Methamphetamine accelerates cellular senescence through stimulation of de novo ceramide biosynthesis

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 P450, 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.

Fig 1. Lipidome-wide profiles in various tissues of rats self-administering D-meth.

(A, B) Heat-maps showing changes in the levels of lipid classes (rows) in (A) brain regions and (A) peripheral organs of rats that self-administered D-meth for 8 days, compared to control rats receiving yoked saline injections. (C) Heat-map showing changes in the levels of various ceramide species (rows) in peripheral tissues of rats self-administering D-meth relative to control rats; columns show data from individual animals. Heat maps were generated by normalizing the data for each lipid species relative to the mean and standard error of the control group, such that the color of each subject’s cell indicates the number of standard errors above (red cells) or below (green cells) the mean of the control group. (D-I) Levels of (D) ceramide, (E) dihydroceramide, (F) sphingomyelin, (G) dihydrosphingomyelin, (H) mRNAs encoding for enzymes of de novo ceramide biosynthesis in skeletal muscle (vastus lateralis), and (I) ceramide synthase activity in skeletal muscle; control (C), open bars; rats self-administering D-meth (M), filled bars. Cer, ceramide; DAG, diacylglycerol; DH-Cer, dihydroceramide; DH-Sphm, dihydrosphingomyelin; MAG, monoacylglycerol; M-FA, monounsaturated fatty acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; P-FA, polyunsaturated fatty acid; PG, phosphatidylglycerol; PI, phosphatidylinositol; PS, phosphatidylserine; S-FA, saturated fatty acid; Sphm, sphingomyelin; TAG, triacylglycerol. Values are expressed as mean±s.e.m. *P<0.05, ***P<0.001, n.s., non significant; two-tailed Student’s t test (n = 6–12).

Fig 2. D-meth stimulates ceramide production in primary MEF cultures.

(A) Concentration- dependence and (B) time-course of the effects of D-meth on ceramide levels. D-meth concentrations are expressed in mM. (C) Effects of various phenethylamines on ceramide levels; D-meth (M, 1 mM) L-meth (LM, 1 mM), D-amphetamine (A, 1mM), and 4-hydroxy-D-methamphetamine (4-OH, 1 mM). (D) Effects of D-meth (1 mM, 24 h) on transcription of enzymes in de novo ceramide biosynthesis. SPT, serine palmitoyl-Coenzyme A transferase; CerS, ceramide synthase. (E-G) Effects of the SPT inhibitors (E) L-cycloserine (L-CS, 30 μM), (F) myriocin (MYR, 10 μM), and (G) the CerS inhibitor fumonisin B₁ (FB1, 50 μM) on ceramide levels in cells exposed to D-meth (M, 1 mM, 24 h) or vehicle. Cells were exposed to the drugs for 24 h. (H-J) Effects of D-meth (0–1 mM, 48 h) on MEF viability as assessed by (H) lactate dehydrogenase (LDH) release, (I) MTT ((3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) reduction and (J) 4′,6-diamidino-2-phenylindole (DAPI) nuclear staining. Values are expressed as mean±s.e.m. of three separate experiments, each performed in triplicate. *P<0.05, **P<0.01, ***P<0.001, ANOVA followed by Bonferroni post hoc test.

Fig 3. Role of cytochrome P₄₅₀ (CYP) and nuclear factor (NF)- κB in D-meth-induced ceramide production.

(A, B) Effects of pan-CYP inhibitor clotrimazole (CLO) on (A) cell-associated D-meth content and (B) ceramide levels. Primary MEF cultures were treated with D-meth (M, 1 mM) for 24 h and rinsed before extraction and quantification of D-meth by LC/MS. (C) Effects of CYP inhibitors on ceramide levels: SKF-525A (SKF, 10 μM), cimetidine (CIM, 10 μM), quinidine (QUI, 10 μM) and HET-0016 (HET, 10 μM). (D-E) Time-course of the effects of (D) D-meth (mM) and (E) 4-hydroxy-D-methamphetamine (4-OH, 1 mM) on ROS production. (F-G) Effects of (F) clotrimazole and (G) SKF-525A, cimetidine, quinidine and HET-0016 on ROS production. (H) Chromatin immunoprecipitation indicating recruitment of NF-κB to the TNF- promoter in primary MEF treated for 24 h with vehicle or D-meth (1 mM). Results are shown as percent of input (i.e., percent of the total amount of chromatin before immunoprecipitation). (I-K) Effects of NF-κB inhibitors (I) thalidomide (THA, 25 M), (J) 5-aminosalicylic acid (ASA, 10 μM) and (K) JSH-23 (JSH, 10 μM) on ceramide levels in MEF treated for 24h with vehicle or D-meth (1 mM). Values are expressed as mean±s.e.m. of three separate experiments, each performed in triplicate. *P<0.05, ***P<0.001, ANOVA followed by Bonferroni post hoc test.

Fig 4. D-meth accelerates replicative senescence.

(A-D) Effects of D-meth (1 mM, closed bars) or vehicle (open bars) on (A) senescence-associated -galactosidase (-Gal) staining, (B) cell morphology, (C) ceramide levels, and (D) expression of CerS5 mRNA in primary MEF cultures. (E-F) Effects of D-meth on replicative capacity assessed by (E) [³H]-thymidine incorporation into DNA and (F) number of cumulative population doublings (vehicle: open squares, D-meth: closed squares). (g-j) Effects of D-meth (M, 1 mM), L-cycloserine (L-CS, 30 μM) and combination of D-meth plus L-CS on transcription of senescence-associated markers: (G) p53, (H) p21, (I) IL-6 and (J) TNF-α. (K, L) Effects of L-CS (30 μM), fumonisin B₁ (FB1, 50 μM) and cell-permeant C8 ceramide (10 μM) on -Gal expression elicited by D-meth (M, 1 mM). ANOVA followed by Bonferroni post hoc test: *P<0.05, **P<0.01, ***P<0.001, vs. vehicle; ^$ P<0.05, ^$$$ P<0.001, vs vehicle at passage 1. ^## P<0.01, vs D-meth.

Fig 5. Enhanced transcription of senescence- and inflammation-associated markers in skeletal muscle of rats exposed to D-meth.

(A–F) mRNA levels in skeletal muscle of control rats (C, open bars) and rats self-administering D-meth (M, closed bars): (A) p53; (B) p21; (C) p16; (D) IGF-1; (E) IL-6; (F) TNF-α. (G) Effects of D-meth self-administration (M), L-cycloserine (L-CS) treatment or a combination of D-meth and L-cycloserine (L-CS) on body weight. (H–M) mRNA levels in skeletal muscle of control rats and rats that received two 10 mg-kg^-1 injections of D-meth in a 2-h period: (H) p53; (I) p21; (J) p16; (K) IGF-1; (L) IL-6; (M) TNF-α. Values are expressed as the mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, two-tailed Student’s t test. *P<0.05, **P<0.01, ***P<0.001, ANOVA followed by Bonferroni post hoc test.

Fig 6. Effects of L-cycloserine (L-CS) on senescence- and inflammation-associated markers in skeletal muscle and brain of rats that self-administer D-meth.

(A) Cumulative D-meth intake in rats receiving daily injections of vehicle (black circles) or L-CS (red squares). (B, C) Effects of D-meth self-administration, L-CS treatment or combination of D-meth plus L-CS on ceramide levels in (B) skeletal muscle and (C) brain dorsal striatum. (D–O) Effects of D-meth self-administration, L-CS treatment or combination of D-meth plus L-CS on (D) body temperature; (E) total food intake; (F–K) skeletal muscle mRNA levels of (F) p53; (G) p21; (H) p16; (i) IGF-1; (J) IL-6; (K) TNF-α; and (L-O) dorsal striatum mRNA levels of (L) p53; (M) p21; (N) IL-6; and (O) IL-1ß. Values are expressed as the mean±s.e.m. *P<0.05, **P<0.01, ***P<0.001, two-tailed Student’s t test. *P<0.05, **P<0.01, ***P<0.001, ANOVA followed by Bonferroni post hoc test.