and IL6 in overnight serum-starved primary human astrocytes pretreated with 10 mM salubrinal and 50 mM 4-PBA for 1 h following treatment with 10 mM cocaine for 12 h. (C) RT-qPCR analysis of mRNA expression profiles of proinflammatory cytokines such as TNF, IL1B, and IL6 in overnight serum-starved human A172 astrocytoma cells pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 mM cocaine for 12 h. (D) RT-qPCR analysis of mRNA expression profiles of proinflammatory cytokines such as TNF, IL1B, and IL6 in overnight serum-starved primary human astrocytes pretreated with 100 nM 3-MA and 100 nM wortmannin for 1 h following treatment with 10 mM cocaine for 12 h. (E) RT-qPCR analysis of mRNA expression profiles of proinflammatory cytokines such as TNF, IL1B, and IL6 in overnight serum-starved human A172 astrocytoma cells transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. (F) RT-qPCR analysis of mRNA expression profiles of proinflammatory cytokines such as TNF, IL1B, and IL6 in overnight serum-starved primary human astrocytes transfected with BECN1 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. (G) RT-qPCR analysis of mRNA expression profiles of proinflammatory cytokines such as TNF, IL1B, and IL6 in overnight serum-starved human A172 astrocytoma cells transfected with EIF2AK3 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. (H) RT-qPCR analysis of mRNA expression profiles of proinflammatory cytokines such as TNF, IL1B, and IL6 in overnight serum-starved primary human astrocytes transfected with EIF2AK3 siRNA and scrambled siRNA following treatment with 10 mM cocaine for 12 h. ACTB was used as an internal control to normalize the gene expression for all experiments. The data are presented as mean § SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. , P < 0.05 vs control group; #, P < 0.05 vs cocaine group.

related neuroinflammatory pathways that trigger astrogliosis are still areas of active investigation. While the role of drugs of abuse, such as morphine and methamphetamine, on induction of apoptosis, HIV disease progression, neuronal injury and neurodegeneration has been well established,34-39 information

in how the drugs influences astrogliosis and the role of autophagy in this process is very limited. Recent research has demonstrated that ER stress and/or autophagy play key roles in the induction of inflammatory responses underlying various pathologies, including neurode-

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Figure 11. Chronic cocaine administration induced the ER stress and autophagy in vivo. (A to E) Representative western blots showing the activation of ER stress marker proteins, such as (A) HSPA5 and (B) p-EIF2S1:EIF2S1, and autophagy marker proteins, such as (C) BECN1, (D) MAP1LC3B-II, and (E) SQSTM1, in the striatum homogenates of mice (n D 6) treated with saline, cocaine, or salubrinal and cocaine, respectively, as explained in the Materials and methods section. (F) Representative western blot showing the upregulation of GFAP protein in the striatal homogenates of mice (n D 6) treated with saline, cocaine, or salubrinal and cocaine, respectively. (G) RT-qPCR analysis of mRNA expression profiles of proinflammatory cytokines such as Tnf, Il1b, and Il6 in the striatal homogenates of mice (n D 6) treated with saline, cocaine, or salubrinal and cocaine, respectively. Actb was used as an internal control for all experiments. The data are presented as mean § SEM from 3 independent experiments. A Student t test was used to determine the statistical significance. , P < 0.05 vs saline group; #, P < 0.05 vs cocaine group. (H) Schematic diagram representing cocainemediated induction of ER stress and autophagy leading to astrogliosis by increased expression of GFAP, which ultimately increases the mRNA expression profiles of proinflammatory cytokines such as TNF, IL1B, and IL6 in human astrocytes.

generative diseases that are often comprised of misfolded and aggregated proteins.40-43 In addition to the known etiological agents, drugs of abuse such as cocaine, morphine and amphetamines, are also known to induce ER stress and autophagy in various cell types of the CNS, thereby exacerbating the disease pathology.14,34,44 Several reports have implicated a central role for ER stress in the induction of autophagy in the pathogenesis of various neurodegenerative diseases mediated by diverse stressors.45-50 In the current study, we demonstrate for the first time a molecular link between ER stress-mediated activation of

autophagy and the induction of astrogliosis that is accompanied with the release of proinflammatory cytokines in cocainetreated human astrocytes. Our findings demonstrate that in both the human A172 astrocytoma cells and primary human astrocytes cocaine exposure results in upstream activation of ER stress and autophagy, ultimately leading to activation of astrocytes. Our findings revealed that cocaine treatment led to increased phosphorylation of ER stress markers, such as EIF2AK3 and EIF2S1, along with a notable increase in the

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expression of other arms of the ER stress signaling proteins such as ATF6, HSPA5, DDIT3, and ERN1, in human astrocytes. Following induction of ER stress mediators, cocaine also mediated a time-dependent induction of autophagy in astrocytes as evidenced by a significant upregulation of autophagy marker proteins, such as BECN1, ATG5 and MAP1LC3B-II, and a concomitant decline in the expression of SQSTM1 protein. Autophagy is a basic cellular mechanism that orchestrates the degradation of defective organelles and misfolded proteins in order to preserve cellular homeostasis. Numerous stress stimuli such as ER stress, hypoxia, infection, and nutrient depletion, play critical roles in the activation of autophagy.51-53 Our findings demonstrated that cocaine treatment of astrocytes significantly increased the expression levels of BECN1 and ATG5 proteins, thereby confirming the formation of autophagosomes and initiation of autophagy in human astrocytes. Following the induction of autophagy, MAP1LC3B-I protein is eventually conjugated into phosphatidylethanolamine and integrated into the autophagic membranes to form MAP1LC3B-II, thereby targeting the cargo for lysosomal degradation.54 Accordingly, cocaine-treated astrocytes significantly demonstrated increased expression of MAP1LC3B-II protein, thereby confirming increased formation of autophagosomes in these cells in response to cocaine. GFP-MAP1LC3B plasmid overexpression studies further confirmed the ability of cocaine to increase MAP1LC3B puncta, i.e., autophagosomes in human astrocytes. Increased accumulation of autophagosomes possibly functions as a pathogenic signal with the early accumulation of autophagosomes likely contributing to induction of cocaine-mediated astrogliosis. In addition to autophagosome accumulation, we also found increase in the autophagic flux when assessed as MAP1LC3B turnover and SQSTM1 degradation, in cocainetreated human astrocytes. We also employed the approaches of pharmacological inhibition that impede either early autophagosome formation (by 3-MA or wortmannin) or late autolysosome formation (by BAF) phases of autophagy signaling in addition to the genetic approach targeting BECN1 knockdown to specifically determine the roles of ER stress and autophagy in mediating the effects of cocaine on astrogliosis. Our findings demonstrated that autophagy inhibitors, 3-MA or wortmannin, or gene-silencing of BECN1 significantly abrogated cocaine-mediated induction of autophagy in human astrocytes, while still maintaining cocainemediated upregulation of ER stress. These findings thus suggested that cocaine-mediated induction of ER stress was upstream of autophagy response that resulted ultimately in increased activation of astrocytes as evidenced by upregulated expression of GFAP and proinflammatory cytokines. Astrogliosis is a multifaceted response that encompasses proliferation, morphological changes and functional adaptation of astrocytes leading to neurodegeneration.25,30-32,55 Herein, using various approaches, we established the potential of a psychostimulant drug such as cocaine in inducing astrogliosis through ER stress-mediated autophagy in human astrocytes. Our results established a close link between ER stress, autophagy, and cocaine-induced overexpression of GFAP and proinflammatory cytokines in human astrocytes, indicating thereby that inhibition of either the ER stress or the autophagy signaling pathway(s) could be considered as an alternative therapeutic approach for the treatment of neuroinflammation

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underlying astrogliosis. It is well established that proinflammatory cytokines secreted from activated astrocytes, microglia, neurons, and lymphocytes, or from damaged cells play a fundamental role in triggering neuroinflammation.3 Accordingly, astrogliosis resulting from cocaine exposure ultimately leads to overexpression of proinflammatory cytokines such as TNF, IL1B, and IL6 in human astrocytes confirming thereby the detrimental role of cocaine in mediating astrogliosis. Our findings are in keeping with those reported by Jackson and colleagues on the neuroinflammatory potential of methamphetamine in primary human astrocytes.56 In summary, our findings have identified the signaling pathway(s) critical for cocaine-mediated upregulation of astrogliosis (Fig. 11H). Whether similar pathways involving ER stress and autophagy are universal and play a role in the induction of astrogliosis by various other mediators such as HIV protein Tat, or other drugs of abuse remains to be established. These novel results provide functional insights into the interaction of cocaine with ER stress, autophagy, astrogliosis, and neuroinflammation and implicate that inhibition of ER stress/autophagy could be developed as an alternative approach for the treatment of cocaine-mediated neuroinflammation.

Materials and methods Reagents The reagent cocaine hydrochloride (C5776), and the autophagy inhibitors wortmannin (W3144) and 3-MA (M9281), were purchased from Sigma-Aldrich. The ER stress inhibitors, salubrinal (sc-202332) and 4-PBA (567616) were bought from Santa Cruz Biotechnology and EMD Millipore Corporation, respectively. Antibodies were obtained from the following sources: BECN1 (sc-11427), ATG5 (sc-33210), p-EIF2AK3 (sc-32577), EIF2AK3 (sc-13073), ERN1 (sc-20790), ATF6 (sc-22799), goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were from Santa Cruz Biotechnology; EIF2S1 (5324), p-EIF2S1 (3398), and DDIT3 (2895) were from Cell Signaling Technology; MAP1LC3B (NB100-2220) was from Novus Biological Company; HSPA5 (610979) was from BD Biosciences and SQSTM1 (PM045) was from MBL International.

In vivo studies C57BL/6N mice were purchased from Charles River Laboratories (Wilmington, MA) and housed in the clean, sterile, polypropylene cages under standard vivarium conditions (12 h light/dark cycle) with ad libitum access to water and food. All animal experiments were approved by the University of Nebraska Animal Care and Use Committee and were in compliance with the Animal Welfare Act (Public Law 91–579) as mandated by the NIH Guide for Care and Use of Laboratory Animals. Experimental mice were divided into 3 groups: saline, cocaine, and salubrinal with cocaine. Salubrinal (1 mg/kg body weight; Sigma, SML0951) was injected intraperitoneally 1 h before cocaine injection. Cocaine (20 mg/kg body weight) was also injected intraperitoneally for 7 consecutive d and the mice were sacrificed 1 h following the last dose of cocaine injection. Brain tissues were dissected and striatal homogenates were

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assessed to determine the levels of ER stress and autophagic marker proteins. Cell culture The human astrocytoma cell line A172 (ATCCÒ CRL1620 ) was purchased from American Type Culture Collection, and cultured as described by the supplier. Briefly, A172 astrocytoma cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM; Corning CellgroÒ , 10-013-CV) containing 10% heatinactivated fetal bovine serum (FBS; Atlanta Biologicals, S11050H), and 10 U/ml penicillin-streptomycin (Life Technologies, 15140–122) in a 5% CO2-humidified incubator at 37 C. A172 astrocytoma cells were used for up to 25 passages and then restarted from frozen stocks to exclude variables related with aging. Primary human primary astrocytes were obtained from ScienCell Research Laboratories (1800) and were cultured in DMEM:F12 (1:1) medium with L-glutamine and 15 mM HEPES (Life Technologies, 11330–032) containing 10% heatinactivated FBS, and 10 U/ml penicillin-streptomycin in a 5% CO2-humidified incubator at 37 C. According to the manufacturer’s instructions, human primary astrocytes were used under 10 passages. The seeding densities of human primary astrocytes were similar to that described for human A172 astrocytoma cells. TM

MTS assay The cell viability of human A172 astrocytoma cells as well as primary human astrocytes treated with various concentration of cocaine were measured by the 3-(4,5-dimethylthiazol-2-yl)5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium (MTS) method as per the manufacturer’s instructions. Astrocytes were cultured in flat-bottomed 96-well plates at a density of 1£104 cells per well for 24 h at 37 C in a humidified, 5% CO2 incubator. Overnight serum-starved astrocytes were then treated with various concentrations of cocaine (1, 10 and 100 mM) for a specified duration. At the end of the experiment, 20 ml of CellTiter 96Ò AQueous One Solution Reagent (Promega, G3582) was added to each well and incubated at 37 C for 4 h in a humidified, 5% CO2 incubator. Last, the cell viability was determined by measuring the absorbance of each well at 490 nm using a SynergyTM Mx Monochromator-Based MultiMode Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA). Small interfering RNA (siRNA) transfection Human A172 astrocytoma cells and primary human astrocytes were transfected with either human BECN1 siRNA (Santa Cruz Biotechnology, sc-29797) or human EIF2AK3 siRNA (Santa Cruz Biotechnology, sc-36213) using LipofectamineÒ 2000 Reagent (Life Technologies, 11668–019) as per the manufacturer’s instructions. Briefly, the astrocytes were seeded in a 6well plate at a density of 3£105 cells per well at 37 C in a humidified, 5% CO2 incubator. At 70% confluence, the culture medium was replaced with 1 ml of Opti-MEMÒ I Reduced Serum Medium (Life Technologies, 31985–070). Meanwhile, LipofectamineÒ 2000 Reagent (3 ml/ml) and 120 pmol/ml of

individual targeted siRNA was incubated separately with OptiMEMÒ I Reduced Serum Medium for 5 min at room temperature. Consequently, the LipofectamineÒ 2000 mix was added to the individual targeted siRNA mix and this mixture was incubated for 20 min at room temperature, after which the combined mixture was added to the cells. The scrambled siRNA mixture also prepared similarly. Then, the culture plate was gently shaken for 5 s and incubated for 24 h at 37 C in a humidified, 5% CO2 incubator. Knockdown efficiencies were determined by western blotting. GFP-MAP1LC3B staining of autophagosomes Human astrocytes were seeded in a 24-well plate containing sterile glass coverslips (11 mm) at a density of 5£104 cells per well at 37 C in a humidified, 5% CO2 incubator. At 70% confluence, the culture medium was replaced with 250 ml of Opti-MEMÒ I Reduced Serum Medium. Cells were transfected with the GFPMAP1LC3B plasmid using LipofectamineÒ 2000 Reagent, according to the manufacturer’s protocol, for 4 h following which the culture medium was replaced with the respective 10% heat-inactivated FBS-DMEM for 24 h. Transfected astrocytes were then treated with 10 mM cocaine for 12 h. After the treatment, the astrocytes were rinsed 2 times with phosphate-buffered saline (PBS; Hyclone Laboratories, SH3025801) at room temperature and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature, followed by a 3 times rinse with PBS. Then the coverslips were mounted on glass slides with ProLongÒ Gold Antifade Reagent with DAPI (Molecular Probes, P36935). Fluorescent images were taken on a Zeiss Observer using a Z1 inverted microscope (Carl Zeiss, Thornwood, NY, USA) and the acquired images were analyzed using the AxioVs 40 Version 4.8.0.0 software (Carl Zeiss MicroImaging GmbH). Analysis of autophagosome maturation Human astrocytes were seeded in a 24-well plate containing sterile glass coverslips (11 mm) at a density of 5£104 cells per well at 37 C in a humidified, 5% CO2 incubator. At 70% confluence, the culture medium was replaced with 250 ml of Opti-MEMÒ I Reduced Serum Medium. Cells were transfected with the tandem fluorescent tagged MAP1LC3B plasmid (ptfLC3; Addgene plasmid #21074)23 using LipofectamineÒ 2000 Reagent, according to the manufacturer’s protocol, for 4 h following which the culture medium was replaced with 10% heat-inactivated FBS-DMEM for 24 h. Transfected astrocytes were then exposed to 10 mM cocaine for 12 h, following which the astrocytes were rinsed 2 times with PBS at room temperature and fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and followed by rinsing with PBS thrice. The coverslips were mounted on glass slides with ProLongÒ Gold Antifade Reagent with DAPI and fluorescent images were taken on a Zeiss Observer using a Z1 inverted microscope. Acquired images were analyzed using the AxioVs 40 Version 4.8.0.0 software. Western blotting At the end of each experiment, the control and treated astrocytes were harvested and lysed using the Mammalian Cell Lysis

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kit (Sigma-Aldrich, MCL1-1KT). Lysates were centrifuged at 12000 £ g for 10 min at 4 C and the protein content of the supernatant fraction was quantified by a BCA assay using the PierceTM BCA Protein Assay Kit (Thermo Fisher Scientific, 23227) according to the manufacturer’s guidelines. Equal amounts of soluble proteins were loaded and electrophoresed in a 10% or 15% sodium dodecyl sulfate-polyacrylamide gel under reducing conditions, followed by blotting onto a polyvinylidene fluoride membrane (Millipore, IPVH00010). Then, the membranes were blocked with 5% nonfat dry milk (in 1£ TTBS buffer) for 1 h at room temperature before an overnight incubation with the indicated primary antibodies at 4 C. After rinsing, the membranes were incubated with a secondary antibody for 1 h at room temperature, and the immunoreactive protein signals were identified using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34078). The intensity of each band was normalized to that of ACTB (actin, b) and the data are presented as a relative fold change by using ImageJ analysis software.57 Real-Time qPCR Total RNA was extracted from the astrocytes using QuickRNATM MicroPrep kit (Zymo Research Corporation, R1051) as per the manufacture’s protocol. Column purified total RNA was quantified and reverse transcribed by using the Verso cDNA Synthesis Kit (Thermo Fisher Scientific, AB-1453/B), according to the manufacturer’s instructions. The reverse transcribed RNA was analyzed with the 7500 Fast Real-Time PCR System (Applied Biosystems, Grand Island, NY) using the RT2 SYBR Green Fluor qPCR Mastermix (Qiagen, 330510). The primer sequences, such as GFAP forward 50 -GGCGCTCAATGCTGGC TTCA-30 ; GFAP reverse 50 -TCTGCCTCCAGCCTCAGGTT-30 ; TNF forward 50 -CAGCCTCTTCTCCTTCCTGAT-30 , TNF reverse 50 -GCCAGAGGGCTGATTAGAGA-30 , IL1B forward 50 -TACCTGTCCTGCGTGTTGAA-30 , IL1B reverse 50 -TCTTT GGGTAATTTTTGGGATCT-30 , IL6 forward 50 -GATGAGTACAAAAGTCCTGATCCA-30 , IL6 reverse 50 -CTGCAGCCACTGGTTCTGT-30 , ACTB forward 50 -GGCACCCAGCAC AATGAA-30 , ACTB reverse 50 -GCCGATCCACACGGAGTACT-30 were designed using OligoPerfectTM Designer, an online-based software (Life Technologies, Carlsbad, CA), and were synthesized from the same company. The primers for mouse Tnf (PPM03113F), Il1b (PPM03109E), Il6 (PPM03015A), and Actb (PPM02945A) were purchased from SABiosciences Corporation. Each reaction was carried out in triplicate and 3 independent experiments were run. Normalization was done with Actb, an internal control and the fold change in expression was obtained by the 2¡DDCT method. The specificity of the RTqPCR was controlled using a non-template control. Immunocytochemistry Human astrocytes were seeded in a 24-well plate containing sterile glass coverslips (11 mm) at a density of 5£104 cells per well at 37 C in a humidified, 5% CO2 incubator for 24 h. Overnight serum-starved astrocytes were then treated with the respective agents for the indicated time period. The astrocytes were next rinsed 2 times with 1£ PBS at room temperature

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and fixed with 4% paraformaldehyde in PBS for 20 min at room temperature, followed by permeabilization with 0.3% Triton X-100 (Fisher Scientific, BP151-500) in PBS. Then, the permeabilized astrocytes were incubated in a blocking buffer for 1 h at room temperature followed by overnight incubation of MAP1LC3B primary antibody at 4 C. After that, Alexa FluorÒ 594 conjugate goat anti-rabbit IgG (HCL) secondary antibody was added for 2 h to detect the expression of indicated protein. Then the coverslips were mounted on glass slides with ProLongÒ Gold Antifade Reagent with DAPI (Molecular Probes, P36935). Fluorescence images were taken on a Zeiss Observer using a Z1 inverted microscope (Carl Zeiss, Thornwood, NY, USA) and the acquired images were analyzed using the AxioVs 40 Version 4.8.0.0 software (Carl Zeiss MicroImaging GmbH).

Statistical analysis All the data are expressed as mean § SEM, and statistical significance was determined by a Student t test for between 2 groups or a one-way ANOVA followed by least significant difference (LSD) post hoc test for multiple groups using the SPSS (version 15.0) software (SPSS Inc.). Values were considered statistically significant when P < 0.05.

Abbreviations ACTB ATF4 ATF6 ATG5 ATG7 ATG8 ATG12 BAF BECN1 CNS DDIT3 DMEM EIF2AK3 EIF2S1 ER ERN1 GFAP HSPA5 IL1B IL6 MAP1LC3B MTS siRNA SQSTM1 TNF VIM

actin, b activating transcription factor 4 activating transcription factor 6 autophagy-related 5 autophagy-related 7 autophagy-related 8 autophagy-related 12 bafilomycin A1 Beclin 1, autophagy related central nervous system DNA-damage inducible transcript 3 Dulbecco’s modified Eagle’s medium eukaryotic translation initiation factor 2 a kinase 3 eukaryotic translation initiation factor 2, subunit 1a endoplasmic reticulum endoplasmic reticulum (ER) to nucleus signaling 1 glial fibrillary acidic protein heat shock protein 5 interleukin 1, b interleukin 6 microtubule-associated protein 1 light chain 3 b 3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium small interfering RNA sequestosome 1 tumor necrosis factor vimentin

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Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed.

[16]

Funding

[17]

This work was supported by grants DA020392, DA040397, DA035203 and DA036157 (SB) from the National Institutes of Health.

[18]

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