Significance Cocaine is one of the most abused drugs in modern society, with overdoses that are frequently lethal. Molecular mechanisms underlying its toxic actions have been obscure. The present study demonstrates that cocaine’s cellular toxicity involves a signaling cascade that utilizes the gasotransmitter nitric oxide, which leads to autophagy, a cellular modification that can cause cell death. Thus, manipulations that impair nitric oxide signaling and autophagy diminish cytotoxic actions of cocaine. By contrast, alterations of apoptosis and other nonautophagic modes of cell death are ineffective. Therapies directed toward the autophagic process may be beneficial in treating cocaine neurotoxicity.

Abstract Cocaine exerts its behavioral stimulant effects by facilitating synaptic actions of neurotransmitters such as dopamine and serotonin. It is also neurotoxic and broadly cytotoxic, leading to overdose deaths. We demonstrate that the cytotoxic actions of cocaine reflect selective enhancement of autophagy, a process that physiologically degrades metabolites and cellular organelles, and that uncontrolled autophagy can also lead to cell death. In brain cultures, cocaine markedly increases levels of LC3-II and depletes p62, both actions characteristic of autophagy. By contrast, cocaine fails to stimulate cell death processes reflecting parthanatos, monitored by cleavage of poly(ADP ribose)polymerase-1 (PARP-1), or necroptosis, assessed by levels of phosphorylated mixed lineage kinase domain-like protein. Pharmacologic inhibition of autophagy protects neurons against cocaine-induced cell death. On the other hand, inhibition of parthanatos, necroptosis, or apoptosis did not change cocaine cytotoxicity. Depletion of ATG5 or beclin-1, major mediators of autophagy, prevents cocaine-induced cell death. By contrast, depleting caspase-3, whose cleavage reflects apoptosis, fails to alter cocaine cytotoxicity, and cocaine does not alter caspase-3 cleavage. Moreover, depleting PARP-1 or RIPK1, key mediators of parthanatos and necroptosis, respectively, did not prevent cocaine-induced cell death. Autophagic actions of cocaine are mediated by the nitric oxide-glyceraldehyde-3-phosphate dehydrogenase signaling pathway. Thus, cocaine-associated autophagy is abolished by depleting GAPDH via shRNA; by the drug CGP3466B, which prevents GAPDH nitrosylation; and by mutating cysteine-150 of GAPDH, its site of nitrosylation. Treatments that selectively influence cocaine-associated autophagy may afford therapeutic benefit.

Cocaine is a well recognized behavioral stimulant, which is widely abused. Its behavioral effects are thought to reflect inhibition of the reuptake inactivation of biogenic amines, especially serotonin and dopamine (1⇓⇓–4). Transcriptional regulation may underlie long-term effects of cocaine, especially the transition from abuse to addiction (5⇓⇓⇓⇓⇓⇓–12). Cocaine is also notably neurotoxic and broadly cytotoxic, but mechanisms for such actions have not been well characterized. Diverse modes of cytotoxicity and cell death have been delineated (13). Apoptosis is well established as a programmed form of cell death (14, 15). Earlier work had regarded necrosis as unprogrammed with cellular integrity disrupted via diverse, seemingly random events. More recently programmed modes of necrosis have been identified. Parthanatos reflects cell death associated with activation of poly(ADP ribose)polymerase-1 (PARP-1) (16). In necroptosis, tumor necrosis factor alpha (TNF-α) typically activates Receptor-interacting protein kinase 1 (RIPK1) (17). Apoptosis is linked to a series of caspases (18⇓–20). Autophagy is a process wherein cells degrade diverse metabolic products (21). Levine and associates (22, 23) delineated a mode of cell death uniquely associated with autophagy, which was designated “autosis.” Understanding the relationship of cocaine toxicity to one or more of these signaling systems might afford novel strategies for treating cocaine toxicity.

Recently we showed that behavioral effects of cocaine reflect a signaling cascade associated with nitric oxide (NO) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (24). Generation of NO is triggered by a variety of stimuli such as glutamate neurotransmission acting via its N-methyl-d-aspartate (NMDA) subtype receptors. The NO nitrosylates GAPDH at a critical cysteine, abolishing its catalytic activity but conferring upon it the ability to bind to the ubiquitin E3 ligase Siah1. Nitrosylated GAPDH associated with Siah1 translocates to the nucleus. With cytotoxic insults nitrosylated GAPDH in the nucleus activates the histone acetylating complex p300/CBP which leads to acetylation and activation of p53 with associated enhancement of transcriptional targets, such as PUMA and Bax, that mediate cell death (25, 26). By contrast, physiologic stimuli associated with neurotrophic actions such as brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF) lead to the nuclear GAPDH–Siah1 complex binding and degrading the histone-methylating enzyme SUV39H1 (27). The diminished histone methylation is associated with augmented histone acetylation with enhancement of cAMP response element-binding protein (CREB)-regulated genes that facilitate nerve outgrowth.

We described a signaling cascade in which the NO/GAPDH/Siah1 system accounts for behavioral stimulant effects of low doses of cocaine as well as neurotoxic actions of high doses (24). The lower doses of cocaine augment CREB genes, whereas higher, neurotoxic, doses activate the cytotoxic p53 system. Evidence that both stimulant and cytotoxic influences involve NO/GAPDH/Siah1 signaling is provided by the use of the drug CGP3466B, an extremely potent inhibitor of GAPDH nitrosylation as well as GAPDH-Siah1 binding, which prevents both the stimulant and neurotoxic actions of cocaine (24, 28).

In the present study, we explored cellular/molecular mechanisms that underlie the cytotoxicity of cocaine. We show that cocaine selectively elicits activation of autophagy, which uniquely mediates cocaine’s cytotoxic actions.

Discussion Autophagic cell death is a relatively recently appreciated but well accepted phenomenon defined by specific criteria. Autophagic death is characterized by autophagic flux and the involvement of at least two autophagic regulatory factors along with the absence of apoptosis (43). Cocaine-elicited cell death fulfills these criteria. Thus, cocaine elicits autophagy as evaluated by several indices: induction of LC3-II, degradation of p62, ultrastructural analysis by TEM, and confocal monitoring of LC3 punctae. Moreover, we have ruled out involvement of cocaine in other modes of programmed cell death such as apoptosis, parthanatos, and necroptosis. Cocaine induces autophagy in cortical cultures at concentrations as low as 1 μM. These levels are clinically relevant because recreational abuse of cocaine is associated with peak serum concentrations between 0.5 and 5 μM. The highest reported cocaine serum concentration in a living person is 120 μM. Cocaine concentrations in the brain are generally higher than those in the blood (44). In human toxicity cases, average brain-to-blood cocaine concentrations ratio is 9.6-fold (45). Recently, other workers have described autophagy associated with cocaine. Guo et al. (46) reported activation of microglia in response to cocaine, which appeared to involve autophagy and inflammatory reactions. Cao et al. (37) detected autophagy in astrocytic cultures following treatment with cocaine. These studies were restricted to single cell lines and did not discriminate autophagy from other modes of cytotoxicity. By contrast, our study systematically rules out a variety of modes of programmed cell death and establishes that cocaine’s toxic actions are uniquely associated with autophagy. Our study also demonstrates that cocaine’s impact upon autophagy involves the NO/GAPDH signaling cascade. Our earlier investigation (24) established a role for the NO/GAPDH system in cell death but failed to delineate the type of cell death involved. In this context, the effects of stimulant and neurotoxic doses of cocaine on NO/GAPDH signaling are blocked by dopamine D1 receptor inhibition. In the current work, cocaine induces NO/GAPDH signaling in primary cortical cultures, which lack the dopaminergic system. This information suggests the existence of another mechanism by which cocaine induces autophagy via the NO/GAPDH cascade. Further work is needed to delineate the upstream molecular events that lead to NO/GAPDH signaling-mediated activation of autophagy in response to high doses of cocaine. Our findings may have therapeutic relevance. Thus, the notion that cocaine toxicity is uniquely associated with autophagy implies that selective and potent inhibitors of autophagy may be therapeutic in treating cocaine users. Such interventions in the autophagic process may also benefit infants born of cocaine-using mothers. In terms of specific agents that might be beneficial, our observations that the NO/GAPDH pathway mediates cocaine actions may have therapeutic ramifications. CGP3466B prevents GAPDH nitrosylation at concentrations as low as 0.1 nM (28) and has been administered in clinical trials of patients with Parkinson’s disease as well as amyotrophic lateral sclerosis (47, 48). In these studies CGP3466B appeared to be safe with negligible side effects. This drug or related agents may be beneficial in the therapy of cocaine abuse.

Materials and Methods Reagents. Neurobasal-A medium (no glucose, no sodium pyruvate), B-27 supplement, and B-27 supplement minus antioxidants were purchased from Life Technologies. Staurosporine, DPQ, Z-VAD-FMK, and Necrostatin-1 were purchased from Santa Cruz Biotachnology. Bafilomycin A1 was purchased from Cayman Chemical. CGP3466B was purchased from Tocris Bioscience. Caspase-3, PARP-1, RIPK1, Beclin-1, and ATG-5 shRNA (verified) lentiviral plasmid, cocaine hydrochloride and poly-l-ornithine hydrobromide were purchased from Sigma-Aldrich. Recombinant murine TNF-α was purchased from Peprotech. N-methyl-N-nitroso-N′-nitroguanidine (MNNG) was purchased from Chem Service. Antibodies. Anti–GAPDH-HRP was purchased from GenScript USA. Anti–MLKL, anti–phospho-MLKL, anti–caspase-3, anti-PARP-1, anti-nNOS, and anti-LC3 were purchased from Cell Signaling Technology. Anti-NeuN was purchased from EMD Millipore. Transfections. HEK-293 cells transfections were performed using the polyfect transfection reagent following the manufacturer’s instructions (Qiagen). Lentivirus Production. Second-generation lentiviral vector packaging was used for lentivirus production. Lentiviral plasmids expressing caspase-3, PARP-1, RIPK-1, beclin-1, or ATG-5 shRNA were cotransfected into HEK-293T cells with packaging system psPAX2 and pMD2 plasmids. The media was changed after overnight incubation. The lentivirus-containing supernatant was collected after 48 h. Floating cells and cell debris were pelleted by centrifugation of the supernatant at 2,000 × g for 10 min at 4 °C. Lentivirus particles were pelleted by ultracentrifugation at 100,000 × g for 2 h at 4 °C. The lentiviral pellet was resuspended in 1/200th volume of OPTI-MEM and stored in −80 °C until use. Primary Neuron Cultures. Animals were housed and cared for in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory (49). Animal experiments were approved by the Johns Hopkins University Animal Care and Use Committee (JHU ACUC). Mouse primary cortical cultures were prepared from gestational day 16–18 fetal CD-1 mice. Briefly, the timed pregnant mice were killed by decapitation, the uterus was quickly dissected out, and pups were decapitated. After removing meninges, the cerebral cortices were dissected and incubated in 0.025% trypsin–EDTA solution for 15 min. The cells were triturated in plating media containing Neurobasal-A media without glucose and sodium pyruvate supplemented with 12.5 mM glucose, 2 mM L-glutamine, and 2% (vol/vol) B-27 minus antioxidants. The cortical cultures were plated on poly-L-ornithine coated plates. Cortical Culture Treatment. Primary cortical cultures were used between day in vitro (DIV) 10 and DIV 15. Half of the media was removed from the cells, mixed with 2× the indicated concentrations of reagents, whereupon the media was returned to the cells. Cultures were incubated in cocaine-containing media for 3 or 48 h, respectively, in autophagy and cell death experiments. Cell Death Assay. Live cultures were stained 48 h after cocaine treatment with 1 μg/mL Hoechst 33342, which stains all cell nuclei, and 7 μM propidium iodide, which stains dead cell nuclei. Images were immediately collected using Carl Zeiss Axiovert 200M microscope. Quantification of cell death in neurons was performed using NIH image-J software. Neuronal nuclei are smaller in size and fluoresce at a significantly higher intensity than glial nuclei. Using these criteria, only neuronal nuclei were counted. Biotin Switch Assay. Primary cortical cultures were lysed on ice in lysis buffer containing 100 mM Hepes, 1 mM EDTA, 0.1 mM neocuproine, pH 8.0, 1% SDS, 1% Nonidet P-40, and 100 mM sodium chloride. DNA was degraded by passing lysate through a 27 gauge needle several times on ice. For each reaction 1 mg of total protein was used. Free thiols in denatured lysates were blocked with methyl methanethiosulfonate (MMTS) at 50 °C for 20 min. MMTS was removed by acetone precipitation. Nitrosylated thiols were reduced using sodium ascorbate and then biotinylated. Streptavidin beads were used to pull down proteins bearing biotinylated thiols for further evaluation by Western blot analysis. Confocal Imaging of Neuronal Nuclei and LC3 Punctae in Neurons. Cortical neurons were transduced with LC3-RFP using LentiBrite RFP-LC3 Lentiviral Biosensor (Millipore) to image LC3 puncta. For immunostaining, cells were fixed with 4% (wt/vol) paraformaldehyde, permeabilized with 0.3% Triton X-100, and blocked with 5% (vol/vol) goat serum. Mouse anti-NeuN antibody was used at a dilution of 1:100 in 0.025% goat serum in PBS overnight at 4 °C. For GAPDH immunostaining, GAPDH antibody was used at a dilution of 1:500. GAPDH immunostaining, LC3 puncta, and neuronal nuclei were imaged under Zeiss Axiovert 200 with 510-Meta confocal module. Images were collected using a 40× (oil) 1.3 NA Zeiss Plan-Neofluar objective. Number of LC3 puncta per cell were counted using Imaris software version 7.7.2. Transmission Electron Microscopy (TEM). Primary cortical neurons were treated with indicated concentrations of cocaine. The cells were fixed in 2.5% (vol/vol) glutaraldehyde, 3 mM MgCl 2 , in 0.1 M sodium cacodylate buffer, pH 7.2, for 1 h at room temperature. After buffer rinse, samples were postfixed in 1% osmium tetroxide in buffer (1 h) on ice in the dark. The cells were stained with 2% (wt/vol) aqueous uranyl acetate (0.22 µm filtered, 1 h in the dark), dehydrated in a graded series of ethanol solutions, and embedded in Eponate 12 (Ted Pella) resin. Samples were polymerized at 37 °C for 2–3 d before moving to 60 °C overnight. Brain Tissue Preparation for TEM. Mice were perfused with 2% (wt/vol) paraformaldehyde (freshly prepared from EM grade prill form), 2% (vol/vol) glutaraldehyde, 3 mM MgCl 2 , in 0.1 M sodium cacodylate buffer, pH 7.2, for 30 min at a rate of 2 mL/min, then postfixed overnight. Regions of interest were dissected and samples were washed in 0.1 M sodium cacodylate buffer with 3 mM MgCl 2 and 3% (wt/vol) sucrose. Samples were postfixed in reduced 2% (wt/vol) osmium tetroxide, 1.6% (wt/vol) potassium ferrocyanide in buffer (2 h) on ice in the dark. Samples were stained with 2% (wt/vol) aqueous uranyl acetate (0.22 µm filtered, 1 h in the dark), dehydrated in a graded series of ethanol propylene oxide solutions, and embedded in Eponate 12 (Ted Pella) resin. Samples were polymerized at 60 °C overnight. Thin sections (60–90 nm) were cut with a diamond knife on a Reichert-Jung Ultracut E ultramicrotome and picked up with 2 × 1 mm copper slot grids. Grids were stained with 2% (wt/vol) uranyl acetate in 50% (vol/vol) methanol and lead citrate at 4 °C and observed with a Hitachi 7600 TEM. Images were captured with an AMT CCD XR50 (2K × 2K) camera. Western Blot Analysis. Cell lysates were prepared using lysis buffer (150 mM NaCl, 0.5% CHAPS, 0.1% Triton, 0.1% BSA, 1 mM EDTA, protease inhibitors, phosphatase inhibitors). Samples were centrifuged at 14,000 × g for 20 min, and the protein concentration of the supernatant was measured (Bio-Rad Protein Assay Dye Reagent Concentrate). Proteins were resolved by SDS-polyacrylamide gel electrophoresis and transferred to PVDF membranes. The membranes were blocked for 2 h at room temperature in 20 mM Tris⋅HCl, pH 7.4, 150 mM NaCl, and 0.02% Tween 20 (Tris-buffered saline/Tween 20) containing 3% (wt/vol) BSA followed by overnight incubation at 4 °C in 1:1,000 dilution of the respective antibodies for LC3, p62, actin, GAPDH, MLKLp, MLKL, caspase 3, PARP, beclin-1, ATG5, and RIPK in 3% (wt/vol) BSA. The membrane was washed three times with Tris-buffered saline/Tween 20, incubated with HRP-conjugated secondary antibody, and the bands visualized by chemiluminescence. The depicted blots are representative replicates selected from at least three experiments. Western Blot Image Quantification and Statistical Analysis. Western blot images were quantified using ImageJ software. Data are presented as means ± SD. Unless otherwise indicated, statistical analysis was performed using Graphpad software with an alpha power level of 0.05. Two-tailed t test was used to perform two group comparisons. One-way analysis of variance (ANOVA) was used to perform multiple comparisons. Data were graphed as means ± SEM.

Acknowledgments We thank Barbara Smith (TEM specialist at Johns Hopkins School of Medicine) for her help in preparation of TEM samples. This work was supported by National Institutes of Health/National Institute on Drug Abuse Grant DA000266.