Research PaperMitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAnnadurai Thangaraj Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USAhttps://orcid.org/0000-0001-7125-2289View further author information, Palsamy Periyasamy Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USAhttps://orcid.org/0000-0002-0386-5611View further author information, Ming-Lei Guo Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USAhttps://orcid.org/0000-0003-2969-0802View further author information, Ernest T. Chivero Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USAhttps://orcid.org/0000-0003-1136-2114View further author information, Shannon Callen Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USAhttps://orcid.org/0000-0001-6241-3335View further author information Shilpa Buch Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, NE, USACorrespondencesbuch@unmc.eduhttps://orcid.org/0000-0002-3103-6685View further author informationReceived 27 Sep 2018Accepted 27 Mar 2019Accepted author version posted online: 16 Apr 2019Published online: 28 Apr 2019Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsIntroductionCocaine is one of the most commonly used illicit drugs in the USA and is known to exert severe stimulatory effects on the central nervous system (CNS). It is estimated that almost 34 million Americans (16.2%) aged 15 or older have used cocaine at least once in their lifetime [1]. Cocaine abuse has been linked to a variety of CNS disorders including an increased risk of stroke and seizures, cognitive impairment, depression, and, in extreme cases, death [2]. Findings from our group have demonstrated that cocaine exposure induced the expression of various pro-inflammatory cytokines and chemokines as well as adhesion molecules, via binding of cocaine to its cognate SIGMAR1 (sigma non-opioid intracellular receptor-1) that is expressed on a variety of cell types [3,4]. Cocaine is a strong inducer of reactive oxygen species (ROS), which in turn, is known to elicit an inflammatory response within the CNS via activation of glial cells such as the microglia [5–8]. Microglia are the resident macrophages of the brain that play a vital role in the regular surveillance of the brain environment, phagocytosis of synapses during normal synaptic development and shaping neural circuit formation, neuronal activity and plasticity [9–11].Under conditions of toxic stimuli, however, microglia elicit the release of pro-inflammatory cytokines as a protective mechanism, however, the unabated release of these cytokines subsequently tips the balance, contributing to a neuroinflammatory milieu that underlies neurodegeneration and behavioral changes [12–14]. Various studies have demonstrated cocaine-mediated induction of innate immune signaling via activation of NFKB (nuclear factor kappa B) signaling in microglia [7,15]. Cocaine has also been shown to induce the release of proinflammatory cytokines via activation of host PRR (pattern recognition receptor) signaling complexes such as TLR2 (toll like receptor 2) and TLR4 (toll like receptor 4) in glial cells [7,16,17]. While there are several mechanisms by which cocaine exerts microglial activation, the role of mitophagy in this process remains less understood.Several studies have implicated that cocaine exerts its toxicity via induction of mitochondrial dysfunction including decreased mitochondrial respiration and altered mitochondrial metabolism [18–20]. Further, dysfunctional mitochondria ultimately affect cellular metabolism and result in increased production of reactive oxygen species that, in turn, triggers inflammation [21–24]. To protect normal cellular functions, the damaged mitochondria are removed by a process called mitophagy in which the damaged mitochondria are sensed by PINK1 (PTEN induced putative kinase 1)-PRKN/Parkin (parkin RBR E3 ubiquitin protein ligase)-OPTN (optineurin) proteins and sequestered by autophagy machinery proteins including BECN1/Beclin1 (beclin1, autophagy-related), MAP1LC3B/LC3B (microtubule-associated protein 1 light chain 3 beta) and SQSTM1/p62 (sequestosome 1). Intriguingly, the cytoplasmic adaptor proteins OPTN (an important receptor for selective autophagy, in particular, mitophagy) and SQSTM1 are recruited to the damaged mitochondria, in turn, binding to the polyubiquitinated cargo via the ubiquitin-binding domain, ultimately leading to the formation of mitophagosome via the MAP1LC3-interacting domain. Mitophagosomes containing the damaged mitochondria are thereby trafficked to the lysosome for efficient clearance. This complex picture of mitophagy involves multiple signaling pathways and routes in which damaged mitochondria are cleared from the cell by lysosomal fusion. However, blockage of mitophagy results in accumulation of damaged mitochondria and aggravates oxidative stress and inflammation [25–28]. Based on these facts, in the present study, cocaine-mediated mitochondrial dysfunction and defective mitophagy signaling and its role in microglial activation were assessed. Our findings established a link between mitochondrial dysfunction, microglial activation and defective removal of dysfunctional mitochondria through mitophagy in response to cocaine exposure. Thus, manipulations that alleviate mitochondrial dysfunction and impaired mitophagy signaling could diminish the cytotoxic actions of cocaine.ResultsCocaine-mediated downregulation of mitochondrial membrane potential and initiation of mitophagy in mouse primary microglial cells (mPMs)To explore the effect of cocaine on mitochondrial membrane potential, mPMs were exposed to varying doses of cocaine (1, 10 and 100 µM) for 24 h, followed by an assessment of mitochondrial membrane potential (Δψm) using the JC-1 assay. In healthy mitochondria, the JC-1 dye forms aggregate inside the mitochondria, leading to the emission of bright red fluorescence. In depolarized mitochondria, however, the dye disperses in cells as a monomer, emitting a green fluorescence [29]. Interestingly, as shown in Figure 1(a), the red to green fluorescence ratio was dose-dependently reduced in mPMs exposed to cocaine, with a significant decrease (P   0.05) in mPMs exposed to 10 and 100 µM of cocaine for 24 h. Next, we sought to do a time course to assess the kinetics of cocaine-mediated changes in mitochondrial membrane potential. Cells were exposed to 10 µM cocaine for varying time points (0, 3, 6, 12 and 24 h) and monitored by the JC-1 assay. As shown in Figure 1(b), red to green fluorescence ratio was significantly reduced (P   0.05) at 6 h and continued to drop time-dependently up to 24 h.Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 1. Cocaine exposure alters mitochondrial membrane potential and initiates mitophagy in mPMs. Cocaine exposure dose- (a) and time- (b) dependently decreased the mitochondrial membrane potential (decreased ratio of the JC-1 aggregate to JC-1 monomers) in mPMs. (c–f) Cocaine exposure dose-dependently upregulated the expression of mitophagy markers, such as PINK1 (c), PRKN (d), DNM1L (e) and OPTN (f) in mPMs. ACTB was probed as a protein loading control for all experiments. The data are presented as mean ± SEM from six independent experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance of multiple groups. *, P  0.05 vs. control.Display full sizeFigure 1. Cocaine exposure alters mitochondrial membrane potential and initiates mitophagy in mPMs. Cocaine exposure dose- (a) and time- (b) dependently decreased the mitochondrial membrane potential (decreased ratio of the JC-1 aggregate to JC-1 monomers) in mPMs. (c–f) Cocaine exposure dose-dependently upregulated the expression of mitophagy markers, such as PINK1 (c), PRKN (d), DNM1L (e) and OPTN (f) in mPMs. ACTB was probed as a protein loading control for all experiments. The data are presented as mean ± SEM from six independent experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance of multiple groups. *, P  0.05 vs. control.Next, we sought to explore the effect of cocaine exposure on initiation of microglial mitophagy. For this expression levels of mitophagy markers such as PINK1, PRKN, DNM1L/Drp1 (dynamin 1 like protein) and OPTN were assessed using western blotting of protein lysates from mPMs exposed to varying doses of cocaine (1, 10 and 100 µM) for 24 h. Interestingly, cocaine dose-dependently upregulated the expression of PINK1 (Figure 1(c)), PRKN (Figure 1(d)), DNM1L (Figure 1(e)) and OPTN (Figure 1(f)) in mPMs. Based on the fact that mitochondrial membrane potential was significantly downregulated in mPMs exposed to 10 µM of cocaine and that the expression of mitophagy marker proteins peaked significantly (P   0.05) in mPMs exposed to 10 µM of cocaine, we chose to expose the cells to 10 µM of cocaine for all further experimentations. Next, we performed a time-course experiment to define the optimal time for cocaine-mediated upregulation of mitophagy marker proteins in mPMs. Cells were exposed to 10 µM for the indicated time points (0, 3, 6, 12 and 24 h) and cell lysates monitored for mitophagy protein expression by western blotting. As shown in Figure 2, the exposure of mPMs to cocaine resulted in significantly (P   0.05) increased expression of PINK1 (Figure 2(a)) and DNM1L (Figure 2(b)) at 6 h with a sustained increase up to 24 h. In addition, the expression of OPTN protein (Figure 2(c)) was significantly upregulated at 12h and 24h post-exposure of cells to cocaine. The expression of PRKN protein, however, was significantly (P   0.05) upregulated only at 24 h in mPMs exposed to cocaine (Figure 2(d)).Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 2. Cocaine exposure time-dependently increases mito/autophagy marker proteins in mPMs. Cocaine exposure time-dependently upregulated the expression of mitophagy markers such as PINK1 (a), DNM1L (b), OPTN (c) and PRKN (d) as well as autophagy markers such as BECN1 (e), MAP1LC3B-II (f), and SQSTM1 (g) in mPMs. ACTB was probed as a protein loading control for all experiments. The data are presented as mean ± SEM from six independent experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance of multiple groups. *, P  0.05 vs. control.Display full sizeFigure 2. Cocaine exposure time-dependently increases mito/autophagy marker proteins in mPMs. Cocaine exposure time-dependently upregulated the expression of mitophagy markers such as PINK1 (a), DNM1L (b), OPTN (c) and PRKN (d) as well as autophagy markers such as BECN1 (e), MAP1LC3B-II (f), and SQSTM1 (g) in mPMs. ACTB was probed as a protein loading control for all experiments. The data are presented as mean ± SEM from six independent experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance of multiple groups. *, P  0.05 vs. control.During the mitophagy process, depolarized mitochondria fail to import and degrade PINK1, which in turn, becomes primed by PRKN proteins and is decorated by the OPTN protein, ultimately leading to its elimination via the BECN1/MAP1LC3B-dependent autophagic process. We thus next sought to examine the expression of proteins involved in the autophagy process including the autophagosome initiation marker, BECN1; the autophagosome formation marker, MAP1LC3B; and the autophagy degradation marker, SQSTM1 in mPMs exposed to cocaine (10 µM) for varying times. As shown in Figure 2(e–g), in mPMs exposed to cocaine there was a significant time-dependent increase in the expression of the autophagy markers, BECN1 (Figure 2(e)), MAP1LC3B-II (Figure 2(f)) and SQSTM1 (Figure 2(g)) for up to 24 h.Cocaine-mediated mitochondrial dysfunction and increased mitophagosome formation in mPMsHaving demonstrated that exposure of mPMs to cocaine significantly downregulated mitochondrial Δψm and increased the initiation of mitophagy, we next sought to determine the impact of cocaine exposure on mitochondrial function. Mitochondrial functioning was assessed by monitoring both the extracellular acidification rate (ECAR) as well as mitochondrial oxygen consumption rate (OCR) using the Seahorse XFp Extracellular Flux Analyzer in mPMs exposed to cocaine (10 µM). Intriguingly, exposure of mPMs to cocaine resulted in mitochondrial dysfunction in mPMs as evidenced by significant downregulation of both OCR (Figure 3(a)) and ECAR (Figure 3(b)). Additionally, mitochondrial basal respiration, ATP production, maximal respiratory capacity (MRC), spare capacity, as well as, non-mitochondrial respiration was also determined by modulating the OCR following inhibition of mitochondrial oxidative phosphorylation complexes at different stages of oxidative phosphorylation. In mPMs exposed to cocaine, there was a significant (P  0.05) decrease in basal mitochondrial respiration, ATP production, MRC, and proton leak compared with control cells (Figure 3(c)). However, spare capacity, and non-mitochondrial respiration in cocaine-exposed mPMs was not significantly altered compared with control cells (Figure 3(c)).Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 3. Cocaine-mediated mitochondrial dysfunction and increased mitophagosome formation in mPMs. (a–c) Cocaine exposure significantly impaired mitochondrial function. (a) mitochondrial OCR and (b) ECAR were determined using a Seahorse XFp Extracellular Flux Analyzer in mPMs exposed to cocaine (10 μM) for 24 h. (c) Bar graph showing individual mitochondrial function parameters calculated from data in Panel A. The data are presented as mean ± SEM from six independent experiments. Wilcoxon test was used to make comparisons between the two groups. *, P  0.05 vs. control. (d) Transmission electron microscopic images of mitochondrial ultrastructure and mitophagosomes in mPMs exposed to cocaine (10 μM) for 24 h. N, nucleus; M, mitochondria; ER, endoplasmic reticulum; AV, autophagic vesicle; AL, autolysosome. Scale bar: 500 nm (e) Representative fluorescence images showing accumulation of mitophagosomes in mPMs transfected with GFP-MAP1LC3B and pLV-mitoDsRed followed by exposure of cells to 10 μM cocaine, and 1 μM rotenone for 24 h. Scale bar: 10 μm.Display full sizeFigure 3. Cocaine-mediated mitochondrial dysfunction and increased mitophagosome formation in mPMs. (a–c) Cocaine exposure significantly impaired mitochondrial function. (a) mitochondrial OCR and (b) ECAR were determined using a Seahorse XFp Extracellular Flux Analyzer in mPMs exposed to cocaine (10 μM) for 24 h. (c) Bar graph showing individual mitochondrial function parameters calculated from data in Panel A. The data are presented as mean ± SEM from six independent experiments. Wilcoxon test was used to make comparisons between the two groups. *, P  0.05 vs. control. (d) Transmission electron microscopic images of mitochondrial ultrastructure and mitophagosomes in mPMs exposed to cocaine (10 μM) for 24 h. N, nucleus; M, mitochondria; ER, endoplasmic reticulum; AV, autophagic vesicle; AL, autolysosome. Scale bar: 500 nm (e) Representative fluorescence images showing accumulation of mitophagosomes in mPMs transfected with GFP-MAP1LC3B and pLV-mitoDsRed followed by exposure of cells to 10 μM cocaine, and 1 μM rotenone for 24 h. Scale bar: 10 μm.After confirming cocaine-mediated mitochondrial dysfunction and upregulation of mito/autophagosomes in mPMs, we next sought to explore the formation and accumulation of autophagic vesicles containing mitochondria in mPMs exposed to cocaine using transmission electron microscopy (TEM). As expected, control cells exhibited the formation of an intact and healthy mitochondrial network. In contrast, exposure of cocaine to mPMs resulted in the formation of autophagic vacuoles containing mitochondria in the perinuclear areas (Figure 3(d)).Further validation of cocaine-mediated formation of mitophagosomes involved dual transfection of mPMs with both pLV-mitoDsRed and GFP-MAP1LC3B for 6 h, followed by exposure of cells to cocaine for 24 h. Transfected mPMs exposed to rotenone for 24 h (a known inducer of mitophagy) were used as a positive control [30]. As shown in Figure 3(e), in transfected control mPMs not exposed to cocaine, there was a clear presence of the red fluorescently labeled mitochondrial network with a low level of MAP1LC3B green puncta. In mPMs exposed to cocaine, on the other hand, there was increased expression of MAP1LC3B green puncta throughout the cell, which notably co-localized with the mitochondrial red puncta (pLV-mitoDsRed), thereby confirming the formation of mitophagosomes. As expected, mPMs exposed to rotenone also showed increased yellow puncta (merged red and green puncta) (Figure 3(e)).Cocaine-mediated defective autophagic flux in mPMsFollowing the induction of autophagy, proteolytically processed cytosolic MAP1LC3B-I is converted to a highly lipophilic form – phosphatidylethanolamine conjugated MAP1LC3B-II – which is subsequently recruited to the phagophore and forms the autophagosome. MAP1LC3B-II then interacts with SQSTM1 for selective degradation and turnover of the cargo. MAP1LC3B-II levels are thus directly correlated with the number of autophagosomes [31]. To assess whether the increased quantity of MAP1LC3B puncta was a result of enhanced autophagosome synthesis or reduced autophagosome turnover (due to delayed trafficking or reduced fusion with the lysosomes), we next performed western blotting for MAP1LC3B-II in control and cocaine-exposed mPMs in the presence or absence of bafilomycin A1 – a known inhibitor of autophagosome fusion [30]. As shown in Figure 4(a), exposure of mPMs to cocaine (10 µM for 24 h) followed by exposure of cells to bafilomycin A1 (400 nM, saturating concentration) for 4 h, resulted in significant accumulation of MAP1LC3B-II, as was also evident in mPMs exposed to bafilomycin A1 alone, suggesting thereby that cocaine exposure inhibited auto-lysosomal fusion and degradation.It has been reported that impaired degradation and accumulation of SQSTM1 protein is directly correlated with the rate of autophagic vesicle degradation [32]. We thus next performed the SQSTM1 degradation assay by measuring the expression of SQSTM1 in mPMs with and without cocaine exposure in the presence or absence of bafilomycin A1. Our findings demonstrated no significant change in the accumulation of SQSTM1 in mPMs exposed to cocaine in the presence or absence of bafilomycin A1 (Figure 4(b)).Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 4. Cocaine increases autophagosome formation and decreases autophagic flux in mPMs. (a and b) Representative western blots showing the expression of MAP1LC3B-II (a) and SQSTM1 (b) in mPMs exposed to 10 μM cocaine for 24 h followed by treatment with 400 nM bafilomycin A1, added during the last 4 h of the 24 h treatment period. ACTB was probed as a loading control for all experiments. (c) mPMs transfected with tandem fluorescent-tagged MAP1LC3B plasmid followed by exposed with either 10 μM cocaine or 100 nM rapamycin for 24 h or 400 nM bafilomycin A1, added during the last 4 h of the 24 h treatment period. Scale bar: 10 μm. (d and e) Bar graph showing the number of autophagosomes (d) and autolysosomes (e) in mPMs transfected with tandem fluorescent-tagged MAP1LC3B plasmid and exposed to 10 μM cocaine or 100 nM rapamycin or 400 nM bafilomycin A1. The data are presented as mean ± SEM from six independent experiments. Non-parametric Kruskal – Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups and Wilcoxon test was used to make comparisons between the two groups: *, P  0.05 vs. control.Display full sizeFigure 4. Cocaine increases autophagosome formation and decreases autophagic flux in mPMs. (a and b) Representative western blots showing the expression of MAP1LC3B-II (a) and SQSTM1 (b) in mPMs exposed to 10 μM cocaine for 24 h followed by treatment with 400 nM bafilomycin A1, added during the last 4 h of the 24 h treatment period. ACTB was probed as a loading control for all experiments. (c) mPMs transfected with tandem fluorescent-tagged MAP1LC3B plasmid followed by exposed with either 10 μM cocaine or 100 nM rapamycin for 24 h or 400 nM bafilomycin A1, added during the last 4 h of the 24 h treatment period. Scale bar: 10 μm. (d and e) Bar graph showing the number of autophagosomes (d) and autolysosomes (e) in mPMs transfected with tandem fluorescent-tagged MAP1LC3B plasmid and exposed to 10 μM cocaine or 100 nM rapamycin or 400 nM bafilomycin A1. The data are presented as mean ± SEM from six independent experiments. Non-parametric Kruskal – Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups and Wilcoxon test was used to make comparisons between the two groups: *, P  0.05 vs. control.Next, to further validate that cocaine-mediated decreased autophagy flux was attributable to impaired fusion, we used a tandem fluorescent-tagged MAP1LC3B plasmid to track autophagosome formation and its fusion with the lysosome. Following autophagosome formation, the plasmid encoding red and green fluorescent protein-tagged MAP1LC3B emits both red and green fluorescence which appears as merged yellow puncta. After fusion with lysosomes, however, the green fluorescent signal is quenched due to the acidic pH of the lysosomes and only emits the red fluorescence. Thus, increased numbers of yellow puncta are an indication of impaired auto-lysosome fusion. Increased accumulation of the red puncta, on the other hand, indicate augmented autophagy flux [33]. Intriguingly, mPMs transfected with the reporter plasmid and exposed to cocaine (10 µM) for 24 h exhibited increased numbers of yellow puncta, thereby indicating cocaine-mediated impairment of autophagosome maturation. Cells exposed to the autophagy inducer rapamycin (100 nM), showed increased numbers of red puncta, as expected. Reciprocally and as expected, mPMs exposed to the autophagy flux inhibitor bafilomycin A1 (400 nM), demonstrated increased numbers of yellow puncta, similar to that observed in cocaine-exposed mPMs (Figure 4(c–e)).Role of mito/autophagosome initiation in cocaine-mediated mitophagy signalingNext, we sought to block autophagy using both pharmacological as well as genetic approaches to unravel the role of this process in cocaine-mediated mitophagy. For the pharmacological approach, mPMs were preexposed to either 5 mM 3-MA or 100 nM wortmannin for 1 h, followed by exposure of cells to 10 µM cocaine for 24 h. Expression of mitophagy and autophagy marker proteins were then determined using western blotting. Our findings demonstrated that cocaine-mediated upregulation of mito/autophagy markers including PINK1 (Figure 5(a)), PRKN (Figure 5(b)), DNM1L (Figure 5(c)), OPTN (Figure S1(a)), BECN1 (Figure 5(d)), MAP1LC3B-II (Figure 5(e)) and SQSTM1 (Figure 5(f)) was significantly (P  0.05) inhibited in mPMs pretreated with either 3-MA or wortmannin. Further validation of the role of mito/autophagy was also done using the gene silencing approach. For this, mPMs were first transfected with either BECN1 or scrambled siRNA followed by exposure of cells to 10 µM cocaine for 24 h and subsequent evaluation of the expression of mito/autophagy marker proteins by western blot. In cells transfected with scrambled siRNA, exposure to cocaine resulted in increased expression of mito/autophagy markers; however, in mPMs transfected with BECN1 siRNA, cocaine failed to upregulate mito/autophagy markers including PINK1 (Figure 5(g)), PRKN (Figure 5(h)), DNM1L (Figure 5(i)), OPTN (Figure S1(b)), BECN1 (Figure 5(j)), MAP1LC3B-II (Figure 5(k)) and SQSTM1 (Figure 5(l)).Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 5. Pharmacological and gene silencing of autophagy markers blocks cocaine-mediated mitophagy. (a–f) Representative western blots showing expression of mitophagy markers such as PINK1 (a), PRKN (b), and DNM1L (c) and autophagy markers such as BECN1 (d), MAP1LC3B-II (e), and SQSTM1 (f) in mPMs pretreated with either 5 mM of 3-methyladenine (3-MA) or 100 nM of wortmannin for 1 h following exposure of cells to 10 μM cocaine for 24 h. (g–l) Representative western blots showing the expression of PINK1 (g), PRKN (h), and DNM1L (i), BECN1 (j), MAP1LC3B-II (k), and SQSTM1 (l) in mPMs transfected with BECN1 siRNA and scrambled siRNA following exposure of cells to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. The data are presented as mean±SEM from six independent experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.Display full sizeFigure 5. Pharmacological and gene silencing of autophagy markers blocks cocaine-mediated mitophagy. (a–f) Representative western blots showing expression of mitophagy markers such as PINK1 (a), PRKN (b), and DNM1L (c) and autophagy markers such as BECN1 (d), MAP1LC3B-II (e), and SQSTM1 (f) in mPMs pretreated with either 5 mM of 3-methyladenine (3-MA) or 100 nM of wortmannin for 1 h following exposure of cells to 10 μM cocaine for 24 h. (g–l) Representative western blots showing the expression of PINK1 (g), PRKN (h), and DNM1L (i), BECN1 (j), MAP1LC3B-II (k), and SQSTM1 (l) in mPMs transfected with BECN1 siRNA and scrambled siRNA following exposure of cells to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. The data are presented as mean±SEM from six independent experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.Next, we sought to evaluate the effect of a specific mitophagy inhibitor (Mdivi-1) on cocaine-mediated mitophagy signaling in mPMs. To assess this, expression of mito/autophagy marker proteins were monitored in mPMs that were pretreated with Mdivi-1 (25 mM) for 1 h followed by exposure to cocaine for 24 h. As expected, in mPMs pretreated with Mdivi1, cocaine did not induce the mitophagy markers, PINK1 (Figure 6(a)), PRKN (Figure 6(b)), DNM1L (Figure 6(c)) and OPTN (Figure S1(c)) nor the autophagy markers, MAP1LC3B-II (Figure 6(d)) and SQSTM1 (Figure 6(e)). Interestingly, cocaine-mediated upregulation of BECN1 (Figure 6(f)) was not inhibited in cells pretreated with Mdivi-1, suggesting thereby that initiation of autophagy was upstream of PINK1/PRKN-mediated priming of the damaged mitochondria. These findings were further validated using the gene silencing approach. Cells were transfected with either PINK1 or scrambled siRNA overnight followed by exposure of mPMs to 10 µM cocaine for 24 h. Cell lysates were then monitored for expression of mito/autophagy marker proteins by western blot. Our results showed that similar to the pharmacological inhibition of mitophagy, genetic silencing also inhibited cocaine-mediated upregulation of mitophagy marker proteins including PINK1 (Figure 6(g)), PRKN (Figure 6(h)), DNM1L (Figure 6(i)) and OPTN (Figure S1(d)), as well as, the autophagy marker proteins, MAP1LC3B-II (Figure 6(j)) and SQSTM1 (Figure 6(k)) but had no effect on the autophagy initiation marker, BECN1 (Figure 6(l)).Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 6. Pharmacological and gene silencing of mitophagy markers blocks cocaine-mediated mitophagy. (a–f) Representative western blots showing expression of mitophagy markers such as PINK1 (a), PRKN (b), and DNM1L (c) and autophagy markers such as MAP1LC3B-II (d), SQSTM1 (e), and BECN1 (f) in mPMs pretreated with 25 μM Mdivi-1 (a mitophagy inhibitor) for 1 h following exposure to 10 μM cocaine for 24 h. (g–l) Representative western blots showing expression of PINK1 (g), PRKN (h), and DNM1L (i), MAP1LC3B-II (j), SQSTM1 (k), and BECN1 (l) in mPMs transfected with either PINK1 siRNA or scrambled siRNA, following exposure of cells to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups.Display full sizeFigure 6. Pharmacological and gene silencing of mitophagy markers blocks cocaine-mediated mitophagy. (a–f) Representative western blots showing expression of mitophagy markers such as PINK1 (a), PRKN (b), and DNM1L (c) and autophagy markers such as MAP1LC3B-II (d), SQSTM1 (e), and BECN1 (f) in mPMs pretreated with 25 μM Mdivi-1 (a mitophagy inhibitor) for 1 h following exposure to 10 μM cocaine for 24 h. (g–l) Representative western blots showing expression of PINK1 (g), PRKN (h), and DNM1L (i), MAP1LC3B-II (j), SQSTM1 (k), and BECN1 (l) in mPMs transfected with either PINK1 siRNA or scrambled siRNA, following exposure of cells to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups.Cocaine-mediated defective mitophagy involves microglial activationNext, we sought to investigate whether cocaine-mediated defective mitophagy played a role in microglial activation and increased production/release of proinflammatory cytokines. Cocaine exposure resulted in increased expression of the microglial activation marker ITGAM as well as proinflammatory cytokines (Figure 7(a–h)). To further confirm the role of auto/mitophagy in this process, cells were pretreated with either 3-MA (5 mM), wortmannin (100 nM) or Mdivi-1 (25 mM) for 1 h and then exposed to 10 µM of cocaine for 24 h and assessed for expression of a) ITGAM by western blotting, as well as, b) mRNAs for proinflammatory cytokines by qPCR. A parallel genetic approach was also done by knockdown of BECN1 or PINK1 using siRNA. Our findings demonstrated that both pharmacological inhibition of auto/mitophagy and genetic knockdown of BECN1 or PINK1, significantly (P  0.05) abrogated cocaine-mediated upregulation of the microglial activation marker ITGAM in mPMs (Figure 7(a–d)). Furthermore, cocaine exposure induced increased mRNA expression of Tnf (tumor necrosis factor), Il1b (interleukin 1 beta) and Il6 (interleukin 6) which was also significantly (P  0.05) abrogated by pharmacological inhibition of either autophagy (Figure 7(e)) or mitophagy (Figure 7(f)). Gene silencing of BECN1 or PINK1 also significantly (P  0.05) attenuated cocaine-mediated upregulation of proinflammatory cytokines (Figure 7(g,h)), thereby underscoring the role of cocaine-mediated induction of auto/mitophagy in activation of microglia.Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 7. Cocaine-mediated defective mitophagy increases microglial activation and elevates the generation of proinflammatory cytokines. (a and b) Representative western blots showing the expression of ITGAM in mPMs pretreated with 5mM 3-MA and 100 nM wortmannin (a), or in cells pretreated with 25 μM Mdivi-1 (b) for 1 h following exposure to 10 μM cocaine for 24 h. (c and d) Representative western blots showing expression of ITGAM in mPMs transfected with either BECN1 siRNA or scrambled siRNA (c) or with PINK1 siRNA or scrambled siRNA (d) following exposure to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. (e and f) Representative bar graphs showing the mRNA expression profile of proinflammatory cytokines such as Tnf, Il1b, and Il6 using qPCR in mPMs pretreated with 5mM of 3-MA and 100 nM of wortmannin (e) or pretreated with 25 μM Mdivi-1 (f) for 1 h following exposure to 10 μM cocaine for 24 h. (g and h) Representative bar graphs showing the mRNA expression profile of proinflammatory cytokines such as Tnf, Il1b, and Il6 using qPCR in mPMs transfected with either BECN1 siRNA or scrambled siRNA (g) or transfected with either PINK1 siRNA or scrambled siRNA (h) following exposure to 10 μM cocaine for 24 h. Gapdh was used as an internal control to normalize the gene expression for all experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.*, P  0.05 vs. control; #, P  0.05 vs. cocaine.Display full sizeFigure 7. Cocaine-mediated defective mitophagy increases microglial activation and elevates the generation of proinflammatory cytokines. (a and b) Representative western blots showing the expression of ITGAM in mPMs pretreated with 5mM 3-MA and 100 nM wortmannin (a), or in cells pretreated with 25 μM Mdivi-1 (b) for 1 h following exposure to 10 μM cocaine for 24 h. (c and d) Representative western blots showing expression of ITGAM in mPMs transfected with either BECN1 siRNA or scrambled siRNA (c) or with PINK1 siRNA or scrambled siRNA (d) following exposure to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. (e and f) Representative bar graphs showing the mRNA expression profile of proinflammatory cytokines such as Tnf, Il1b, and Il6 using qPCR in mPMs pretreated with 5mM of 3-MA and 100 nM of wortmannin (e) or pretreated with 25 μM Mdivi-1 (f) for 1 h following exposure to 10 μM cocaine for 24 h. (g and h) Representative bar graphs showing the mRNA expression profile of proinflammatory cytokines such as Tnf, Il1b, and Il6 using qPCR in mPMs transfected with either BECN1 siRNA or scrambled siRNA (g) or transfected with either PINK1 siRNA or scrambled siRNA (h) following exposure to 10 μM cocaine for 24 h. Gapdh was used as an internal control to normalize the gene expression for all experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.*, P  0.05 vs. control; #, P  0.05 vs. cocaine.Silencing of PINK1 or BECN1 failed to prevent cocaine-mediated mitochondrial dysfunction in mPMsNext, we sought to study the role of mitophagy in mitochondrial functioning. For this, cells were transfected with either PINK1 or BECN1 siRNAs overnight and then exposed to cocaine and assessed for mitochondrial functions. OCR and ECAR were determined both at baseline, as well as, following sequential injection of oligomycin (mitochondrial oxidative phosphorylation complex V inhibitor), FCCP-Carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone (complex I inhibitor), and rotenone and antimycin A (complex I and V inhibitors, respectively) using a Seahorse XFe96 Analyzer. Our findings demonstrated that mPMs transfected with either PINK1 or BECN1 siRNA failed to block cocaine-mediated downregulation of both OCR (Figure 8(a,b)) and ECAR (Figure 8(c,d)). In mPMs transfected with either scrambled, PINK1 or BECN1 siRNA, exposure to cocaine continued to downregulate basal respiration, ATP production rate, MRC of the mitochondria and proton leak (Figure 8(e,f)), thereby suggesting that cocaine-mediated mitochondrial dysfunction was upstream of the mitophagy process.Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 8. Gene silencing of PINK1 and BECN1 partially inhibited cocaine-mediated mitochondrial dysfunction in mPMs. (a and b) Graphical representation of the OCR measurement over time in mPMs transfected with either PINK1 siRNA and scrambled siRNA (a) or transfected with either BECN1 siRNA or scrambled siRNA (b) following exposure of cells to 10 μM cocaine for 24 h. (c and d) Graphical representation of the ECAR measurement over time in mPMs transfected with either PINK1 siRNA and scrambled siRNA (c) or transfected with either BECN1 siRNA and scrambled siRNA (d) following exposure of cells to 10 μM cocaine for 24 h. (e and f) Bar graphs showing the relative parameters of the mitochondrial respiratory function in mPMs transfected with either PINK1 siRNA (e) or BECN1 siRNA (f) calculated from respective Panel A or Panel B. The data are presented as mean ± SEM from six independent experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.Display full sizeFigure 8. Gene silencing of PINK1 and BECN1 partially inhibited cocaine-mediated mitochondrial dysfunction in mPMs. (a and b) Graphical representation of the OCR measurement over time in mPMs transfected with either PINK1 siRNA and scrambled siRNA (a) or transfected with either BECN1 siRNA or scrambled siRNA (b) following exposure of cells to 10 μM cocaine for 24 h. (c and d) Graphical representation of the ECAR measurement over time in mPMs transfected with either PINK1 siRNA and scrambled siRNA (c) or transfected with either BECN1 siRNA and scrambled siRNA (d) following exposure of cells to 10 μM cocaine for 24 h. (e and f) Bar graphs showing the relative parameters of the mitochondrial respiratory function in mPMs transfected with either PINK1 siRNA (e) or BECN1 siRNA (f) calculated from respective Panel A or Panel B. The data are presented as mean ± SEM from six independent experiments. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.Cocaine-mediated defective mitophagy and microglial activation involves SIGMAR1Cocaine exerts its toxic and stimulatory actions on cells via binding to SIGMAR1, a well-documented protein target for cocaine [34]. In keeping with this, several studies have reported that protective doses of SIGMAR1 antagonists prevented cocaine-mediated gene dysregulation, behavioral changes, and psychomotor stimulant effects [35–37]. To understand the role of SIGMAR1 in cocaine-mediated auto/mitophagy effects in mPMs, cells were pretreated with the SIGMAR1 antagonist, BD1047 (10 µM) for 1 h, then exposed to cocaine for 24 h, followed by assessment of cell lysates for expression of auto/mitophagy markers and the microglial activation marker ITGAM by western blot. As shown in Figure 9(a–g), cocaine-mediated induction of mitophagy markers such as PINK1 (Figure 9(a)), PRKN (Figure 9(b)), DNM1L (Figure 9(c)) and OPTN (Figure 9(d)), as well as, autophagy markers, BECN1 (Figure 9(e)), MAP1LC3B-II (Figure 9(f)) and SQSTM1 (Figure 9(g)) were significantly (P  0.05) inhibited in mPMs pretreated with BD1047, which was comparable to control unexposed mPMs. We have also checked the protein expression of the SIGMAR1. Intriguingly, exposure of mPMs to cocaine significantly (P  0.05) upregulated SIGMAR1 expression (Figure 9(h)) which was prevented by treatment with BD1047 prior to cocaine exposure. In addition, cocaine exposure mediated increased protein expression of the microglial activation marker ITGAM (Figure 9(i)), as well as, elevated mRNA expression of proinflammatory cytokines, such as Tnf (Figure 9(j)),Il1b (Figure 9(k)) and Il6 (Figure 9(l)); effects which were also averted in mPMs pretreated with BD1047 (Figure 9(j–l)).Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 9. Pharmacological blocking of SIGMR1 inhibited cocaine-mediated mitophagy and microglial activation. (a–i) Representative western blots showing expression of mitophagy markers such as PINK1 (a), PRKN (b), DNM1L (c) and OPTN (d) and autophagy markers such as BECN1 (e), MAP1LC3B-II (f), and SQSTM1 (g), SIGMAR1 (h) and microglial activation marker ITGAM (i) in mPMs pretreated with 10 μM BD1047 (an SIGMR1 inhibitor) for 1 h following exposure of cells to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. (j–l) Representative bar graph showing the mRNA expression profile of proinflammatory cytokines such as Tnf (j), Il1b (k), and Il6 (l) using qPCR in mPMs pretreated with 10 μM BD1047 for 1 h following exposure of cells to 10 μM cocaine for 24 h. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.Display full sizeFigure 9. Pharmacological blocking of SIGMR1 inhibited cocaine-mediated mitophagy and microglial activation. (a–i) Representative western blots showing expression of mitophagy markers such as PINK1 (a), PRKN (b), DNM1L (c) and OPTN (d) and autophagy markers such as BECN1 (e), MAP1LC3B-II (f), and SQSTM1 (g), SIGMAR1 (h) and microglial activation marker ITGAM (i) in mPMs pretreated with 10 μM BD1047 (an SIGMR1 inhibitor) for 1 h following exposure of cells to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. (j–l) Representative bar graph showing the mRNA expression profile of proinflammatory cytokines such as Tnf (j), Il1b (k), and Il6 (l) using qPCR in mPMs pretreated with 10 μM BD1047 for 1 h following exposure of cells to 10 μM cocaine for 24 h. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.Mitigation of ROS abrogated cocaine-mediated defective mitophagy and microglial activationSince cocaine is known to induce the generation of ROS, the next step was to explore the involvement of ROS in cocaine-mediated induction of mitophagy and microglial activation. Cells were pretreated with either TEMPOL, a non-specific superoxide scavenger which is a precursor for most ROS [38] or MitoTEMPO, a mitochondrial-specific superoxide scavenger [39] for 1 h followed by exposure of mPMs to 10 µM of cocaine for 24 h and assessed for auto/mitophagy markers by western blot. As shown in Figure 10(a–g), exposure of cells to cocaine resulted in the upregulation of mitophagy markers PINK1 (Figure 10(a)), PRKN (Figure 10(b)) DNM1L (Figure 10(c)) and OPTN (Figure 10(d)), as well as, autophagy markers, BECN1 (Figure 10(e)), MAP1LC3B-II (Figure 10(f)) and SQSTM1 (Figure 10(g)). Expression of all these mediators was significantly (P   0.05) inhibited in cells pretreated with TEMPOL (20 µM). Furthermore, expression of the microglial activation marker ITGAM (Figure 10(h)), as well as the proinflammatory cytokines (Tnf, Il1b, and Il6; Figure 10(i)), was also abrogated in mPMs that were pretreated with TEMPOL followed by exposure to cocaine. Intriguingly, pretreatment of mPMs with MitoTEMPO (10 μM) also blocked cocaine-mediated upregulated expression of mito/autophagy markers (Figure 11(a–g)), microglial activation (Figure 11(h)) and upregulation of proinflammatory cytokines (Figure 11(i)), thereby underscoring the role of cocaine-mediated mitochondrial ROS in defective mitophagy and activation of microglia.Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 10. ROS scavenger TEMPOL abrogated cocaine-mediated mitophagy and microglial activation. (a–h) Representative western blots showing expression of mitophagy markers – PINK1 (a), PRKN (b), DNM1L (c) and OPTN (d) and autophagy markers – BECN1 (e), MAP1LC3B-II (f), and SQSTM1 (g) and microglial activation marker ITGAM (h) in mPMs pretreated with 20 μM TEMPOL (total cellular superoxide dismutase mimetic) for 1 h following exposure of cells to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. (i) Representative bar graph showing the mRNA expression profile of proinflammatory cytokines such as Tnf, Il1b, and Il6 using qPCR in mPMs pretreated with 20 μM TEMPOL for 1 h following exposure of cells with 10 μM cocaine for 24 h. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.Display full sizeFigure 10. ROS scavenger TEMPOL abrogated cocaine-mediated mitophagy and microglial activation. (a–h) Representative western blots showing expression of mitophagy markers – PINK1 (a), PRKN (b), DNM1L (c) and OPTN (d) and autophagy markers – BECN1 (e), MAP1LC3B-II (f), and SQSTM1 (g) and microglial activation marker ITGAM (h) in mPMs pretreated with 20 μM TEMPOL (total cellular superoxide dismutase mimetic) for 1 h following exposure of cells to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. (i) Representative bar graph showing the mRNA expression profile of proinflammatory cytokines such as Tnf, Il1b, and Il6 using qPCR in mPMs pretreated with 20 μM TEMPOL for 1 h following exposure of cells with 10 μM cocaine for 24 h. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 11. ROS scavenger MitoTEMPO abrogated cocaine-mediated mitophagy and microglia activation. (a–h) Representative western blots showing expression of mitophagy markers – PINK1 (a), PRKN (b), DNM1L (c) and OPTN (d) and autophagy markers – BECN1 (e), MAP1LC3B-II (f), and SQSTM1 (g) and microglial activation marker ITGAM (h) in mPMs pretreated with 10 μM MitoTEMPO (mitochondrial ROS scavenger) for 1 h following exposure of cells to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. (i) Representative bar graph showing the mRNA expression profile of proinflammatory cytokines such as Tnf, Il1b, and Il6 using qPCR in mPMs pretreated with 10 μM MitoTEMPO for 1 h following exposure of cells to 10 μM cocaine for 24 h. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.Display full sizeFigure 11. ROS scavenger MitoTEMPO abrogated cocaine-mediated mitophagy and microglia activation. (a–h) Representative western blots showing expression of mitophagy markers – PINK1 (a), PRKN (b), DNM1L (c) and OPTN (d) and autophagy markers – BECN1 (e), MAP1LC3B-II (f), and SQSTM1 (g) and microglial activation marker ITGAM (h) in mPMs pretreated with 10 μM MitoTEMPO (mitochondrial ROS scavenger) for 1 h following exposure of cells to 10 μM cocaine for 24 h. ACTB was probed as a protein loading control for all experiments. (i) Representative bar graph showing the mRNA expression profile of proinflammatory cytokines such as Tnf, Il1b, and Il6 using qPCR in mPMs pretreated with 10 μM MitoTEMPO for 1 h following exposure of cells to 10 μM cocaine for 24 h. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to determine the statistical significance among multiple groups. *, P  0.05 vs. control; #, P  0.05 vs. cocaine.Cocaine-mediated increased expression of mito/autophagy markers, microglial activation and proinflammatory cytokines in vivoHaving demonstrated that cocaine-mediated defective mitophagy and microglial activation in vitro, we next sought to assess whether cocaine administration in vivo could also result in defective auto/mitophagy and microglial activation. Mice were divided into two groups (n = 4/group) with one group of mice given saline and the other group administered cocaine once a day (20 mg/kg, I.P.) for 7 consecutive days. One hour following the last cocaine injection, mice were sacrificed, brains were removed and the striatal tissue homogenates assessed for the expression of mito/autophagic markers, a microglial activation marker and proinflammatory cytokines. Our results demonstrated that chronic cocaine administration resulted in significant upregulation of mitophagy markers [PINK1 (Figure 12(a)), PRKN (Figure 12(b)), DNM1L (Figure 12(c)) and OPTN (Figure 12(d))], as well as, autophagy markers [BECN1 (Figure 12(e)), MAP1LC3B-II (Figure 12(f)) and SQSTM1 (Figure 12(g))] in the striatal brain tissue compared with saline-injected controls. Additionally, chronic cocaine administration also resulted in significant upregulation of both ITGAM (Figure 12(h)) and mRNA expression of the proinflammatory cytokines, such as Tnf, Il1b and Il6 (Figure 12(i)) compared with the saline administered mice.Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 12. Cocaine-mediated upregulation of mito/autophagy markers and proinflammatory cytokines in vivo. (a–h) Representative western blots showing the activation of mitophagy markers – PINK1 (a), PRKN (b), DNM1L (c) and OPTN (d) and autophagy markers – BECN1 (e), MAP1LC3B-II (f), and SQSTM1 (g) and microglial activation marker ITGAM (h) in the striatum of saline or cocaine administered mice (n = 4). ACTB was probed as a protein loading control for all experiments. (i) Representative bar graphs showing the mRNA expression profile of proinflammatory cytokines such as Tnf, Il1b, and Il6 using qPCR in the striatum of saline or cocaine administered mice (n = 4). Gapdh was used as an internal control to normalize the gene expression for all experiments. The data are presented as mean ± SEM. Unpaired Student t test was used to determine the statistical significance. *, P  0.05 vs. saline group.Display full sizeFigure 12. Cocaine-mediated upregulation of mito/autophagy markers and proinflammatory cytokines in vivo. (a–h) Representative western blots showing the activation of mitophagy markers – PINK1 (a), PRKN (b), DNM1L (c) and OPTN (d) and autophagy markers – BECN1 (e), MAP1LC3B-II (f), and SQSTM1 (g) and microglial activation marker ITGAM (h) in the striatum of saline or cocaine administered mice (n = 4). ACTB was probed as a protein loading control for all experiments. (i) Representative bar graphs showing the mRNA expression profile of proinflammatory cytokines such as Tnf, Il1b, and Il6 using qPCR in the striatum of saline or cocaine administered mice (n = 4). Gapdh was used as an internal control to normalize the gene expression for all experiments. The data are presented as mean ± SEM. Unpaired Student t test was used to determine the statistical significance. *, P  0.05 vs. saline group.To further validate the link between cocaine-mediated defective mitophagy and microglial activation, we performed double immunofluorescence staining for mito/autophagy markers PINK1 and DNM1L, and AIF1 (a microglia marker) in the brain sections of mice administered either cocaine or saline for 7 consecutive days. Our results showed that the stain intensity of PINK1 (Figure 13(a,b)) and DNM1L (Figure 13(c,d)), the number of puncta (green), as well as, the number of cells expressing AIF1 (red; Figure 13(e)) was significantly increased in the striatal regions of cocaine administered mice compared with the saline group. In the saline group, AIF1 labeling exhibited a homogenous distribution throughout the microglial cell body, as well as, in the cellular processes, revealing a ramified structure with multiple long, thin highly branched processes (Figure 13(a–e)). In contrast, AIF1 labeling of brain sections from cocaine administered mice demonstrated a rounded morphology with a significantly reduced length of the cell processes that was accompanied with increased AIF1 intensity (Figure 13(a–e)) compared to the saline group. Intriguingly, in cocaine administered mice, the mito/autophagy markers (green) also were found to colocalize with AIF1 (red; Figure 13(a–d)), suggesting thereby that cocaine administration resulted in defective mitophagy in microglia, which in turn, resulted in increased activation of microglia with a concomitantly increased pro-inflammatory response.Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 13. Cocaine-mediated upregulation of mito/autophagy markers and microglial activation in vivo. (a) Immunofluorescence staining for PINK1 (green), AIF1, microglial activation marker (red), and DAPI (blue) in the striatum of saline or cocaine-administered mice. (b) Bar graph showing the percentage colocalization of PINK1 with AIF1 and fluorescence intensity of PINK1 in the striatal regions of saline and cocaine administered mice. (c) Immunofluorescence staining for DNM1L (green), AIF1, microglial activation marker (red), and DAPI (blue) in striatal regions of saline or cocaine-administered mice. (d) Bar graph showing the percentage colocalization of DNM1L with AIF1 and fluorescence intensity of DNM1L in the striatal regions of saline and cocaine administered mice. (e) Bar graph showing the mean microglial cell processes length and number AIF1 positive microglial cells in the striatal regions of saline and cocaine administered mice. Scale bar: 10 μm. *, P  0.05 vs. control.Display full sizeFigure 13. Cocaine-mediated upregulation of mito/autophagy markers and microglial activation in vivo. (a) Immunofluorescence staining for PINK1 (green), AIF1, microglial activation marker (red), and DAPI (blue) in the striatum of saline or cocaine-administered mice. (b) Bar graph showing the percentage colocalization of PINK1 with AIF1 and fluorescence intensity of PINK1 in the striatal regions of saline and cocaine administered mice. (c) Immunofluorescence staining for DNM1L (green), AIF1, microglial activation marker (red), and DAPI (blue) in striatal regions of saline or cocaine-administered mice. (d) Bar graph showing the percentage colocalization of DNM1L with AIF1 and fluorescence intensity of DNM1L in the striatal regions of saline and cocaine administered mice. (e) Bar graph showing the mean microglial cell processes length and number AIF1 positive microglial cells in the striatal regions of saline and cocaine administered mice. Scale bar: 10 μm. *, P  0.05 vs. control.DiscussionMicroglia belong to the monocyte/macrophage lineage and are the predominant resident immune cells of the CNS, playing a significant role in brain tissue repair, neurogenesis and mediating immune responses [10,40,41]. Several reports have documented the activation of microglia under conditions of drug addiction, including exposure to cocaine, resulting in the release of a plethora of proinflammatory cytokines and chemokines that in turn, contribute to neurodegeneration [7,16,42–44]. Findings from our group have also demonstrated that cocaine exposure can activate microglial cells via activation of the TLR2 signaling cascade [45]. Another study has also demonstrated that cocaine-mediated the induction of microglial activation and ensuing inflammation, which involved impaired autophagy signaling [46]. Interestingly, there is a growing body of evidence implicating the role of neuroinflammation (activation of glial cells) as a contributor to the development and maintenance of addictive behaviors involving cocaine and other abused substances [47–49].Cocaine has been reported to induce alterations in spine density and neuronal morphology in both the hippocampal and cortical neurons via its binding to SIGMAR1 via transactivation of the NTRK2/TrkB (neurotrophic receptor tyrosine kinase 2) pathway [50]. In another study, cocaine was shown to activate SIGMAR1, in turn, resulting in sustained Ca2+ efflux from the endoplasmic reticulum involving the inositol 1,4,5-trisphosphate receptors, leading to activation of D1-neurons in the nuclear accumbens. Additionally, repeated cocaine administration was shown to significantly modulate the dopamine neuron function, in turn, resulting in drug-induced alterations in synaptic plasticity, circuit remodeling and drug-adaptive behavior [51]. Several studies have also implicated cocaine-mediated activation of microglia and its sequelae of neuroinflammation, as contributors of neuronal plasticity changes, and alterations in behavioral responses associated with drug addiction [48,52,53].Microglia have been implicated in the maturation of neuronal circuitry during development. Furthermore, in the adult brain, microglia have also been shown to play a role in remodeling of neuronal circuitry, alterations in synaptic plasticity, pruning of synapses in response to the synaptic transmission, maintenance of biochemical homeostasis and providing nourishment to the neurons with neurotrophic factors [54–56]. Additionally, activated microglia have also been shown to be involved in phagocytosis of the damaged synaptic terminals of motor neurons [57]. Microglia have thus been implicated to play critical roles in drug-induced physiological and morphological changes in neuronal plasticity via multiple mechanisms, involving the release of proinflammatory cytokines, via direct synaptic remodeling or phagocytosis [43,48]. Collectively, microglial-neuron interactions play a vital role in drug abuse-mediated changes in synaptic plasticity, drug seeking and addictive behaviors. Further investigations are warranted to explore the interactions between microglia and neurons in cocaine addicts and also in identifying the molecular mechanism(s) underlying cocaine-mediated microglial activation.Cocaine exposure has been associated with mitochondrial dysfunction in the brain and heart by mediating alterations in the mitochondrial respiratory chain and the mitochondrial apoptotic pathway [19,58,59]. Mitochondria are cellular organelles that maintain the energetic status of the cell by producing ATP via the mitochondrial electron transfer chain and are also a source of cellular ROS due to electron leakage [60,61]. In recent years, several experimental in vivo studies have demonstrated that mitochondria significantly contribute to cocaine-mediated oxidative stress, increased ROS production, as well as, inflammation [5,62,63]. Mitophagy is a cellular process wherein the damaged mitochondria are primed, sequestered and removed via the lysosomal degradation, thereby, maintaining mitochondrial homeostasis and reduced ROS production and inflammation [64–66]. Recent reports have implicated that incomplete removal of the damaged mitochondria via the mitophagy underlies several neurodegenerative disorders [67–70]. Based on these findings, the present study was aimed at evaluating the role of defective mitophagy in cocaine-mediated activation of microglia.In this study, we demonstrated that exposure of mPMs to cocaine causes downregulation of mitochondrial membrane potential with alterations in the mitochondrial functions including basal and maximal respiration rate, ATP production rate, spare capacity, and the overall OCR. We observed that a physiologically relevant dose of cocaine (10 μM) could cause significant mitochondrial dysfunction while also inducing mitophagy. The concentration of cocaine in the serum of cocaine abusers has been reported to be between 0.3 µM and 1 mM [71]. The concentration of cocaine in the brain is generally higher than that in the blood. In fact, human toxicity studies have demonstrated brain/blood ratios of cocaine concentration was 9.6 and its metabolite benzoylecgonine concentrations 0.36 [35]. Our range of cocaine concentration is thus in line with reported human studies.In the current study, the mitophagy markers proteins PINK1, PRKN, DNM1L and OPTN, as well as the autophagy machinery proteins, BECN1, MAP1LC3B-II, and SQSTM1, were significantly upregulated following exposure of mPMs to cocaine. This was further validated by TEM and, mitoDsRed and GFP-MAP1LC3B plasmid overexpression studies, which demonstrated the formation and accumulation of mitophagosomes in mPMs exposed to cocaine. These findings suggested that cocaine exposure commenced the initiation of the mitophagy pathway in mPMs. Following the induction of mitophagy, the formed mitophagosomes are continually degraded by lysosomes via the lysosomal fusion pathway. Accumulation of mitophagosomes in mPMs suggested a possible stress-mediated induction of mitophagy with a blockade of mitophagosome maturation and its clearance. Interestingly, our results showed a time-dependent upregulation of SQSTM1 protein following exposure of mPMs to cocaine, suggesting thereby increased accumulation of mito/autophagosomes that was accompanied by the impaired maturation of the same. The MAP1LC3B turnover assay and SQSTM1 degradation assay further confirmed the blockage of autophagic flux, as evidenced by the accumulation of MAP1LC3B-II and SQSTM1 in cocaine-exposed mPMs. Overexpression of the tandem fluorescently-tagged MAP1LC3B plasmid also validated increased accumulation of autophagosomes but not autolysosomes in the presence of cocaine.We also employed pharmacological, and gene silencing approaches to further confirm the role of mitophagy in cocaine-mediated microglial activation. Our results demonstrated that pharmacological inhibition of autophagy using 3-MA and wortmannin, and specific inhibition of mitophagy using Mdivi-1 or by gene silencing of BECN1 or PINK1, significantly abrogated cocaine-mediated induction of mitophagy as evidenced by inhibition of mito/autophagy marker proteins, microglial activation (demonstrated by decreased ITGAM expression) and neuroinflammation (evidenced by a significant reduction in proinflammatory cytokines) in cocaine-exposed mPMs. Intriguingly, inhibiting priming of damaged mitochondrial did not affect cocaine-mediated induction of BECN1 or initiation of autophagy, suggesting thereby that initiation of autophagy was upstream of the priming of damaged mitochondria. Reciprocally, however, inhibition of autophagy initiation or gene silencing of BECN1 significantly inhibited cocaine-mediated induction of PINK1/PRKN. We also performed mitochondrial functional analyses by measuring OCR in mPMs transfected with either BECN1 or PINK1 siRNAs, followed by exposure to cocaine. Our findings demonstrated that the cocaine-mediated downregulation of mitochondrial OCR was not blocked by silencing of either BECN1 and PINK1, suggesting thereby that the damage was persistent and was not reversed by blocking the mito/autophagy pathway.Cocaine-mediated effects have been shown to be elicited via SIGMAR1 activation, which in turn, functions as a molecular chaperone for a diverse complement of proteins and modulates multiple signaling pathways, which contribute to oxidative and nitrosative stress, inflammation and behavioral changes [4,36,72–74]. Several studies have identified the potential of SIGMAR1 antagonists as promising pharmacotherapy agents for combating cocaine abuse [7,75,76]. It has been reported that SIGMAR1 interacts at both the molecular and functional level with the DRD2 (dopamine receptor D2) and that, cocaine binds to SIGMAR1-DRD2 receptor heteromers thereby, modulating dopaminergic signaling in both cultured neuronal cells and the striatum [77]. Further, it has been well reviewed that SIGMAR1 ligands are involved in modulating multiple neurodegenerative processes either directly or indirectly, including calcium dysregulation, excitotoxicity, endoplasmic reticulum, and mitochondrial dysfunction, oxidative stress, astrogliosis and inflammation [78].In the current study, we assessed the role of SIGMAR1 activation in cocaine-mediated mitophagy and microglial activation and demonstrated that pretreatment of mPMs with the SIGMAR1 antagonist abrogated cocaine-mediated impairment of mito/autophagy signaling and microglial activation. Furthermore, our results also demonstrated that pretreatment of cells with the superoxide mimetics – TEMPOL as well as MitoTEMPO (both with superoxide radical scavenging properties) significantly blocked cocaine-mediated induction of mitophagy, suggesting thereby that cocaine-mediated generation of cellular and mitochondrial ROS plays a role in mitochondrial damage and defective mitophagy.Furthermore, our in vivo studies also validated cocaine-mediated upregulation of mitophagy markers (PINK1, PRKN, DNM1L, and OPTN), as well as, autophagy machinery proteins (BECN1, MAP1LC3B-II and SQSTM1) in the striatum of cocaine (20mg/kg) administered mice compared to wild-type controls. The expression of both proinflammatory cytokine RNA, as well as the microglial activation marker ITGAM was also found to be increased in the striatum of mice, administered cocaine compared with the wild-type mice. The rationale for choosing striatum is based on its ability to coordinate signals from other brain regions including the prefrontal cortex, ventral tegmental area and in regulating the behavioral output such as motor planning, decision-making, motivation, and reward. Importantly, striatum serves as a central interface of the brain circuit, and the neuroplastic changes within this region are believed to be crucial for drug seeking and addictive behaviors [79–82].In summary our findings underpin cocaine-induced microglial activation as a link to mitochondrial damage and ineffective clearance of dysfunctional mitochondria, owing to defective mitophagy (Figure 14). These findings also underscore the role of potent ROS scavengers and selective inhibitors of mito/autophagy as potential therapeutic agents for the treatment of cocaine abuse.Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsAll authorsAnnadurai Thangaraj , Palsamy Periyasamy , Ming-Lei Guo , Ernest T. Chivero , Shannon CallenShilpa Buch https://doi.org/10.1080/15548627.2019.1607686Published online:28 April 2019Figure 14. Schematic diagram outlining cocaine-mediated defective mitophagy and microglial activation. Exposure of microglia to cocaine decreases mitochondrial membrane potential, leading in turn, to mitochondrial dysfunction, that is followed by the initiation of mitophagy and mitophagosome formation. Exposure to cocaine, however, blocks mitophagosome maturation, thereby leading to impaired clearance of damaged mitochondria. Accumulation of mitophagosome due to defective mitophagy results in microglial activation and increased expression of proinflammatory cytokines, leading ultimately to neuroinflammation.Display full sizeFigure 14. Schematic diagram outlining cocaine-mediated defective mitophagy and microglial activation. Exposure of microglia to cocaine decreases mitochondrial membrane potential, leading in turn, to mitochondrial dysfunction, that is followed by the initiation of mitophagy and mitophagosome formation. Exposure to cocaine, however, blocks mitophagosome maturation, thereby leading to impaired clearance of damaged mitochondria. Accumulation of mitophagosome due to defective mitophagy results in microglial activation and increased expression of proinflammatory cytokines, leading ultimately to neuroinflammation.Materials and methodsReagentsCocaine hydrochloride (C5776), 3-MA (M9281), wortmannin (W3144), rapamycin (R0395), bafilomycin A1 (B1793), Mdivi-1 (M0199), rotenone (557368), MitoTEMPO (SML0737) were purchased from Sigma-Aldrich. Anti-PINK1 (ab23707) was purchased from Abcam. Anti-BECN1 (sc-11427), anti-PRKN (sc-32282), anti-OPTN (sc-271549), PINK1 siRNA (sc-44599), BECN1 siRNA (sc-29798), scrambled siRNA (sc-37007), and TEMPOL (sc-200825) were purchased from Santa Cruz Biotechnology. Anti-DNM1L (611112) was obtained from BD Biosciences. Anti-MAP1LC3B (NB100-2220) and anti-ITGAM (NB110-89474) were purchased from Novus Biological Company. Anti-SQSTM1 (PM045) was purchased from MBL International. Peroxidase-AffiniPure goat anti-mouse IgG (H + L) (111-035-003) and peroxidase-conjugated AffiniPure goat anti-rabbit IgG (H + L) (115-035-003) were from Jackson ImmunoResearch Inc. TEMPOL was purchased from Chem CruzAnimalsC57BL/6N mice purchased from Charles River Laboratories (Wilmington, MA) were used in this study. The animals were housed in clean polypropylene cages under standard vivarium conditions (12 h light/dark cycle) with constant temperature and humidity, during which time they had free access to food and water ad libitum. All the experimental procedures involving animal subjects were conducted according to the protocols reviewed and approved by the University of Nebraska Animal Care and Use Committee and were in accordance with guidelines. Experimental mice were divided into two groups: saline and cocaine. Both the groups of animals were injected with either saline or cocaine (20 mg/kg body weight) for 7 consecutive days intraperitoneally, and the mice were sacrificed 1 h following the last injection. Brain tissues were dissected out, and homogenates of striatum were used to determine the protein levels of mito/autophagic markers and microglial activation markers. Mice injected with saline served as controlsmPMs isolationC57BL/6N pregnant mice were purchased from Charles River Laboratories (Wilmington, MA USA). 1- to 3-d-old newborn pups (C57BL/6 strain) were used to prepare mouse primary mixed glial cultures, as described previously [16,83]. Briefly, Cerebral cortices from neonatal mice were dissected out. The blood vessels and meninges were removed from cerebral cortices of newborn pups and followed by mild trypsinization using 0.25% trypsin (Invitrogen, 25300-054) the medium containing cell mixture was passed through a 40 µm nylon mesh to remove cells debris. The cell pellet was obtained by centrifugation at 1000 x g for 5 min and the pellet was re-suspended in Dulbecco modified Eagle medium (DMEM; Corning Cellgro®, 10-013-CV) supplemented with 10% fetal bovine serum (FBS; Atlanta Biologicals, S11150H), penicillin (100 U/ml)-streptomycin (100 μg/ml) (Gibco, 15140122), 0.25 ng/ml of CSF1/macrophage colony-stimulating factor (Millipore Sigma, GF026) and OPI media supplement (Sigma-Aldrich, O5003-1VL). The cells (2 X 107) were then added to 20 mL culture medium and were plated to 75 cm2 culture flasks containing. The flask was maintained in a humidified incubator gassed with 5% CO2 at 37°C. On the third day, the whole media was changed with fresh 20 ml of culture media and about 5–10 ml of additional culture medium was added every two days to promote cell growth. Approximately 12–14 days after the flask containing mixed glial cultures were subjected to shaking at 37°C at 220 g for 2 h, to promote microglial detachment from the astrocytes bed. The media containing detached microglia was collected, centrifuged at 1000 x g for 5 min and plated in 6-well or 24 wells with a coverslip for further experimental use.Analysis of mitochondrial membrane depolarizationmPMs exposed to cocaine was monitored to assess the change in mitochondrial membrane potential using the JC-1 Mitochondrial Membrane Potential Assay Kit (Cayman Chemicals, 10009172) according to the manufacturer’s instructions. Briefly, mPMs were seeded at a density of 0.01 × 106 cells per well in a 96-well plate. The cells were then exposed to cocaine (10 μM) followed by treatment with JC-1 reagent (100 µl/ml of medium) diluted in serum-free culture medium (1:10 dilution) and incubated for 20 min at 37°C in 5% CO2 incubator. After that, the cells were rinsed once in 1× rinsing buffer provided in the kit. The red fluorescence intensities of JC-1 aggregates (λexcitation, 535 nm; λemission, 585 nm) and the green fluorescence intensities of monomers (λexcitation, 485 nm; λemission, 535 nm) were determined using a Synergy™ Mx Monochromator-Based Multi-Mode Microplate Reader (BioTek Instruments, Inc. Winooski, VT, USA). All experiments were repeated at least six times.Staining of mitophagosome formationmPM cells were seeded at a density of 5 × 104 per well onto sterile glass coverslips positioned in a 24-well plate and incubated at 37°C in a humidified, 5% CO2 incubator. The culture medium was replaced with Reduced Serum Medium Opti-MEM® I (Life Technologies, 31985070). The cells were then transfected with pLV-mitoDsRed plasmid [84] (a gift from Prof. Tsoulfas; Addgene, 44386) and the GFP-MAP1LC3B plasmid [85] (a gift from Prof. Kirkegaard; Addgene, 11546) using Lipofectamine® 2000 Reagent (Invitrogen, 12566014) according to the manufacturer’s protocol. After 6 h, the transfection medium was removed, and fresh normal culture medium (DMEM) supplemented with antibiotics and 10% heat-inactivated FBS was added and incubated for overnight at 37°C in a humidified, 5% CO2 incubator. Transfected mPMs were then exposed with either cocaine (10 µM) or rotenone (1 μM; as a positive control) for 24 h. At the end, the cells were fixed in 4% paraformaldehyde for 15 min at room temperature. The fixed cells were then washed 3 times with PBS (Hyclone Laboratories, SH3025801), the coverslips and each coverslip were mounted in a glass slide with ProLong Gold Antifade Reagent with 4,6-diamidino-2-phenylindole (DAPI; Molecular Probes, P36935). Images were acquired on a Zeiss Observer using a Z1 inverted fluorescence microscope. Red and green puncta colocalization in the images were analyzed using the Axio Vs 40 Version 4.8.0.0 software. All experiments were repeated at least six times.Transmission electron microscopymPMs were seeded in 12-well plates containing sterile glass coverslips at a density of 5 × 104 cells/well and placed overnight in a humidified, 5% CO2 incubator at 37°C. After overnight starvation, mPMs on coverslip were exposed to cocaine (10 µM) for 24 h. At the end of the experiment, the cells were washed with PBS twice and fixed with EM grade glutaraldehyde fixative buffer (containing 2% glutaraldehyde, 2% paraformaldehyde and 0.1 M cacodylate) for 30 min at room temperature. The fixed cells were stored at 4°C until processing for electron microscopy. The images were taken on FEI Tecnai G2 Spirit transmission electron microscope (FEI, Houston, TX, USA).Analyses of autophagosome formation and maturationAt a density of 5 × 104 cells per well, mPMs were seeded onto 11 mm sterile glass coverslips that were placed in a 24-well plate and incubated at 37°C in a humidified, 5% CO2 incubator. The tandem fluorescent-tagged MAP1LC3B plasmid (ptfLC3; a gift from Tamotsu Yoshimori; Addgene, 21074) [86] were transfected into the mPMs using Lipofectamine® 2000 Reagent according to the manufacturer’s protocol. To attain transfection of plasmid, the cells were incubated for 6–8 h at 37°C in a humidified, 5% CO2 incubator and at the end, the culture medium was replaced with DMEM containing 10% heat-inactivated FBS and kept in an incubator with same conditions for overnight. The transfected mPMs were then exposed to either cocaine (10 µM) or rapamycin (an autophagy inducer; 100 nM), for 24 h or bafilomycin A1 (an autophagosome-lysosome fusion inhibitor; 400 nM) for last 4 h at the end of experimental period. The fluorescence images were acquired on a Zeiss Observer using a Z1 inverted microscope (Carl Zeiss, Thornwood, NY, USA) and the images were analyzed by ImageJ software, the number of colocalized red and green fluorescence dots (autophagosomes) and the number of red fluorescence dots (autolysosome). The numbers of RFP-GFP-MAP1LC3B or RFP-MAP1LC3B dots were counted in 12 random non-overlapping fields. All experiments were repeated six times.MAP1LC3B turnover and SQSTM1 degradation assaysThe mPMs were collected from glial mixture flask and seeded at a density of 5 × 105 cells/well in a 6-well plate. The plates were then incubated in a humidified, 5% CO2 incubator at 37°C for attachment. Cells were then starved overnight in the serum-free culture medium. Next day, the mPMs were exposed to either cocaine (10 µM) for 24 h alone or the cells also exposed to 400 nM bafilomycin A1 in the last 4 h of the 24 h cocaine treatment period. After 24 h, protein samples were prepared from the cells and used for western blotting analysis. All experiments were repeated at least six times.siRNA transfectionGenetic knockdown of BECN1 or PINK1 was attained in mPMs using transfection of either with BECN1 or PINK1 siRNAs. Briefly, mPMs were seeded at a density of 5 × 105 cells/well into 6-well plate and allow them to attach at the bottom of the plate and incubated at 37°C with 5% CO2 in a humidified incubator. Before transfecting the cells, the culture medium was replaced with reduced Serum containing Medium Opti-MEM®. The individual targeted siRNA (either BECN1 or PINK1; 120 pmol/mL) was transfected into the mPMs using Lipofectamine® 2000 reagent according to manufacturer’s protocol. The cells also transfected with scrambled siRNA as a control. After 6–8 h, to attain efficient genetic knockdown, the transfected cells were cultured with DMEM supplemented with 10% heat-inactivated FBS for overnight. The transfected mPMs were then exposed to cocaine (10 µM) for 24 h. After 24 h, protein samples were prepared from the cells and used for western blotting analysis. The transfection efficiency was also analyzed by western blotting. All experiments were repeated at least six times.Western blottingAt the end of each experiment, harvested cells or brain tissue were lysed in RIPA buffer supplemented with Protease Inhibitor Cocktail (Thermo Fisher Scientific, 78429) and Phosphatase Inhibitor Cocktail (Thermo Fisher Scientific, 78426) using Fisherbrand™ Q125 Sonicator. The Cell lysates were then centrifuged at 12,000 x g for 15 min at 4°C. The supernatant was collected, and the protein concentration in those samples was then estimated by a BCA assay using the Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific, 23227) according to the manufacturer’s guidelines. The denatured protein samples were used to determine the relative expression levels of proteins such as PINK1, PRKN, DNM1L, OPTN, BECN1, MAP1LC3B-II, SQSTM1, and ITGAM by immunoblotting analyses. Briefly, equal amounts of soluble proteins samples with loading (10 μg) were subjected to separation based on molecular weight by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The protein bands in the gels were then transferred to a polyvinylidene fluoride membrane followed by blocking the membrane with 5% non-fat dry milk in 1× TTBS buffer (1.21 g Tris [Fisher Scientific, BP152-5], 8.77 g NaCl [Fisher Scientific, BP358-212], 500 μL Tween-20 [Fisher Scientific, BP337-500], pH 7.6 for 1 L) for 1 h at room temperature. The membrane was then probed with specific primary antibodies for overnight at 4°C, followed by the membrane was washed with 1× TTBS buffer (pH 7.6) for three times (each 5 min). Next, the membrane was incubated with species-specific secondary antibody conjugated to horseradish peroxidase for 1 h, followed by washing as described earlier. The Immunoreactive fluorescent bands were identified using Super Signal West Pico Chemiluminescent Substrate (Thermo Fisher Scientific, 34078). ACTB (actin beta; Sigma-Aldrich, A5316) was used as an internal control. Densitometry of each band was calculated using ImageJ analysis software [86]. The fold was calculated and presented as a graph. All experiments were repeated at least six times.Real-time qPCRTotal RNA was extracted from the mPMs and the brain tissues using Quick-RNATM MicroPrep kit (Zymo Research, R1051) as per the manufacturer’s protocol. The column purified total RNA was used as a template to synthesize cDNA via reverse transcription using Verso cDNA Synthesis Kit (Thermo Fisher Scientific, AB-1453/B), according to the manufacturer’s instructions. The expression profile of specific gene was successively analyzed by real time-PCR (Applied Biosystems, Grand Island, NY) using the RT2 SYBR Green Fluor qPCR Mastermix (Qiagen, 330510) using relevant mouse primer sequences, such as Tnf forward 5′-CAGCCTCTTCTCCTTCCTGAT-3′, Tnf reverse 5′-GCCAGAGGGCTGATTAGAGA-3′, Il1b forward 5′-TACCTGTCCTGCGTGTTGAA-3′, Il1b reverse 5′-TCTTTGGGTAATTTTTGGGATCT-3′, Il6 forward 5′-GATGAGTACAAAAGTCCTGATCCA-3′, Il6 reverse 5′-CTGCAGCCACTGGTTCTGT-3′, Gapdh (glyceraldehyde-3-phosphate dehydrogenase) forward 5′-GGCACCCAGCACAATGAA-3′, Gapdh reverse 5′-GCCGATCCACACGGAGTACT-3′. The amplification protocol comprised 1 cycle at 95°C for 3 min followed by 40 cycles at 95°C for 20 s, 60°C for 30 s, and then 72°C for 30 s. Normalization was done with Gapdh, an internal control. Each reaction was carried out in triplicate, and six independent experiments were run. The fold change in expression was then obtained by the 2−ΔΔCT method.Analysis of mitochondrial functionOxygen consumption rate and ECAR were measured using a Seahorse XFp Extracellular Flux Analyzer or Seahorse XFe96 Analyzer (Seahorse Bioscience, Billerica, MA, USA). MPMs were cultured in Seahorse XFp or XFe96 well cell culture plate at a density of 2 × 105 cells/well in DMEM media containing antibiotics and 10% heat-inactivated FBS. Following exposure to cocaine (10 µM) for 24 h, the culture medium was replaced with unbuffered DMEM containing 10 mM glucose, 2 mM pyruvate, and 2mM L-glutamine. At the same time, ‘Flux Pak’ cartridge was hydrated with XF Calibrant solution by overnight incubation in a non-CO2 incubator at 37°C. Oxygen consumption was measured, and the respiration rate was analyzed with step by step injections of mitochondrial complex inhibitors such as 10 μM oligomycin A (20 μl), 20 μM FCCP (22 μl), and 10 μM rotenone–antimycin A cocktail (25 μl) according to manufacturer’s protocol. These mitochondrial complex inhibitors were provided with the Agilent Seahorse XF Cell Mito Stress Test Kit (Seahorse Bioscience, 103015-100). Results were normalized to total protein determined using Bradford assay. Analysis of data was done using the Seahorse Wave 2.2.0 software package (Seahorse Bioscience).Microglial immunostaining and analysisDouble immunofluorescence staining (either PINK1 with AIF1 or DNM1L with AIF1) was performed in the whole brain, sagittal section from mice injected with either saline or cocaine (20 mg/kg body weight) for 7 consecutive days intraperitoneally. Briefly, formalin-fixed, paraffin-embedded brain slides were baked at 55°C overnight. The sections were deparaffinized using xylene and rehydrated by incubating the slide in graded series (100, 95 and 70%; each 5 min) of alcohol. Next, the slides were subjected to antigen retrieval by boiling them in Tris/EDTA buffer (pH 9) for about 20 min. The 10% goat serum in PBS was used to block the tissue, followed by the slides were co-incubated with primary antibody PINK1 and AIF1 or DNM1L and AIF1 for overnight at 4ºC. Next day, the slides were washed with PBS for three times, followed by incubated with corresponding secondary Alexa Fluor 488 goat anti-mouse IgG (Invitrogen, A-11008) or Alexa Fluor 594 goat anti-rabbit (Invitrogen, A-11032) for 2 h. Finally, the slides were again washed three times with PBS and mounting with ProLong Gold Antifade Reagent with DAPI. Fluorescence images were taken on a Zeiss Observer using a Z1 inverted microscope (Carl Zeiss, Thornwood, NY, USA). The acquired images were analyzed for the intensity of red and green fluorescence, colocalization and microglial process length using the Axio Vs 40 Version 4.8.0.0 software (Carl Zeiss MicroImaging GmbH). All experiments were repeated at least three times. AIF1-positive microglial somas in striatal region of both control and cocaine administered group (n = 4 per group; 6 fields per animal; total 48 images analyzed) were quantified using the Axio Vs 40 Version 4.8.0.0 software (Carl Zeiss MicroImaging GmbH) manual counting tool. To visualize the microglia processes, maximum intensity confocal images were converted to binary images and then skeletonized for analyzing the length of microglial processes. Numbers of microglial process endpoints and process lengths were measured and normalized with number of cell soma using the ImageJ software. Total length of the microglial process was summarized for statistical comparisons using Analyze Skeleton plugin by ImageJ software. The percent area and mean fluorescence intensity for each threshold image were multiplied to calculate the total fluorescence intensity for each image, as described previously [87,88].Statistical analysisThe values are expressed as mean±SEM. Non-parametric Kruskal-Wallis One-way ANOVA followed by Dunn’s post hoc test was used to compare the multiple experimental groups, and Wilcoxon matched-pairs signed rank test was used to compare between two groups. For the in vivo experiments, an unpaired Student t test was used for comparing between the two groups. All the statistical analyses were assessed using the GraphPad Prism software (Version 6.01). Values were considered statistically significant when P  0.05.Supplemental materialSupplemental MaterialDownload MS Word (154 KB)Related Research DataMitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimeticsSource:Figshare Linking provided by AcknowledgmentsThis work was supported by grants DA036157, DA043138, DA044586 (SB) from the National Institutes of Health. The support by the Nebraska Center for Substance Abuse Research is acknowledged. We are grateful to Dr. Ashutosh Tripathi, Dr. Guoku Hu and Dr. Susmita Sil for their useful discussions and Dr. Kelly L. Stauch, Dr. Venkata Sunil Bendi, Mr. Ke Liao, Ms. Fang Niu, Ms. Dan Feng, Ms. Maria Burkovetskaya, and Mr. Jiayu Shao for their technical assistance. The authors would like to thank Tom Bargar and Nicholas Conoan of the Electron Microscopy Core Facility (EMCF) at the University of Nebraska Medical Center for technical assistance. The EMCF is supported by state funds from the Nebraska Research Initiative (NRI) and the University of Nebraska Foundation, and institutionally by the Office of the Vice Chancellor for Research.Disclosure statementNo potential conflict of interest was reported by the authors.Additional informationFundingThis work was supported by the National Institute on Drug Abuse [DA043138]; National Institute on Drug Abuse [DA036157]; National Institute on Drug Abuse [DA044586]; National Institute on Drug Abuse [DA047156].ReferencesDegenhardt L, Chiu WT, Sampson N, et al. Toward a global view of alcohol, tobacco, cannabis, and cocaine use: findings from the WHO World Mental Health Surveys. PLoS Med. 2008 Jul 1;5(7):e141. PMID: 18597549. PMC2443200. [Crossref], [PubMed], [Web of Science ], [Google Scholar]Spear LP, Kirstein CL, Bell J, et al. Effects of prenatal cocaine exposure on behavior during the early postnatal period. Neurotoxicol Teratol. 1989 Jan–Feb;11(1):57–63. 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