Abstract Methamphetamine is a highly addictive psychostimulant that causes profound damage to the brain and other body organs. Post mortem studies of human tissues have linked the use of this drug to diseases associated with aging, such as coronary atherosclerosis and pulmonary fibrosis, but the molecular mechanism underlying these findings remains unknown. Here we used functional lipidomics and transcriptomics experiments to study abnormalities in lipid metabolism in select regions of the brain and, to a greater extent, peripheral organs and tissues of rats that self-administered methamphetamine. Experiments in various cellular models (primary mouse fibroblasts and myotubes) allowed us to investigate the molecular mechanisms of systemic inflammation and cellular aging related to methamphetamine abuse. We report now that methamphetamine accelerates cellular senescence and activates transcription of genes involved in cell-cycle control and inflammation by stimulating production of the sphingolipid messenger ceramide. This pathogenic cascade is triggered by reactive oxygen species, likely generated through methamphetamine metabolism via cytochrome P 450 , and involves the recruitment of nuclear factor-κB (NF-κB) to induce expression of enzymes in the de novo pathway of ceramide biosynthesis. Inhibitors of NF-κB signaling and ceramide formation prevent methamphetamine-induced senescence and systemic inflammation in rats self-administering the drug, attenuating their health deterioration. The results suggest new therapeutic strategies to reduce the adverse consequences of methamphetamine abuse and improve effectiveness of abstinence treatments.

Citation: Astarita G, Avanesian A, Grimaldi B, Realini N, Justinova Z, Panlilio LV, et al. (2015) Methamphetamine Accelerates Cellular Senescence through Stimulation of De Novo Ceramide Biosynthesis. PLoS ONE 10(2): e0116961. https://doi.org/10.1371/journal.pone.0116961 Academic Editor: Stefano L. Sensi, University G. D’Annunzio, ITALY Received: October 21, 2014; Accepted: December 17, 2014; Published: February 11, 2015 Copyright: © 2015 Astarita et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: These studies were funded by the National Institute on Drug Abuse (RC2 DA028902 to D.P.) and by the Intramural Research Program of the National Institute on Drug Abuse, NIH, DHHS. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors disclose the following conflict of interest: GA, AA and DP are inventors on the following US patent application by the Regents University of California and Fondazione Istituto Italiano di Tecnologia: Methods of treatment, diagnosis and monitoring for methamphetamine toxicity which target ceramide metabolic pathways and cellular senescence, WO 2013/149250.

Introduction The abuse of methamphetamine (D-meth) is a major health concern in industrialized countries, where a socially diverse group of users seeks the drug for its desirable psychological and physiological effects—a combination of euphoria, heightened arousal, reduced appetite and decreased fatigue [1]. Due to its highly addictive properties, D-meth also initiates an escalation in frequency and intensity of use, which brings about a host of negative symptoms such as panic and psychosis [1]. The pharmacological mechanism underlying these diverse actions is well understood—D-meth acts in the central nervous system to interrupt the reuptake of dopamine and other amine neurotransmitters, and facilitate their release into the synaptic space [1]. With prolonged drug exposure, these neurochemical alterations can lead to long-lasting damage to the brain, especially in structures containing dopaminergic axon terminals, which contribute to the emotional and cognitive problems experienced by D-meth addicts [2,3,4]. This transition to pathology has been attributed to a series of concurring events that include disruption of neuronal redox homeostasis [5,6,7,8], activation of apoptotic and necrotic processes [9], and recruitment of pro-inflammatory pathways dependent on the transcription nuclear factor NF-κB [7,10,11,12]. In addition to being neurotoxic, D-meth exerts widespread harmful effects throughout the body. Perhaps most striking among them is the extreme tooth decay (‘meth mouth’) that exacerbates the prematurely aged physical appearance typical of D-meth addicts [13]. While several abused drugs may increase the speed of organismal decline [14,15], post mortem studies have specifically linked the protracted use of D-meth to various pathologies characteristic of old age, including coronary artery atherosclerosis, pulmonary fibrosis and liver steatosis [16,17,18]. Nevertheless, the molecular mechanism through which D-meth might accelerate the emergence of age-related diseases remains unknown. Here we report that D-meth promotes cellular senescence and activates transcription of genes involved in inflammation and aging through a mechanism that requires increased biosynthesis of the sphingolipid messenger ceramide. These results shed new light on the molecular mechanism underlying D-meth toxicity and identify potential therapeutic targets to attenuate the life-threatening consequences of D-meth exposure in recovering addicts.

Materials and Methods Chemicals D-Methamphetamine (D-meth), L-methamphetamine (L-meth), L-cycloserine, myriocin, thalidomide, cimetidine, quinidine, SKF-525A and clotrimazole were purchased from Sigma Aldrich (St. Louis, Missouri). Fumonisin B 1 (FB1), C6 and C8 ceramide, and HET-0016 were from Cayman Chemicals (Ann Arbor, Michigan). 5′-aminosalicylic acid and JSH-23 were from Santa Cruz Biotechnology (Santa Crux, CA). Animals Adult male C57BL/6 mice (25–30 g; Charles River, Wilmington, MA) and Sprague-Dawley rats (360–440 g; Charles River) were kept in a temperature-controlled environment with a 12 h light/12 h dark cycle, with standard chow and water ad libitum. Rats were individually housed. All procedures met the National Institutes of Health guidelines for the care and use of laboratory animals, as well as the Guidelines for the Care and Use of Mammals in Neuroscience and Behavioral Research (National Research Council 2003), and were approved by the Institutional Animal Care and Use Committees of the University of California, Irvine, and the Intramural Research Program of the National Institute on Drug Abuse (NIDA). Methamphetamine self-administration Surgery Silastic intravenous catheters were implanted into the external jugular vein under anesthesia with a mixture of ketamine and xylazine (60 and 10 mg-kg-1, i.p., respectively). Catheters exited the skin behind the ear. After catheter implantation, a nylon bolt glued to an acrylic mesh was implanted subcutaneously in the midscapular region. The nylon bolt served as a tether, preventing the catheter from being pulled out during self-administration sessions. Following surgery, the catheters were flushed daily during the first week with 0.2–0.3 ml of a solution containing cephalosporin (100 mg-ml-1; cefazolin USP; Hospira Inc., Lake Forest, IL, USA) and flushed with saline before and after each daily session to maintain patency. Apparatus We used 18 standard operant-conditioning chambers (Coulbourn Instruments, Lehigh Valley, PA), which contained a white house light and two holes with nose-poke operanda on either side of a food trough. Upon activation, each nose poke produced a brief feedback tone. One hole was defined as active (left in 9 chambers, right in the remaining 9) and the other hole as inactive. D-meth or saline were delivered through Tygon tubing, protected by a metal spring and suspended through the ceiling of the experimental chamber from a single-channel fluid swivel. The tubing was attached to a syringe pump (Harvard Apparatus, South Natick, MA), which was programmed to deliver 2 s injections. The injected volume was adjusted for every animal to deliver a D-meth dose of 0.1 mg-kg-1 per injection. Experimental events were controlled by microcomputers using MED Associates interfaces and software (Med Associates Inc., East Fairfield, VT). Procedure Each experimental group of rats was divided into 2 subgroups, which were tested at the same time. Subgroup 2 served as yoked control and passively received a saline injection each time a response-contingent injection of D-meth was self-administered by a Subgroup 1 rat. Nose-poke responses by yoked control rats were recorded, but had no programmed consequences. Eight consecutive 15 h sessions were always conducted between 4 pm and 8 am. At the start of each session, a white light was turned on and a priming injection of D-meth (0.1 mg-kg-1) or saline sufficient to fill the “dead” space of the catheter, was automatically delivered. Rats learned to self-administer D-meth under one-response, fixed ratio schedule (FR1). Each nose-poke response in the active hole (FR1) produced a delivery of D-meth injection (0.1 mg-kg-1) followed by a 30 s timeout, during which the chamber was dark and responses in either hole had no programmed consequences. Responses in the inactive hole were recorded, but had no programmed consequences. Tissue collection The rats were killed by decapitation 2 h after the end of the eighth session. Brain, liver, heart, kidney (left), spleen, pancreas, testis, epididymal fat, skeletal muscle (vastus lateralis), and skin (hind paw) were harvested from each rat. The tissues were rinsed in a mix of RNase-free water with DEPC-treated PBS and blotted with sterile gauze. Brains and livers were snap-frozen in isopentane. All other tissues were snap-frozen in liquid nitrogen. Tissues were wrapped in aluminum foil and stored in -80°C. Acute methamphetamine administration Three groups of male Sprague-Dawley rats were used. Group 1 received two injections of D-meth (10 mg-kg-1, intraperitoneal, i.p., n = 6) or saline (n = 6), one every 2 h. Group 2 received two injections of saline (n = 6) or D-meth (1.5 mg-kg-1, i.p., n = 6), one every 2 h. Group 3 received two i.p. injections of saline (n = 6) or D-meth (1.5 mg-kg-1, n = 5; 5 mg-kg-1, n = 5; 10 mg-kg-1, n = 6), one every 2 h. Animals were killed by decapitation 2 h after the last injection. Tissues were harvested, snap-frozen in liquid nitrogen and stored at -80°C for analyses. Cell cultures Immortalized mouse embryonic fibroblasts (MEF) were purchased from American Type Culture Collection (Manassas, VA), murine C2C12 cells were a kind gift of Dr. Maria Pennuto’s group (Italian Institute of Technology, Genova, Italy), primary MEF cultures were prepared from C57BL/6 mouse embryos, as described [19]. See Supplemental information for detailed culture methods. Lipid extractions and analysis Lipid extractions were carried out as described [20]. A detailed description of the extraction procedure and LC/MS conditions used for lipid analysis is reported in Supporting Material and Methods. D-Methamphetamine measurements A detailed description of the extraction procedure and LC/MS conditions used for D-Methamphetamine measurements is reported in Supporting Material and Methods. Ceramide synthase activity Ceramide synthase activity was measured as described [21]. A detailed description of assay conditions is reported in the Supporting Material and Methods. Senescence and cell toxicity assays Senescence-associated β-Gal staining was performed as previously reported [22]. DNA replication assay, Crystal Violet Staining, Population Doublings counting, MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) and LDH (lactate dehydrogenase) assays were used to measure cell viability and are fully described in the Supporting Material and Methods. Gene expression and silencing See S1 Materials and Methods and S8 Table for detailed method. Reactive Oxygen Species (ROS) Production ROS production was measured using the fluorescent probe CM-H2DCFDA (Invitrogen). MEFs were grown in Dulbecco’s Modified Eagle’s Medium (DMEM) without phenol red. Cells were plated to subconfluence in 12-well plates, washed 3 times with pre-warmed Phosphate Buffered Saline (PBS) and loaded for 30 min at 37°C/5% CO 2 with 5 mM CM-H2DCFDA in DMEM without phenol red (loading medium). The loading medium was removed and pre-warmed fresh medium containing the different cytochromes P450 (CYP) inhibitors in presence or absence of D-meth was added. Fluorescence (excitation at 485 nm, emission at 530 nm) was analyzed immediately, then cells were incubated at 37°C and 5% CO 2 , and fluorescence was measured at the indicated time points. ROS rate versus control (%) was calculated subtracting the percentage of ROS increasing from time zero in the D-meth-treated samples to the percentage of ROS increasing from time zero in the vehicle treated samples. Chromatin immunoprecipitation (ChIP) We used the two-step cross-linking method described in Nowak et al. [23]. See Supporting Material and Methods for detailed description. Statistical analyses See S1 Materials and Methods for details.

Discussion While conducting a survey of lipidomic abnormalities associated with self-administration of D-meth, we found that ceramide production is strongly increased in rats that voluntarily take the drug. This effect was clearly detectable in select regions of the brain, such as the dorsal striatum, but was particularly pronounced in peripheral organs known to undergo pathological changes following prolonged exposure to D-meth (e.g., skeletal muscle, heart and liver) [16,17,18]. In those organs, accrued ceramide biosynthesis was associated with activation of a senescence-like transcription program—characterized by enhanced expression of genes involved in cell-cycle control (e.g., p21, p16) and chronic inflammation (e.g., IL-6 and TNF-α)—which could be recapitulated in vitro by treating mouse fibroblasts or differentiated C2C12 myoblasts with D-meth. Importantly, pharmacological inhibitors of ceramide formation prevented the occurrence of these molecular events, and lowered D-meth-induced cellular and systemic toxicity. These studies suggest that modulation of ceramide signaling might be utilized therapeutically to attenuate the serious, often fatal health consequences of D-meth abuse and enhance the success of behavior-based abstinence programs. Our lipidomic analyses show that D-meth self-administration in rats—a model that realistically captures abuse of the drug in humans [24]—is accompanied by changes in the levels of ceramide and its precursor dihydroceramide, which are suggestive of an up-regulation of the de novo pathway of ceramide biosynthesis. This possibility is supported by two additional observations: (i) D-meth increased transcription of genes encoding for two key enzyme families in that pathway, SPT and CerS; and (ii) pharmacological interference with the activities of those enzymes blocked D-meth-induced ceramide formation. How may D-meth influence SPT and CerS expression? Pharmacological evidence implicates CYP-mediated metabolism, which is responsible for the metabolic clearance of D-meth in humans and rodents [33,34]. Experiments with mouse fibroblasts show that (i) D-meth stimulates ROS generation, NF-κB activation and ceramide production; (ii) genetic or pharmacological interference with CYP or NF-κB activities prevents these effects; (iii) structural analogs of D-meth, such as L-meth and 4-hydroxy-D-methamphetamine, which are comparatively poor substrates for CYP-mediated oxidation, only partially mimic the effects of D-meth. Thus, an economical interpretation of the results outlined above is that D-meth metabolism via CYP generates ROS, which in turn activate SPT and CerS transcription through an NF-κB-dependent mechanism. This model is consistent with the greater vulnerability of human CYP2D6 extensive metabolizers to D-meth neurotoxicity [45], but does not exclude the possibility that D-meth might also stimulate ceramide biosynthesis through other mechanisms. Ceramide plays an important role in the transition of cells into replicative senescence [26,28,46], a state in which mitotic cells irreversibly stop dividing and start secreting pro-inflammatory cytokines and tissue-modifying factors [39,40]. Our in vitro experiments indicate that the increased ceramide biosynthesis evoked by D-meth accelerates progression of a senescent phenotype characterized by lowered proliferation, altered cell morphology and heightened expression of β-Gal and other senescence-associated markers. Among such markers is a group of genes encoding for protein regulators of cell cycle (p53, p16, p21 and IGF-1) and inflammation (IL-6 and TNF-α), which we found to be abnormally high in skeletal muscle and other peripheral tissues of rats that voluntarily take D-meth. Importantly, this same panel of genes is known to be elevated in tissues of elderly persons [47,48,49] and is thought to participate in chronic inflammatory conditions that are typical of old age [40]. The premature occurrence of such conditions is a hallmark of D-meth abuse in humans, which cannot be explained by the psychostimulant and sympathomimetic properties of this drug [17,18], but is consistent with its ability to elevate ceramide levels. Indeed, overactive ceramide signaling has been implicated in several age-related pathologies—including coronary artery atherosclerosis, liver steatosis and pulmonary fibrosis [50,51,52]—which are frequently documented in autopsy reviews of deceased D-meth users [16,17,18]. Collectively, the available data suggest that the rapid health decline caused by D-meth is due to a ceramide-mediated acceleration of genetic programs that are also engaged during chronic inflammation and aging. Pharmacological strategies aimed at normalizing ceramide signaling—for example by modulating NF-κB recruitment (which include drugs that are already available in the clinic, such as 5-aminosalicylic acid and thalidomide) or SPT activity—might slow down this pathogenic process and facilitate detoxification of D-meth users.

Acknowledgments This article is dedicated to the memory of Steven R. Goldberg. We thank Daniel Aiello, Sveti Patel, Opeyemi Oluyemi, Chanel Barnes, and Jordan Adair for help with the experiments. We also thank Drs. Maria E. Secci, Cesar Quiroz and Marco Orru for their assistance with tissue harvesting. The contribution of Agilent Technologies/University of California Irvine Analytical Discovery Facility is gratefully acknowledged.

Author Contributions Conceived and designed the experiments: DP GA AA NR BG ZJ. Performed the experiments: GA AA NR ZJ BG. Analyzed the data: GA AA NR ZJ AB BG LVP. Wrote the paper: DP. Supervised experiments: DP SRG.