Mitigation of cocaine-mediated mitochondrial damage, defective mitophagy and microglial activation by superoxide dismutase mimetics

Abstract

Although cocaine exposure has been shown to potentiate neuroinflammation by upregulating glial activation in the brain, the role of mitophagy in this process remains an enigma. In the present study, we sought to examine the role of impaired mitophagy in cocaine-mediated activation of microglia and to determine the ameliorative potential of superoxide dismutase mimetics in this context. Our findings demonstrated that exposure of mouse primary microglial cells (mPMs) to cocaine resulted in decreased mitochondrial membrane potential, that was accompanied by increased expression of mitophagy markers, PINK1 and PRKN. Exposure of microglia to cocaine also resulted in increased expression of DNM1L and OPTN with a concomitant decrease in the rate of mitochondrial oxygen consumption as well as impaired mitochondrial functioning. Additionally, in the presence of cocaine, microglia also exhibited upregulated expression of autophagosome markers, BECN1, MAP1LC3B-II, and SQSTM1. Taken together, these findings suggested diminished mitophagy flux and accumulation of mitophagosomes in the presence of cocaine. These findings were further confirmed by imaging techniques such as transmission electron microscopy and confocal microscopy. Cocaine-mediated activation of microglia was further monitored by assessing the expression of the microglial marker (ITGAM) and the inflammatory cytokine (Tnf, Il1b, and Il6) mRNAs. Pharmacological, as well as gene-silencing approaches aimed at blocking both the autophagy/mitophagy and SIGMAR1 expression, underscored the role of impaired mitophagy in cocaine-mediated activation of microglia. Furthermore, superoxide dismutase mimetics such as TEMPOL and MitoTEMPO were shown to alleviate cocaine-mediated impaired mitophagy as well as microglial activation.Abbreviations: 3-MA: 3-methyladenine; Δψm: mitochondrial membrane potential; ACTB: actin, beta; AIF1: allograft inflammatory factor 1; ATP: adenosine triphosphate; BAF: bafilomycin A₁; BECN1: beclin 1, autophagy related; CNS: central nervous system; DNM1L: dynamin 1 like; DMEM: Dulbecco modified Eagle medium; DAPI: 4,6-Diamidino-2-phenylindole; DRD2: dopamine receptor D2; ECAR: extracellular acidification rate; FBS: fetal bovine serum; FCCP: Trifluoromethoxy carbonylcyanide phenylhydrazone; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; IL1B: interleukin 1, beta; IL6: interleukin 6; ITGAM: integrin subunit alpha M; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; mPMs: mouse primary microglial cells; MRC: maximal respiratory capacity; NFKB: nuclear factor kappa B; NLRP3: NLR family pyrin domain containing 3; NTRK2: neurotrophic receptor tyrosine kinase 2; OCR: oxygen consumption rate; OPTN: optineurin; PBS: phosphate buffered saline; PINK1: PTEN induced putative kinase 1; PRKN: parkin RBR E3 ubiquitin protein ligase; ROS: reactive oxygen species; siRNA: small interfering RNA; SQSTM1: sequestosome 1; TNF: tumor necrosis factor.

Keywords: Cocaine; MitoTEMPO; TEMPOL; microglial activation; mitochondria; mitophagy; neuroinflammation.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.

Figure 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.