Methamphetamine (METH) is an illicit psychoactive drug that can cause a variety of detrimental effects to the nervous system, especially dopaminergic pathways. We hypothesized that DNA damage-inducible transcript 4 (DDIT4) is involved in METH-induced dopaminergic neuronal autophagy and apoptosis. To test the hypothesis, we determined changes of DDIT4 protein expression and the level of autophagy in rat catecholaminergic PC12 cells and human dopaminergic SH-SY5Y cells, and in the hippocampus, prefrontal cortex, and striatum of Sprague Dawley rats exposed to METH. We also examined the effects of silencing DDIT4 expression on METH-induced dopaminergic neuronal autophagy using fluorescence microscopy and electron microscopy. Flow cytometry and Western blot were used to determine apoptosis and the expression of apoptotic markers (cleaved caspase-3 and cleaved PARP) after blocking DDIT4 expression in PC12 cells and SH-SY5Y cells with synthetic siRNA, as well as in the striatum of rats by injecting LV-shDDIT4 lentivirus using a stereotaxic positioning system. Our results showed that METH exposure increased DDIT4 expression that was accompanied with increased autophagy and apoptosis in PC12 cells (3 mM) and SH-SY5Y cells (2 mM), and in the hippocampus, prefrontal cortex, and striatum of rats. Inhibition of DDIT4 expression reduced METH-induced autophagy and apoptosis in vitro and in vivo. However, DDIT4-related effects were not observed at a low concentration of METH (1 μM). These results suggest that DDIT4 plays an essential role in METH-induced dopaminergic neuronal autophagy and apoptosis at higher doses and may be a potential gene target for therapeutics in high-dose METH-induced neurotoxicity.

Methamphetamine (METH) is an illegal and widely abused psychoactive stimulant. METH exposure causes detrimental effects on multiple organ systems, primarily the nervous system, especially dopaminergic pathways, in both laboratory animals and humans. In this study, we hypothesized that Nuclear protein 1 (Nupr1/com1/p8) is involved in METH-induced neuronal apoptosis and autophagy through endoplasmic reticulum (ER) stress signaling pathway. To test this hypothesis, we measured the expression levels of Nupr1, ER stress protein markers CHOP and Trib3, apoptosis-related protein markers cleaved-caspase3 and PARP, as well as autophagy-related protein markers LC3 and Beclin-1 in brain tissues of adult male Sprague-Dawley (SD) rats, rat primary cultured neurons and the rat adrenal pheochromocytoma cells (PC12 cells) after METH exposure. We also determined the effects of METH exposure on the expression of these proteins after silencing Nupr1, CHOP, or Trib3 expression with synthetic small hairpin RNA (shRNA) or siRNA in vitro, and after silencing Nupr1 in the striatum of rats by injecting lentivirus containing shRNA sequence targeting Nupr1 gene to rat striatum. The results showed that METH exposure increased Nupr1 expression that was accompanied with increased expression of ER stress protein markers CHOP and Trib3, and also led to apoptosis and autophagy in rat primary neurons and in PC12 cells after 24 h exposure (3.0 mM), and in the prefrontal cortex and striatum of rats after repeated intraperitoneal injections (15 mg/kg × 8 injections at 12 h intervals). Silencing of Nupr1 expression partly reduced METH-induced apoptosis and autophagy in vitro and in vivo. These results suggest that Nupr1 plays an essential role in METH-caused neuronal apoptosis and autophagy at relatively higher doses and may be a potential therapeutic target in high-dose METH-induced neurotoxicity.

Methamphetamine (METH) abuse has been a serious global public health problem for decades. Previous studies have shown that METH causes detrimental effects on the nervous and cardiovascular systems. METH-induced cardiovascular toxicity has been, in part, attributed to its destructive effect on vascular endothelial cells. However, the underlying mechanism of METH-caused endothelium disruption has not been investigated systematically. In this study, we identified a novel pathway involved in endothelial cell apoptosis induced by METH. We demonstrated that exposure to METH caused mitochondrial apoptosis in human umbilical vein endothelial cells and rat cardiac microvascular endothelial cells in vitro as well as in rat cardiac endothelial cells in vivo. We found that METH mediated endothelial cell apoptosis through Nupr1–Chop/P53–PUMA/Beclin1 signaling pathway. Specifically, METH exposure increased the expression of Nupr1, Chop, P53 and PUMA. Elevated p53 expression raised up PUMA expression, which initiated mitochondrial apoptosis by downregulating antiapoptotic Bcl-2, followed by upregulation of proapoptotic Bax, resulting in translocation of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria to cytoplasm and activation of caspase-dependent pathways. Interestingly, increased Beclin1, upregulated by Chop, formed a ternary complex with Bcl-2, thereby decreasing the dissociative Bcl-2. As a result, the ratio of dissociative Bcl-2 to Bax was also significantly decreased, which led to translocation of cyto c and initiated more drastic apoptosis. These findings were supported by data showing METH-induced apoptosis was significantly inhibited by silencing Nupr1, Chop or P53, or by PUMA or Beclin1 knockdown. Based on the present data, a novel mechanistic model of METH-induced endothelial cell toxicity is proposed. Collectively, these results highlight that the Nupr1–Chop/P53–PUMA/Beclin1 pathway is essential for mitochondrion-related METH-induced endothelial cell apoptosis and may be a potential therapeutic target for METH-caused cardiovascular toxicity. Future studies using knockout animal models are warranted to substantiate the present findings.

Methamphetamine (METH) is an extremely addictive stimulant drug that is widely used with high potential of abuse. Previous studies have shown that METH exposure damages the nervous system, especially dopaminergic neurons. However, the exact molecular mechanisms of METH-induced neurotoxicity remain unclear. We hypothesized that caspase-11 is involved in METH-induced neuronal apoptosis. We tested our hypothesis by examining the change of caspase-11 protein expression in dopaminergic neurons (PC12 and SH-SY5Y) and in the midbrain of rats exposed to METH with Western blotting. We also determined the effects of blocking caspase-11 expression with wedelolactone (a specific inhibitor of caspase-11) or siRNA on METH-induced apoptosis in PC12 cells and SH-SY5Y cells using Annexin V and TUNEL staining. Furthermore, we observed the protein expression changes of the apoptotic markers, cleaved caspase-3 and cleaved poly(ADP-ribose) polymerase 1 (PARP), after silencing the caspase-11 expression in rat midbrain by injecting LV-shcasp11 lentivirus using a stereotaxic positioning system. Results showed that METH exposure increased caspase-11 expression both in vitro and in vivo, with the effects in vitro being dose- and time-dependent. Inhibition of caspase-11 expression with either wedelolactone or siRNAs reduced the number of METH-induced apoptotic cells. In addition, blocking caspase-11 expression inhibited METH-induced activation of caspase-3 and PARP in vitro and in vivo, suggesting that caspase-11/caspase-3 signal pathway is involved in METH-induced neurotoxicity. These results indicate that caspase-11 plays an essential role in METH-induced neuronal apoptosis and may be a potential gene target for therapeutics in METH-caused neurotoxicity.

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Methamphetamine (Meth) is a widely abused psychoactive drug that primarily damages the nervous system, notably causing dopaminergic neuronal apoptosis. CCAAT-enhancer binding protein (C/EBPβ) is a transcription factor and an important regulator of cell apoptosis and autophagy. Insulin-like growth factor binding protein (IGFBP5) is a proapoptotic factor that mediates Meth-induced neuronal apoptosis, and Trib3 (tribbles pseudokinase 3) is an endoplasmic reticulum (ER) stress-inducible gene involved in autophagic cell death through the mammalian target of rapamycin (mTOR) signaling pathway. To test the hypothesis that C/EBPβ is involved in Meth-induced IGFBP5-mediated neuronal apoptosis and Trib3-mediated neuronal autophagy, we measured the protein expression of C/EBPβ after Meth exposure and evaluated the effects of silencing C/EBPβ, IGFBP5, or Trib3 on Meth-induced apoptosis and autophagy in neuronal cells and in the rat striatum after intrastriatal Meth injection. We found that, at relatively high doses, Meth exposure increased C/EBPβ protein expression, which was accompanied by increased neuronal apoptosis and autophagy; triggered the IGFBP5-mediated, p53-up-regulated modulator of apoptosis (PUMA)-related mitochondrial apoptotic signaling pathway; and stimulated the Trib3-mediated ER stress signaling pathway through the Akt-mTOR signaling axis. We also found that autophagy is an early response to Meth-induced stress upstream of apoptosis and plays a detrimental role in Meth-induced neuronal cell death. These results suggest that Meth exposure induces C/EBPβ expression, which plays an essential role in the neuronal apoptosis and autophagy induced by relatively high doses of Meth; however, relatively low concentrations of Meth did not change the expression of C/EBPβ in vitro. Further studies are needed to elucidate the role of C/EBPβ in low-dose Meth-induced neurotoxicity.-Xu, X., Huang, E., Luo, B., Cai, D., Zhao, X., Luo, Q., Jin, Y., Chen, L., Wang, Q., Liu, C., Lin, Z., Xie, W.-B., Wang, H. Methamphetamine exposure triggers apoptosis and autophagy in neuronal cells by activating the C/EBPβ-related signaling pathway.

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