Cocaine-mediated microglial activation involves the ER stress-autophagy axis

Abstract

Cocaine abuse leads to neuroinflammation, which, in turn, contributes to the pathogenesis of neurodegeneration associated with advanced HIV-1 infection. Autophagy plays important roles in both innate and adaptive immune responses. However, the possible functional link between cocaine and autophagy has not been explored before. Herein, we demonstrate that cocaine exposure induced autophagy in both BV-2 and primary rat microglial cells as demonstrated by a dose- and time-dependent induction of autophagy-signature proteins such as BECN1/Beclin 1, ATG5, and MAP1LC3B. These findings were validated wherein cocaine treatment of BV-2 cells resulted in increased formation of puncta in cells expressing either endogenous MAP1LC3B or overexpressing GFP-MAP1LC3B. Specificity of cocaine-induced autophagy was confirmed by treating cells with inhibitors of autophagy (3-MA and wortmannin). Intriguingly, cocaine-mediated induction of autophagy involved upstream activation of 2 ER stress pathways (EIF2AK3- and ERN1-dependent), as evidenced by the ability of the ER stress inhibitor salubrinal to ameliorate cocaine-induced autophagy. In vivo validation of these findings demonstrated increased expression of BECN1, ATG5, and MAP1LC3B-II proteins in cocaine-treated mouse brains compared to untreated animals. Increased autophagy contributes to cocaine-mediated activation of microglia since pretreatment of cells with wortmannin resulted in decreased expression and release of inflammatory factors (TNF, IL1B, IL6, and CCL2) in microglial cells. Taken together, our findings suggest that cocaine exposure results in induction of autophagy that is closely linked with neuroinflammation. Targeting autophagic proteins could thus be considered as a therapeutic strategy for the treatment of cocaine-related neuroinflammation diseases.

Abbreviations

3-MA

3-methyladenine

ATF6

activating transcription factor 6

ATG5

autophagy-related 5

BCL2

B-cell CLL/lymphoma 2

BECN1

Beclin 1, autophagy related

Baf1

bafilomycin A₁

CCL2

chemokine (C-C motif) ligand 2

DAPI: 4

6-diamidino-2-phenylindole, dihydrochloride

DDIT3

DNA-damage-inducible transcript 3

EGFP

enhanced green fluorescent protein

EIF2AK3

eukaryotic translation initiation factor 2-α kinase 3

EIF2S1

eukaryotic translation initiation factor 2, subunit 1 α, 35kDa

ER

endoplasmic reticulum

ERN1

endoplasmic reticulum to nucleus signaling 1

HIV

human immunodeficiency virus

IL1B

interleukin 1, β

IL6

interleukin 6

MAP1LC3B

microtubule-associated protein 1 light chain 3

METH

methamphetamine

MTOR

mechanistic target of rapamycin

PPP1R3A

protein phosphatase 1, regulatory subunit 3A

NFKB1

nuclear factor of kappa light polypeptide gene enhancer in B-cells 1

PBN

N-tert-butyl-α-phenylnitrone

PtdIns3K

class III phosphatidylinositol 3-kinase

ROS

reactive oxygen species

rPMCs

rat primary microglial cells

RPS6

ribosomal protein S6

TLR4

toll-like receptor 4

TNF

tumor necrosis factor

wort

wortmannin

Introduction

Cocaine, one of the most commonly abused drugs, has long been known to stimulate the brain reward system by elevating synaptic dopamine concentration through binding with the dopamine transporter (blocking dopamine reuptake).^1-3 Recent emerging evidence suggests that addictive drugs such as cocaine^4-7 and ethanol^8,9 are capable of stimulating inflammatory responses by activating innate and adaptive immune responses in the brains of addicts. Microglia, the resident macrophages of the central nervous system and estimated to comprise 10% to 15% of all the brain cells, are one of the main targets of these abused drugs.^10-11 Following activation, microglia undergo associated changes in morphology and proliferation,¹² leading to production and secretion of a plethora of cytokines, chemokines, and neurotoxic factors,¹³ which in turn, play critical roles in regulating neuronal activities and functioning. Intriguingly, these cytokines have been implicated in the pathogenesis of various neuroinflammatory diseases such as Alzheimer and Parkinson diseases^14,15 and multiple sclerosis.¹⁶ Among the cocaine-mediated secretory cytokines, upregulation of inflammatory factors such as IL1B, IL6, and TNF has been demonstrated both in the in vitro and in vivo studies.^4-6 Detailed molecular mechanism(s) underlying this phenomenon however, are still largely elusive although ER stress-¹⁷ and oxidative stress-^18,19 related pathways have been alluded to in this process.

Autophagy and the innate immune responses, are highly conserved evolutionary processes in virtually all eukaryotic cells.^20-23 Autophagy is a cellular homeostatic process that involves the sequestration of unfolded cytosolic proteins within double-membrane-bound compartments (autophagosomes), which in turn, can fuse with lysosomes to form autolysosomes that are eventually destined for protein degradation.²¹ This process is critical for elimination of aggregated and unfolded proteins and for the removal of excess or damaged organelles in the cells. Basal and constitute levels of autophagy aid in regulating quality control of proteins while also protecting cells from damaged protein aggregation.^22,23 Under cellular stress conditions such as nutrient depletion or temperature and oxidative stress, autophagy can be induced to maintain cell survival and, upon extended insult, can also lead to autophagic cell death.²⁴ Autophagy is an important player in various diseases such as cancer and those involving neurodegenerative processes.^21-23

At the molecular level, autophagy is highly regulated by sequential expression of autophagy-related (ATG) genes and their protein products.²⁵ Briefly, BECN1, a phylogenetically conserved protein that is essential for the initiation of autophagy, interacts with proteins including ATG14 and PIK3C3 to generate the class III phosphatidylinositol 3-kinase (PtdIns3K) that forms an initiation complex; this complex subsequently promotes the recruitment of certain ATG proteins. Following this is the formation of the ATG12–ATG5-ATG16L1 protein complex, which facilitates addition of a phosphatidylethanolamine (PE) group to the carboxyl terminus of the cytosolic MAP1LC3B-I (a well-characterized mammalian ortholog of yeast Atg8) such that it gets anchored to the membrane as MAP1LC3B-II. The conversion to MAP1LC3B-II is widely recognized and employed as a quantification marker for autophagosome formation.^24,25

Autophagy is recognized as an important component of the innate immune response as evidenced by the fact that lipopolysaccharide (LPS) induces autophagy in macrophage through TLR4 (toll-like receptor 4)-mediated pathways.²⁶ Autophagy and innate immune responses comprise the first line of defense against bacterial infection. Both of these processes also regulate each other reciprocally.^23-25 Addictive drugs including methamphetamine (METH), morphine, and cannabinoid compounds induce neuroinflammation.^27-29 Among these, the psychostimulant METH has the ability to induce autophagy in neuron-derived cell lines.²⁷ Opiates such as morphine induce autophagy in human neuroblastoma SH-SY5Y cells and in the rat hippocampus.²⁸ Cannabinoids also induce autophagy in human glioma cells.²⁹ However, to date there are no functional studies addressing the relationship between cocaine and autophagy. It has been known that autophagy has a close functional link with activation of the upstream ER stress pathway.³⁰ Individual ER stress pathway(s), which are responsible for autophagy upregulation, are cell-type dependent. For example, in SK-N-SH neuroblastoma cells, activation of autophagy following ER stress is ERN1-dependent;³¹ however, in human lymphoma cells, ER-stress induced autophagy is dependent on EIF2AK3-mediated stress signaling.³²

Microglia, the resident macrophages within the CNS, are usually maintained in a quiescent state. These cells constitute the first line of defense in response to injurious stimuli such as viral infection, neuron injury, ischemic stroke, or exposure to drug abuse.^10,11 Following stimulation with external stimuli, microglia undergo morphological changes and/or proliferation, resulting in production and secretion of a plethora of cytokines, chemokines, and neurotoxic factors. These factors, in turn, play critical roles in regulating neuronal activity and function. Microglia play key role in CNS homeostasis by mediating a protective response via immunological surveillance to repair brain injuries. However, prolonged exposure to toxic stimuli such as chronic exposure to either cocaine or its secreted cytokines, can lead to significant neuronal dysfunction and cognitive impairment, thereby resulting in exacerbated damage within the CNS.^33,34

While autophagy levels have been shown to be regulated in microglial cells by various stimuli such as IL1B and TLR2 agonist,^35,36 there is limited information on psychostimulant-induced autophagy in these cells. In this report, we demonstrate that exposure of microglial cells to cocaine leads to increased autophagy with the implication of ER stress related pathways in this process. Functionally, cocaine-induced autophagy modulated the production of inflammatory mediators such as TNF, IL1B, IL6, and CCL2 in microglial cells. These results suggest that autophagy plays an important role in psychostimulant-induced neuroinflammation and that inhibition of autophagy could be a useful therapeutic strategy for the treatment of drug abuse-mediated neuroinflammation.

Results

Time- and dose-dependent effects of cocaine on autophagy markers in BV-2 microglial cells

It is well recognized that cocaine is a strong inducer of oxidative stress and cell activation.^37,38 Based on studies indicating that drugs such as METH and morphine can induce autophagy^27,28 and also that cocaine induces endoplasmic reticulum (ER) stress, we sought to examine whether cocaine also has the ability to regulate microglial autophagy. We monitored the expression levels of 3 different autophagic markers (BECN1, ATG5, and MAP1LC3B) in BV-2 cells exposed to cocaine for various times. BV-2 cells were exposed to cocaine (10 µM) for various times (0, 3, 6, 12, and 24 h), lysed and subjected to western blotting for detecting the abundance of autophagic markers. As shown in Figure 1A, cocaine induced a significant increase in BECN1 levels starting at 12 h (2.62 ± 0.23 fold, P < 0.05) with enhanced expression persisting up to 24 h (2.78 ± 0.39 fold, P < 0.05). Interestingly, no significant effects of cocaine on BECN1 levels were observed during early treatment (3 and 6 h). Another autophagic marker, ATG5 also showed a similar time-dependent expression as BECN1 following cocaine exposure. In response to cocaine ATG5 levels demonstrated an increased expression at 12 h (1.50 ± 0.14 fold, P < 0.05) post-treatment with a further increase in expression at 24 h (1.71 ± 0.21 fold, P < 0.05). Cocaine also substantially increased MAP1LC3B-II levels from 12 to 24 h shown in Figure 1C. As expected, untreated cells did not demonstrate upregulation of the 3 autophagic markers (BECN1, ATG5, and MAP1LC3B-II) at the times tested (0 to 24 h) seen in Figure 1A, 1B, and 1C. These findings thus demonstrated that cocaine had the ability to enhance autophagy in BV-2 microglia.

Figure 1.

Cocaine induced BECN1, ATG5, and MAP1LC3B levels in BV-2 cells in a time- and dose- dependent manner. BV-2 cells were seeded into 6-well plates and treated with the indicated doses of cocaine (Coc) for the indicated time periods. Cocaine significantly induced BECN1 (A), ATG5 (B), and MAP1LC3B-II (C) levels at 12 and 24 h after treatment (10 µM). Untreated control cells did not exhibit upregulation of the 3 autophagy markers (A, B, and C). Cocaine significantly induced BECN1, ATG5, and MAP1LC3B-II levels at the concentrations of 10 and 100 µM (D). All experiments were done 4 times independently. For all western blots, ACTB served as a protein loading control. Representative western blots are shown left of the quantification of western blots (A to D). Data (4 independent experiments) are expressed as means ± SEM and were analyzed using ANOVA (* P < 0.05 versus control).

The next step was to determine the dose curve of cocaine exposure for optimal expression of autophagic markers. BV-2 cells were exposed to varying doses of cocaine (1, 10, 100 µM) for 24 h and assessed for expression of autophagic markers by western blotting. As shown in Figure 1D, cocaine exposure of BV-2 cells resulted in enhanced expression of BECN1 (2.32 ± 0.37 and 2.24 ± 0.14 fold at 10 and 100 µM cocaine respectively; P < 0.05). Similarly there was increased expression of ATG5 in cells exposed to cocaine (2.15 ± 0.18 and 2.70 ± 0.11 fold at 10 and 100 µM cocaine respectively, P < 0.05). Levels of MAP1LC3B-II also exhibited a similar dose-dependent trend in response to cocaine with an increase of 1.67 ± 0.16 and 1.78 ± 0.24 fold at 10 and 100 µM cocaine exposure respectively (P < 0.05). A relatively low dose of cocaine (1 µM) had no effect on the expression levels of microglial autophagy. Taken together, these findings demonstrated that cocaine mediates induction of autophagic markers (BECN1, ATG5, and MAP1LC3B) in a time- and dose- dependent manner in vitro.

Time- and dose-dependent effects of cocaine on autophagic markers in rat primary microglial cells

We next explored whether cocaine could also mediate upregulation of autophagic markers in rat primary microglial cells (rPMCs). rPMCs were isolated from newborn pup brains and exposed to cocaine for various times and subsequently with varying concentrations as described above for BV-2 cells. Similar to BV-2 cells, rPMCs also demonstrated cocaine-mediated temporal upregulation of BECN1, ATG5, and MAP1LC3B-II with maximal induction at 12 to 24 h. As shown in Figure 2A–C, 12 h of cocaine exposure resulted in induction of BECN1, ATG5, MAP1LC3B-II (2.32 ± 0.54, 2.69 ± 0.68, and 1.80 ± 0.17 fold, respectively; P < 0.05,) compared to untreated cultures. Cocaine exposure for 24 h resulted in further induction of these autophagy proteins (BECN1 – 2.86 ± 0.30, ATG5 – 3.23 ± 0.51, and MAP1LC3B-II – 2.10 ± 0.10 fold; P < 0.05). Similar to the findings in BV-2 cells, untreated rPMCs did not reveal any change in the expression levels of the 3 autophagic markers.

Figure 2.

Cocaine induced BECN1, ATG5, and MAP1LC3B levels in rat primary microglial cells in a time- and dose-dependent manner. Rat primary microglial cells were isolated and seeded into 6-well plates and treated with the indicated doses of cocaine (Coc) at the indicated time periods (A to C). Cocaine significantly induced BECN1 (A), ATG5 (B), and MAP1LC3B-II (C) levels at 12 and 24 h post-cocaine treatment (10 µM). Untreated control cells did not exhibit upregulation of the 3 autophagy markers (A, B, and C). Cocaine significantly induced BECN1, ATG5, and MAP1LC3B-II levels at 10 and 100 µM cocaine concentrations (D). All experiments were done 4 times independently. For all western blots, ACTB served as protein loading control. Representative western blots are shown left of the quantification of western blots (A to D). Data (4 independent experiments) are expressed as means± SEM and were analyzed using ANOVA (* P < 0.05 vs. control).

Exposure of rPMCs to varying doses of cocaine also resulted in a significant induction of all 3 proteins (BECN1 – 2.48 ± 0.22 and 3.13 ± 0.44 fold; ATG5 – 2.44 ± 0.35 and 2.07 ± 0.10 fold; and MAP1LC3B-II – 2.01 ± 0.15 and 2.20 ± 0.17 at 10 and 100 µM cocaine exposure respectively; P < 0.05) compared to controls in Figure 2D. Cocaine exposure thus resulted in upregulation of autophagy in a time- and dose-dependent manner in rat PMCs.

Cocaine increases autophagosome formation without affecting cell viability

Having demonstrated biochemically that cocaine increased autophagy markers in both primary and microglial cell lines, we next wanted to validate our findings by transfecting the cells with the GFP-MAP1LC3B plasmid and assessing the cells for MAP1LC3B-II-derived puncta following exposure to cocaine. For this the GFP-MAP1LC3B plasmid was overexpressed in BV-2 cells followed by exposure of cells to 10 µM cocaine. Rapamycin, a classical inducer of autophagy was employed as a positive control. Figure 3A demonstrated that as expected, rapamycin treatment of BV-2 cells resulted in induction of the autophagy markers BECN1, ATG5, and MAP1LC3B-II. We next sought to examine the formation of MAP1LC3B-II puncta in BV-2 cells transfected with GFP-MAP1LC3B construct and treated with either cocaine, rapamycin, and cocaine with bafilomycin A₁ (Baf1), followed by assessment of observed MAP1LC3B-II puncta formation by fluorescence microscopy. As shown in Figure 3B, similar to rapamycin, cocaine and coexposure of cocaine with Baf1 treatments also resulted in increased formation of GFP-MAP1LC3B-II-derived puncta in BV-2 cells overexpressing the MAP1LC3B-II construct. Having determined that cocaine induced the formation of autophagosomes in MAP1LC3B-II-overexpressing cells, we next wanted to determine whether cocaine treatment could also induce endogenous formation of the MAP1LC3B-II autophagosomes. BV-2 cells were exposed to cocaine for 24 h and assessed for MAP1LC3B-II puncta formation using antibody specific for MAP1LC3B-II. As shown in Figure 3C, exposure of BV-2 cells to cocaine, resulted in increased formation of endogenous MAP1LC3B-II puncta similar to that observed in rapamycin-treated cultures. Further validation of autophagosome formation was assessed by electron microscopy analysis. Typically, autophagosome morphology is presented by vacuoles containing a lamellar, vesicular structure with a surrounding double membrane. As shown in Figure 3D, in the BV-2 cells with cocaine and coexposure of cocaine with Baf1, large vacuoles (arrows) appeared in the cytoplasm, and these membrane compartments contained multiple cellular organelles, which were not seen in control samples. These morphological features clearly reflect the classical autophagic characteristics demonstrating cocaine has the ability to induce autophagosome formation in microglial cells. Next we assessed whether cocaine had any effect on toxicity of BV-2 cells. As shown, cocaine did not significantly affect cell viability in both BV-2 cells and rPMCs within a 24-h period in Figure 3E and F (P > 0.05). Based on this we rationalized that autophagy could initiate a prosurvival response following cocaine exposure. To test this we treated BV-2 cells with cocaine and an autophagy inhibitor 3-methyladenine (3-MA). Cell viabilities were then detected by MTS assays and compared within groups receiving either a single agent or in combination. Our findings in Figure 3G showed that the viability of cells with coexposure was significantly less compared with the cells exposed to a single agent. These results thus implied that autophagy played a prosurvival role in BV-2 cells exposed to cocaine.

Figure 3.

Cocaine increased autophagosome formation in BV-2 cells. (A) BV-2 cells were seeded into 6-well plates and treated with the indicated doses of rapamycin. After 24 h, cell lysates were collected and subjected to western blotting. The levels of BECN1, ATG5, and MAP1LC3B-II were upregulated by rapamycin. (B) BV-2 cells were transfected with GFP-MAP1LC3B plasmid and treated with cocaine and/or Baf1. Cells were observed under a fluorescence microscope for MAP1LC3B-II puncta formation. As shown in the middle panel, cocaine exposure resulted in increased formation of puncta in GFP-MAP1LC3B-transfected BV-2 cells. Rapamycin-treated cells (right panel) served as controls. Scale bar: 5 µm. (C) BV-2 cells were seeded into 24-well plates and following various treatment cells were fixed and analyzed by immunofluorescence microscopy to detect endogenous MAP1LC3B puncta formation. Scale bar: 20 µm. (D) BV-2 cells were seeded into 10-cm diameter plates and treated with cocaine (10 µM) for 24 h, following which the cells were fixed and observed for autophagosomes under the electron microscope. Scale bar: 1 µm. BV-2 cells (E) and rat primary microglial cells (F) were seeded into 96-well plates treated with or without cocaine and assessed for cell viability using the MTS assays. (G) BV-2 cells were seeded into 96-well plates followed with various treatments. At the indicated time points, cell viabilities were assessed by MTS assays. Experiments were done 4 times independently. Data (4 independent experiments) are expressed as means ± SEM and were analyzed using ANOVA.

MAP1LC3B-II can be increased either by enhanced autophagy initiation or by inhibiting lysosomal degradation.³⁹ To explore the underlying mechanisms responsible for MAP1LC3B-II upregulation following cocaine exposure, we treated BV-2 cells with cocaine in the presence or absence of Bfa1. Then cells were collected and homogenates were prepared for detection of MAP1LC3B-II levels. Our findings in Figure S1A showed that BV-2 cells exposed to cocaine in the presence of Bfa1, demonstrated a significant elevation of MAP1LC3B-II levels compared with the cells receiving either agent alone, indicating thereby that cocaine increased MAP1LC3B-II levels primarily through enhancing the initiation of autophagy.

Under normal cellular homeostasis, the anti-apoptotic protein BCL2 binds with BECN1 to inhibit the autophagic process.⁴⁰ Recent work has demonstrated that this interaction can be disrupted in the presence of METH, leading to initiation of autophagy.⁴¹ To determine whether such a mechanism was also responsible for cocaine-induced autophagy, we investigated the binding status of BCL2 with BECN1 following cocaine treatment. Using co-immunoprecipitation assays, it was demonstrated that the amount of BCL2 in the BECN1-immunoprecipitated protein complex decreased while the amount of total BCL2 remained unchanged in cocaine-treated cultures seen in Figure S1B. These results thus indicated that cocaine has the ability to interrupt BCL2-BECN1 binding, thereby leading to autophagy initiation.

Cocaine-induced autophagy can be blocked by PtdIns3K inhibitors, as well as by Becn1 and Atg5 siRNA

At the initiation step, autophagy is strictly controlled by BECN1 interacting with the regulatory serine/threonine protein kinase ULK1/2 and PtdIns3K to form a protein complex that integrates upstream signals and induces the downstream ATG conjugate cascade. As an essential part of this protein complex, the PtdIns3K plays central roles in autophagy regulation. Therefore, we rationalized that the lipid kinase activity of PtdIns3K also takes part in the cocaine-induced autophagy and inhibiting PtdIns3K could lead to inhibition of autophagy. To explore this possibility, cells were pretreated with the classic PtdIns3K inhibitors 3-MA or wortmannin (wort), followed by exposure to cocaine and assessment for expression of the autophagy marker MAP1LC3B-II. As shown in Figure 4A and B, pretreatment of BV-2 cells with either 3-MA or wort resulted in abrogation of cocaine-mediated induction of autophagy marker MAP1LC3B-II. Corroboration of these findings was also performed by assessing inhibition of cocaine-mediated formation of MAP1LC3B-II puncta in cells pretreated with 3-MA or Wort in Figure 4C. These findings thus underlined the role of PtdIns3K-mediated pathways in cocaine-mediated induction of autophagy.

Figure 4.

Cocaine-induced autophagy is blocked by the PtdIns3K inhibitors 3-MA or wortmannin. (A) BV-2 cells were treated with cocaine (Coc) in the presence or absence of 3-MA. Whole cell lysates were subjected to western blots to detect MAP1LC3B-II levels. (B) BV-2 cells were treated with cocaine in the presence or absence of wortmannin (wort). Whole cell lysates were subjected to western blots to detect MAP1LC3B-II levels. Representative western blots are shown on the left of the quantification of western blots. Data (4 independent experiments) are expressed as means ± SEM and were analyzed using ANOVA (*P < 0.05 versus control). (C) BV-2 cells were seeded into 24-well plates and treated with cocaine in the presence of absence 3-MA or wortmannin. Following treatment, cells were fixed with 4% paraformaldehyde and subjected to immunofluorescence microscopy to detect endogenous MAP1LC3B puncta formation. Scale bar: 5 µm. (D) BV-2 cells were transfected with Becn1 siRNA and scrambled siRNA followed with cocaine treatment. Then cells were collected and the supernatant fractions were subjected to various western blots. (E) BV-2 cells were transfected with Atg5 siRNA and scrambled siRNA followed with cocaine treatment. Then cells were collected and the supernatants were subjected to various western blots.

In addition to using the pharmacological inhibitors 3-MA and wort, cocaine-induced autophagy was also validated using a genetic approach. BV-2 cells were transfected with either Becn1 or Atg5 siRNAs or scrambled siRNA for 24 h following which, the cells were exposed to cocaine. Cell lysates were examined for expression of MAP1LC3B-II levels by western blotting. As shown in Figure 4D and E, cells knocked down for either BECN1 or ATG5 failed to exhibit cocaine-induced autophagy.

Cocaine-induced autophagy involves ROS-ER stress pathways

Generation of autophagy in a cell can involve upstream ER stress pathways.^30,31 To explore the involvement of ER stress pathways in cocaine mediated-autophagy, the levels of 3 ER stress pathways were monitored in BV-2 cells exposed to cocaine. As shown in Figure 5A, exposure of BV-2 cells to cocaine resulted in time-dependent upregulation of phosphorylated EIF2AK3 (eukaryotic translation initiation factor 2-α kinase 3) and EIF2S1 (eukaryotic translation initiation factor 2, subunit 1 α, 35kDa) proteins, with no change in the total levels of these proteins. Levels of ERN1 (endoplasmic reticulum to nucleus signaling 1), a protein mediating yet another ER stress pathway, were also increased in cells treated with cocaine. Consistent with these findings, levels of the ER stress sensor protein HSPA5 (heat shock 70kDa protein 5 [glucose-regulated protein 78kDa]) were also significantly upregulated in response to cocaine shown in Figure 5B. Intriguingly, the ER stress pathway mediated by ATF6 (activating transcription factor 6), remained unaffected in cells treated with cocaine. Prolonged activation of ER stress often leads to cellular apoptosis that is reflected by increased levels of the protein DDIT3 (DNA-damage-inducible transcript 3).^42,43 Treatment of BV-2 cells with cocaine for 24 h did not show increased expression of DDIT3, thereby implicating lack of cellular apoptosis in cells treated with cocaine. These findings are in agreement with our results demonstrating failure of cocaine to impact cell viability within 24 h of treatment in Figure 3E and F.

Figure 6.

Autophagy facilitates induction of TNF, IL1B, IL6, and CCL2 by cocaine in BV-2 cells. (A) BV-2 cells were seeded and treated with cocaine in the presence or absence of wortmannin (wort) or salubrinal (salu). After 12 h, total RNA was extracted and the mRNA levels of Tnf, Il6, Il1b, and Ccl2 were detected by quantitative RT-PCR. (B) BV-2 cells were seeded and treated with cocaine in the presence or absence of wortmannin, 3-MA, or salubrinal. After 24 h, the levels of secreted TNF protein in the supernatant were monitored by ELISA assays. (C) BV-2 cells were seeded and treated with cocaine in the presence or absence of wortmannin, 3-MA, or salubrinal. Twenty-four h post-treatments, levels of secreted CCL2 protein in the supernatant fractions were monitored by ELISA assays. Data are expressed as means ± SEM (4 independent experiments) and were analyzed using ANOVA (*P < 0.05 vs. control; #P< 0.05 versus cocaine-treated group).

Figure 5.

Cocaine-induced autophagy involves EIF2AK3- and ERN1-mediated ER stress pathways. BV-2 cells were treated with cocaine at the indicated times and whole cell lysates were subjected to western blots to detect the levels of ER stress proteins. (A) Cocaine-mediated time-dependent phosphorylation of EIF2AK3 and EIF2A proteins. (B) Cocaine mediated time-dependent phosphorylation of ERN1 and HSPA5 proteins, but not ATF6 and DDIT3. (C) BV-2 cells were treated with cocaine in the presence or absence of salubrinal followed by assessment by western blots of whole cell lysates for BECN1, ATG5, and MAP1LC3B protein levels. Representative western blots are shown left to the quantification of western blots. Data are expressed as means ± SEM (4 independent experiments) and were analyzed using ANOVA (*P < 0.05 vs. control; #P< 0.05, coexposure of cocaine and salubrinal versus cocaine only). (D) BV-2 cells were treated with cocaine in the presence or absence of salubrinal and cells were fixed and assessed for MAP1LC3B immunofluorescence. Quantification was shown on the right. Scale bar: 5 µm.

Further confirmation of role of ER stress in cocaine-mediated induction of autophagy was examined in cells pretreated with the ER stress inhibitor salubrinal. Salubrinal acts as a phosphatase inhibitor and increases EIF2S1 phosphorylation levels leading to global translation decrease, subsequently to lessen ER stress level.⁴⁴ As shown in Figure 5C and D, pretreatment of cells with salubrinal resulted in abrogation of cocaine-mediated induction of autophagy evidenced by decreased expression of MAP1LC3B-II levels compared to cocaine-treated cells without pretreatment with the inhibitor. Similar to findings with MAP1LC3B-II, levels of BECN1 and ATG5 were also downregulated in cells pretreated with salubrinal, thereby confirming that the ER stress response is upstream of autophagy in cocaine-treated BV2 cells shown in Figure 5C. This was further validated by cocaine-mediated formation of MAP1LC3B-II puncta that was inhibited in salubrinal-pretreated BV-2 cells in Figure 5D. These findings thus imply that upstream activation of 2 ER stress (EIF2AK3- and ERN1-dependent) pathways plays a role in cocaine-mediated induction of autophagy and that inhibition of ER stress pathway blocks cocaine-induced autophagy. We have demonstrated that salubrinal can reverse cocaine-induced autophagy in microglial cells. To further validate the role of ER stress in cocaine-mediated induction of autophagy induction, we employed a genetic approach targeting PPP1R3A (protein phosphatase 1, regulatory subunit 3A), which is responsible for EIF2S1 dephosphorylation. BV-2 cells were transfected with either Ppp1r3a siRNA or scrambled siRNA for 24 h followed by exposure of cells to cocaine. Cell lysates were then assessed for expression of MAP1LC3B-II levels by western blotting. Our findings in Figure S2 demonstrated that BV-2 cells knocked down for PPP1R3A failed to exhibit cocaine-mediated increase of autophagy. Taken together these findings thus underpin the role of ER stress responses in cocaine-induced autophagy.

Having determined that cocaine-mediated autophagy involved ER stress, we next sought to determine the molecular link between cocaine exposure and ER stress. It has been well documented that cocaine can upregulate the expression of reactive oxygen species (ROS) in human pulmonary microvascular endothelial cells⁴⁵ and also that upregulation of ROS plays a role in ER stress response.⁴⁶ Based on this, we first sought to examine the expression of ROS in cells exposed to cocaine. As shown in Figure S3A and B following cocaine exposure there was increased expression of ROS at 15 min, which peaked at 30 min to about 3 fold. Blocking ROS generation resulted in abrogation of cocaine-mediated activation of EIF2AK3 and ERN1 seen in Figure S3C and autophagy markers seen in Figure S3D. These findings this suggested that ROS is a molecular linker between cocaine exposure and EIF2AK3 and ERN1 activation.

It is well known that autophagy is regulated by the MTOR (mechanistic target of rapamycin) pathway. To assess whether cocaine-mediated induction of autophagy involved the MTOR pathway, cells were exposed to cocaine and assessed for activation of AKT and RPS6 (ribosomal protein S6), markers for the MTOR pathway. As shown in Figure S4A and B both AKT and RPS6 phosphorylation levels decreased in BV-2 cells exposed to cocaine. These results thus suggested that similar to rapamycin cocaine exposure also inhibited MTOR activity, thereby implying the involvement of MTOR pathway in cocaine-mediated induction of autophagy in microglial cells.

Autophagy facilitates cocaine-medicated induction of TNF, IL1B, IL6, and CCL2 in microglial cells

Autophagy regulates inflammatory responses.^23-25 Psychostimulants such as cocaine activate cells of myeloid origin,^4-6 leading to increased production and secretion of proinflammatory factors including TNF, IL1B, IL6, and CCL2.⁴⁷ To investigate whether cocaine-mediated induction of ER stress and autophagy played a role in microglial inflammatory responses, BV-2 cells were pretreated with either salubrinal or the autophagy inhibitors wort or 3-MA, followed by exposure to cocaine and then assessed for expression of activation markers. As shown in Figure 6A, exposure of BV-2 cells to cocaine resulted in significantly increased mRNA expression of mediators such as Tnf, Il1b, Il6, and Ccl2 and pretreatment of cells with either salubrinal or autophagy inhibitors wort or 3-MA abrogated cocaine-mediated induction of inflammatory cytokines and chemokines. Consistent with mRNA levels, pretreatment of cells with the respective inhibitors also resulted in blocking cocaine-mediated upregulation of TNF and CCL2 protein levels as evidenced by ELISA shown in Figure 6B and C. Autophagy inhibitors decrease cocaine-induced cytokines release from BV-2 microglial cells. To further validate that autophagy can modulate inflammatory responses, a genetic approach involving Atg5 siRNA was employed. BV-2 cells were transfected with either siRNA specifically targeting Atg5 or a scrambled siRNA for 24 h followed by exposure of cells to cocaine for another 24 h. Culture supernatant fractions were then collected and assessed for expression of secreted TNF and CCL2 by ELISA. As shown in Figure S5 cells transfected with Atg5 siRNA failed to exhibit cocaine-mediated induction of cytokine secretion. Taken together, our findings provide evidence that autophagy contributes to cocaine-induced cytokine release in microglia.

Chronic administration of cocaine increases autophagic markers in vivo

Having determined the role of cocaine in mediating ER-stress and autophagy in vitro in microglial cells, we next sought to determine whether cocaine could also induce autophagy in microglial in vivo. Groups of mice (n = 6) were treated with cocaine once a d (20 mg/kg, I.P.) for 7 consecutive d and, 1 h following the last cocaine injection, mice were sacrificed, brains were removed, and striatal homogenates were assessed for levels of autophagic markers. Mice injected with saline served as controls. Our findings demonstrated that chronic cocaine administration resulted in a significant increase in expression of autophagic markers (BECN1, ATG5, and MAP1LC3B-II levels) in the striatum of mice in Figure 7A–C compared with saline-injected controls. To further examine whether microglial cells in the CNS contributed to increased autophagy brain sections from cocaine- and saline-treated mice were double immunostained for autophagy markers and the microglial cell marker AIF1 (allograft inflammatory factor 1). Our findings demonstrated that in sections of cocaine-treated mice AIF1 (red) colocalized with increased endogenous MAP1LC3B puncta (green) in Figure 7E and increased BECN1 in Figure 7D. We already showed that cocaine can induce autophagy in vivo. To further confirm that ER stress response also plays a role in vivo, mice were pretreated with the ER stress inhibitor salubrinal (I.P., 1 mg/kg), followed with cocaine administration for 7 d. Mice were sacrificed at 1 h following the last injection, brains were removed, and the homogenate assessed for expression of BECN1, ATG5, and MAP1LC3B-II using western blots. As shown in Figure S6, mice exposed to cocaine demonstrated upregulation of autophagic makers, which is in agreement with our previous findings. Interestingly, mice pretreated with salubrinal did not exhibit cocaine-mediated upregulation of autophagy markers in the brain. These results this imply that ER stress lies upstream of cocaine-mediated induction of autophagy.

Figure 8.

Schematic description of cocaine-mediated induction of ER stress and autophagy, leading to microglial activation. Cocaine exposure activates the ER stress pathways, leading to induction of autophagy and ultimately, to activation of microglial cells. Pp1, Ppp1r3a.

Figure 7.

Chronic administration of cocaine increased BECN1, ATG5, and MAP1LC3B levels in mouse brains. Mice (n = 6) received injections of cocaine (20 mg/kg) or saline for consecutive 7 d. One h following the last injection, mice were sacrificed and brains were removed, lysed and homogenates were subjected to western blots for detection of BECN1 (A), ATG5 (B), and MAP1LC3B-II (C) levels. Representative western blots (4 independent experiments) are shown left of the quantification of western blots. Double immunostaining of striatal brain sections from cocaine-treated and control mice for BECN1 (D) or MAP1LC3B-II (E) (green) with the microglial cell marker AIF1 (red). Scale bar: 10 µm.

Discussion

Herein, using both in vitro and in vivo approaches we provided evidence that the psychostimulant drug cocaine has the ability to induce autophagy in BV-2 microglia as well as rat primary microglial cells. We also demonstrated that ER stress plays a role in cocaine-mediated autophagy, which in turn, leads to activation and induction of inflammatory responses. This is a first report addressing the functional relationship between cocaine and autophagy in microglial cells. Our results demonstrated a close link between autophagy and cocaine-induced microglial inflammatory responses and indicate that inhibition of autophagy could possibly be considered as an alternative approach for the treatment of neuroinflammatory diseases induced by activation of microglia.

There is a growing list of addictive drugs that induce autophagy in both in vitro and in vivo studies.^27-29 For example, METH is shown to induce autophagy in human umbilical vein endothelial cells, and primary human brain microvascular endothelial cells, indicating that an early autophagic response induced by METH is a prosurvival response preceding the apoptotic endothelial cell death.³⁴ Similarly another drug, morphine also enhances autophagy in human neuroblastoma SH-SY5Y cells and in the rat hippocampus.²⁸ Autophagy also plays a role in drug addiction, wherein morphine induced-autophagy resulting in decreased mitochondrial DNA, was linked to opiate addiction.⁴⁸ Intriguingly, autophagy has also been implicated to play a neuroprotective role in the context of alcohol⁴⁹ and is shown to involve TLR-mediated pathways.⁵⁰ Additionally, cannabinoids also induce autophagy in human glioma cells.²⁹ Our findings for the first time suggest that yet another drug, cocaine can induce autophagy in microglial cells. Cocaine-mediated induction of autophagy was an early event following cocaine exposure (12 h post-treatment) and involved very low levels of cocaine exposure (10 µM). Furthermore, we also demonstrated that cocaine-mediated upregulation of ER stress pathways preceded the autophagic response, which resulted in increased activation of microglia. These findings could have clinical ramifications as increased neuroinflammation has been demonstrated in the context of cocaine abuse in individuals abusing this drug.^51,52 While an initial inflammatory response by microglia can be envisioned as a protective response, prolonged activation can lead to further damage within the CNS, due to enhanced neurotoxicity. Under conditions of prolonged stimulus of activation, microglia secrete a number of cytokines and chemokines, which in turn, can lead to neuronal injury. Overactivation of microglia has been implicated as an underlying mechanism in various neurodegenerative diseases such as Parkinson and Alzheimer diseases and depression.^14,15 Intriguingly, neuroinflammation plays a role in addictive behavior.⁵³

In this report, we provide evidence that inhibition of autophagy resulted in abrogation of microglial activation, underlining thereby that autophagy plays a key role in initiation and persistence of microglial activation. Our findings are in agreement with previous reports demonstrating a functional link between autophagy and inflammation.^23-25 For example in lung inflammation induced by influenza H5N1 pseudotyped virus, autophagy enhanced NFKB1 and MAPK14 (mitogen-activated protein kinase 14) signaling pathways to exacerbate H5N1pp-induced lung inflammation.⁵⁴ Furthermore, inhibition of autophagy by pretreatment of microglial cells with tetracycline in the ischemic-stroke brain suppressed the inflammatory process via inhibiting the activation of the NFKB1 pathway.⁵⁵ TLR7-mediated pathways can stimulate inflammation and autophagy simultaneously although the relationship between autophagy and inflammation was not explored.⁵⁶ A mechanistic study implicating the role of autophagy on secretion of the proinflammatory cytokine IL1B demonstrates that the export pathway depends on Atg5 as well as the inflammasome, and other protein complexes.⁵⁷ Also, a recent research in lung cancer cells has also shown that LPS- or poly(I:C)-induced autophagy facilitates the production of inflammatory mediators such as IL6, CCL2, and CCL20, and that inhibition of autophagy results in downregulation of these mediators.⁵⁸

Another aspect of this study is the implication in diseases such as HIV/AIDS. Abuse of illicit drugs such as cocaine is common in HIV-infected individuals.^59,60 Interestingly HIV proteins such as transactivator of transcription (Tat) and the envelope protein gp120 have also been implicated to independently regulate autophagic pathways.^61,62 In fact, induction of autophagy has been demonstrated in the brains of patients with HIV-induced CNS disease involving extensive microglial activation.^63,64 Cocaine can thus accelerate the incidence and progression of (HIV)-1 associated neurological disorders via exacerbation of the autophagy-mediated microglial activation response.^65,66

We have shown that cocaine can induce autophagy in microglial cells, which in turn, can lead to microglial activation. However, the effects of rapamycin-induced autophagy on microglial activation remain elusive and controversial. Rapamycin has been reported to inhibit microglial activation for functional recovery after spinal cord injury,⁶⁷ contribute to glioma- induced microglial activation through polarizing its M1 phenotype,⁶⁸ or have no effect at all on microglial activation in rat models of temporal lobe epilepsy.⁶⁹ These inconsistencies could be attributable to the different disease models or the different doses of rapamycin used.

In summary, our findings demonstrated that cocaine is able to induce autophagy in microglial cells and this process is involved in ROS generation and ER stress responses. Cocaine-mediated autophagy contributes to the microglial activation, which ultimately leads to neuroinflammation. These novel findings provide insights into the functional linkage between cocaine, autophagy, and neuroinflammation, suggesting thereby that inhibiting autophagy could provide an alternative target for treatment of cocaine-mediated neuroinflammation.

Materials and Methods

Reagents

Reagents including cocaine hydrochloride (C5776), autophagy inhibitors wortmannin (W3144), 3-MA (M9281) were from Sigma. ER-stress inhibitor salubrinal (sc-202332) was bought from Santa Cruz. Antibodies were obtained from the following sources: BECN1 (sc-11427), ATG5 (sc-332), pEIF2AK3 (sc-32577), EIF2AK3 (sc-13073), BCL2 (sc-7382), BECN1 (sc-48341), and ERN1 (sc-20790) were from Santa Cruz Biotechnology; EIF2S1 (5324), pEIF2S1 (3398), HSPA5 (3183), DDIT3 (2895), p-AKT (9271), AKT (9272), pRPS6 (2211), and RPS6 (2217) were from Cell Signaling Technology; MAP1LC3B (NB100-2220) was from Novus Biologicals; goat anti-rabbit (sc-2004) and goat anti-mouse (sc-2005) were from Santa Cruz Biotechnology. Becn1 siRNA (sc-29798), Atg5 siRNA (sc-41446), Ppp1r3a siRNA (43533), and scrambled siRNA (sc-37007) were from Santa Cruz Biotechnology. N-tert-Butyl-α-phenylnitrone (PBN, B7263), and bafilomycin A₁ (B1793) were from Sigma. LIVE Green Reactive Oxygen Species Detection Kit (I36007) was from Molecular Probes.

Animals

Fischer 344 rats and C57BL/6N mice were purchased from Charles River Laboratories (Wilmington, MA USA). All the animals were housed under conditions of constant temperature and humidity on a 12-h light, 12-h dark cycle, with lights on at 0700 h. Food and water were available ad libitum. All animal procedures were performed according to the protocols approved by the Institutional Animal Care and Use Committee of the University of Nebraska Medical Center and the National Institutes of Health. Three groups of C57BL/6N mice were subjected to 1 of the following treatments: saline, cocaine, and salubrinal with cocaine. Salubrinal (1 mg/kg; Sigma, SML0951) was injected 1 h before cocaine administration. Mice were treated with cocaine once a day (20 mg/kg, I.P.) for 7 consecutive d and 1 h following the last cocaine injection, mice were sacrificed, brains were removed and striatal homogenates were assessed for levels of autophagic markers. Mice injected with saline served as controls.

Chronic cocaine administration

Three groups of C57BL/6N mice were subjected to one of the following treatments: saline, cocaine, and salubrinal with cocaine. Salubrinal (1 mg/kg; Sigma, SML0951) was injected 1 h before cocaine administration. Mice were treated with cocaine once a d (20 mg/kg, I.P.) for 7 consecutive d and 1 h following the last cocaine injection, mice were sacrificed, brains were removed and striatal homogenates were assessed for levels of autophagic markers. Mice injected with saline served as controls.

Primary rat microglial cell isolation

Primary microglia cells were obtained from 1- to 3-d-old C57BL/6 or Sprague-Dawley newborn pups. After digestion and dissociation of the dissected brain cortices in Hank's buffered salt solution (Invitrogen, 14025076) supplemented with 0.25% trypsin (Invitrogen, 25300-054), mixed glial cultures were prepared by resuspending the cell suspension in DMEM (Invitrogen, 11995-065) supplemented with 10% heat-inactivated fetal bovine serum (Invitrogen, 16000-044) with 100 U/ml penicillin, and 0.1 mg/ml streptomycin. Cells were plated at 20 × 10⁶ cells/flask density onto 75 cm² cell culture flasks. The cell medium was replaced every 5 d, and after the first medium change, macrophage colony-stimulating factor (Invitrogen, PHC9504) 0.25 ng/ml was added to the flasks to promote microglial proliferation. When confluent (7 to 10 d), mixed glial cultures were subjected to shaking at 37°C at 220 g for 2 h, to promote microglia detachment from the flasks. The cell medium, containing the released microglia cells, was collected from each flask and centrifuged at 1000 g for 5 min to collect cells, then plated on cell culture plates for all subsequent experiments. The purity of microglial cultures was evaluated by immunohistochemical staining using the antibody specific for AIF1 (Wako Pure Chemical Industries, 019-19741) and was routinely >95% pure.

BV-2 cell culture

The BV-2 immortalized cell line was obtained from Dr. Sanjay Maggirwar (University of Rochester Medical Center, Rochester, NY, USA) and was grown and routinely maintained in DMEM with 10% fetal bovine serum (FBS) at 37°C and 5% CO2 and used up to passage 20.

SiRNA transfection

BV-2 cells were seeded into 6-well plates to grow about 80% confluent. The next day, individual targeted siRNA and scrambled siRNA (30 pM) were mixed with lipofectamine 2000 (2 µl) in 100 µl opti-MEM (Life technologies, 31985062). After 30 min incubation at room temperature, mixed liquids were dropped into cell culture medium (serum free) and were incubated for 4 h. Then the medium was changed to 10% FBS-containing medium for 20 h incubation. The transfected cells were then ready for the following experiments.

ROS detection

This experiment was performed according to the manufacturer's (Life Technologies, D-339) recommended protocol. Basically, cells were seeded into cover slips in 24-well plates one d before the experiment. Then cells were washed with Hank's balanced salt solution and covered by 25 μM carboxy-H2DCFDA working solution. The plate was put into an incubator for 30 min at 37°C. Susbequently, the cells were washed again with Hank's balanced salt solution and mounted with DAPI (Invitrogen, 36935). The cells were observed under a Zeiss Observer. A Z1 inverted microscope (Carl Zeiss MicroImaging) was used; images were processed using AxioVs 40 4.8.0.0 software (Carl Zeiss MicroImaging).

Immunoprecipitation

To investigate the interaction between BECN1 with BCL2, cells with cocaine treatment were lysed with RIPA buffer and supernatant fractions were collected after centrifugation. Cellular protein (500 µg) was incubated with BECN1 antibody (rabbit, 2 µg) overnight at 4°C and precipitated with protein A/G beads (Santa Cruz Biotechnology, 2003). The beads were washed and heated for 10 min with 40 µl 2X SDS loading buffer. Then the protein complexes were detected using BCL2 antibody (mouse). Also the input protein (without antibody addition) served as control to show the equal amount of total protein used.

Western blotting

Treated cells were lysed using the Mammalian Cell Lysis kit (Sigma, MCL1-1KT). Equal amounts of the proteins were electrophoresed in a sodium dodecyl sulfate-polyacrylamide gel (12%) under reducing conditions followed by transfer to PVDF membranes (Millipore, IPVH00010). The blots were blocked with 5% nonfat dry milk in phosphate-buffered saline (PBS; 137 mM NaCl, 2.7 mM KCl, 10 mM Na₂HPO₄, 2 mM KH₂PO₄). The western blots were then probed with antibodies recognizing the indicated proteins. The protein amounts loaded were normalized according to the ACTB/β-actin signal, using an anti-ACTB antibody (Sigma, A5441). The secondary antibodies were HRP conjugated to goat anti-mouse/rabbit IgG (Santa Cruz Biotechnology, sc-2005 and sc-2004).

Immunocytochemistry

For immunocytochemistry, primary rat microglia or BV-2 cells were plated on coverslips. The next day cells were fixed with 4% paraformaldehyde for 15 min at room temperature, followed by permeabilization with 0.3% Triton X-100 (Fisher scientific, BP151-1) in PBS. Cells were then incubated with a blocking buffer containing 10% NGS in PBS for 1 h at room temperature followed by addition of rabbit anti-MAP1LC3B (1:300) antibody and incubated overnight at 4°C. Finally, the secondary Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, A-11008) were added at a 1:500 dilution for 2 h to detect MAP1LC3B. Cells were washed 3 times in buffer and mounted with prolong gold antifade reagent with 4,6-diamidino-2-phenylindole (Invitrogen, 36935).

Immunohistochemistry

C57BL/6N mice (25 to 30 g) were randomly separated into 2 groups (n = 6). One group was administered cocaine (20 mg/kg, I.P.) daily for 7 d and sacrificed 1 h post the final injection. Mice treated with saline of the same volume served as controls. Animals were perfused and immunohistochemical procedures were performed as described below. Frozen sections (30 µm) were coincubated with primary anti-AIF1 antibody (Abcam; ab15690) and MAP1LC3B anti-rabbit antibody (Novus Biologicals, NB100-2220) or BECN1 anti-rabbit (Santa Cruz Biotechnology, sc-11427) overnight at 4°C. Secondary Alexa Fluor 488 goat anti-rabbit IgG (Invitrogen, A-11008) or Alexa Fluor 594 goat anti-mouse (Invitrogen, A-11032) was added for 2 h to detect AIF1 and MAP1LC3B, followed by mounting of sections with DAPI (Invitrogen, 36935). Fluorescent images were acquired at room temperature on a Zeiss Observer. A Z1 inverted microscope (Carl Zeiss, German) was used; images were processed using AxioVs 40 4.8.0.0 software (Carl Zeiss MicroImaging). Photographs were acquired using an AxioCam MRm digital camera (Carl Zeiss, German).

RNA extraction, reverse transcription, and quantitative polymerase chain reaction (qPCR)

Total RNA was extracted using Trizol reagent (Invitrogen, 15596-018). Briefly, monolayer cells in 6-well plates were washed with PBS and lysed directly adding 1 ml Trizol. Cell lysate was aspirated into new 1.5 ml microcentrifuge tubes and 0.2 ml of chloroform was added. After extensive vortexing, the samples were centrifuged at 10,000 g for 15 min at 4°C. The upper aqueous phase was transferred to a new tube and 500 µl isopropyl alcohol was added. Samples were incubated for 10 min and centrifuged again to precipitate total RNA. The total RNA was dissolved in DEPC-treated H₂O and quantified. Reverse transcription reactions were performed using a Verso cDNA kit (Invitrogen, AB-1453/B). The reaction system (20 µl) included 4 µl 5X cDNA synthesis buffer, 2 µl dNTP mix, 1 µl RNA primer, 1 µl RT enhancer, 1 µl Verso enzyme Mix (Invitrogen, AB-1453/B), total RNA template 1 µg, and a variable volume of water. Reaction conditions were set at 42°C for 30 min. QPCRs were performed by using SYBR Green ROX qPCR Mastermix (Qiagen, 330510). Reaction systems were set up as follows: 10 µl SYBR Green Mastermix, 0.5 µl forward primers, 0.5 µl reverse primers, and 9 µl distilled H₂O. 96-well plates were placed into a 7500 fast real-time PCR system (Applied Biosystems, Grand Island, NY) for program running. Mouse primers for Tnf, Il6, Il1b, and Ccl2 were purchased from Invitrogen (Mm00443258, Mm00446190, Mm00434200, and Mm00441242).

MTS assay

Microglial cell viability was measured by the (3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium) method (Promega, G3582). Briefly, BV-2 cells or rat primary microglial cells were collected from culture flasks and seeded in 96-well plates. Different seeding densities were optimized at the beginning of the experiments and incubated at 37°C in 5% CO₂ for 24 h. Serum-starved cells were then treated with cocaine (10 μM) for the indicated time period. After incubation for up to 24 h, 20 μl MTS reagent dissolved in DMEM at a final concentration of 5 mg/ml was added to each well and incubated in a CO₂ incubator for 4 h. Finally, the absorbance of each well was obtained using a plate counter at 490 nm.

TNF and CCL2 analyses by ELISA

Supernatants collected from BV-2 cells that were treated with cocaine in the presence or absence of the indicated inhibitors were examined for secreted TNF and CCL2 protein levels using a commercially available ELISA kit (R&D Systems, MTA00B and MJE00). The data represent results obtained from 3 independent experiments.

Electron microscopy

Following experimental treatments, BV-2 cells were washed with 1X PBS twice to remove excess protein derived from the culture medium and flooded with 2.5% EM grade glutaraldehyde fixative buffer (Sigma, G7651) for 30 min at room temperature. Cells were removed by scraping from the plate into the microcentrifuge tube, centrifuged for 200 g for 5 min to pellet the cells and suspended in the fixative solution. Samples were stored at 4°C until ready for electron microscopy.

Plasmid preparation and transfection

DH5α bacteria containing GFP-MAP1LC3B plasmid (Addgene, 22405) were cultured in LB broth overnight at 37°C. GFP-MAP1LC3B plasmids were extracted by using the PureLink™ quick plasmid miniprep kit (Invitrogen, K210010) and dissolved into distilled water. For transfection experiments, cells were seeded into 6-well plates and maintained with 10% FBS DMEM overnight. Next day, cells were exposed to serum-free DMEM followed by treatment with GFP-MAP1LC3B plasmid (dissolved in 100 µl opti-MEM) that was mixed with Lipofectamine 2000 (Invitrogen, 11668-019), for 30 min at room temperature. Following addition of the DNA-lipo mixture to the serum-free medium, the cells were incubated at 37°C for 4–5 h, after which the medium was replaced with 10% FBS DMEM. After 24 h, cells were treated with cocaine for the indicated times and observed under a fluorescence microscope for exogenous MAP1LC3B puncta formation.

Statistics

The results are presented as means ± SEM, and were evaluated using a one-way analysis of variance followed by a Bonferroni (Dunn) comparison of groups using least squares-adjusted means. Probability levels of <0.05 were considered statistically significant.

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We are grateful to Drs. Han Chen and You “Joe” Zhou at the microscopy core research facility of University of Nebraska-Lincoln for assistance with the TEM.

Funding

This work was supported by NIH grants DA033614, DA035203, DA036157, and DA033150.