Role Of DDIT4 In Methamphetamine-induced Neurotoxicity And Cardiotoxicity

Background:Methamphetamine (METH), also known as "ice" for its ice-like and crystalline appearance, belongs to amphetamine-type stimulants (ATS). METH is one of the most popular drugs which have brought about great hazard to the society. The production and processing of METH is relatively simple, and the price is much cheaper than other drugs. METH gradually replace traditional drugs using poppy as the main raw material, and quickly spread throughout the world, at the end of the twentieth century.2014 annual report of China Food and Drug Administration (CFDA) notes that the prevalence of traditional drugs such as heroin was brought under control, but the number of new synthetic drugs abusers such as METH has increased rapidly. Violence and death associated with drug use is always a problem of great concern to forensic work. However, the detailed mechanism underlying METH-induced organ damage and death has not been reported.A major target of METH is the central nervous system, especially the central dopaminergic circuitries. METH enters dopaminergic neurons via dopamine (DA) transporter, subsequently enters synaptic vesicles through vesicular monoamine transporter-2 and causes DA release into the cytoplasm, where DA undergoes auto-oxidation, producing toxic metabolites and resulting in oxidative stress, mitochondrial dysfunctions, and ultimately neuronal death.Previous studies show that METH induced neurotoxicity can cause disease recessive neuropathies such as Alzheimer’s (AD) and Parkinson’s disease (PD) in the brain. Our recent study showed that METH exposure caused dopaminergic neuronal apoptosis via activation of the mitochondria-related caspase-3/PARP apoptosis pathway by up-regulating IGFBP5 (insulin-like growth factor binding protein 5) and caspase-11. Additionally, autophagy has been shown to play a role in METH-induced neuronal death. However, the detailed mechanism underlying METH-induced neuronal autophagy remains to be investigated.In recent years, increasing reports have shown that METH can also cause adverse and potentially fatal effects on the cardiovascular system. METH abuse, ranging from episodes of binge abuse to chronic abuse over several years, can cause a variety of myocardial damages in humans.Cardiovascular pathology, typically coronary artery atherosclerosis, was detected in 54% of 371 METH-related deaths occurring between July 2000 and June 2005in Australia, highlighting the role of cardiotoxicity in METH-induced death.Our previous study showed that METH exposure induced apoptosis in cardiomyocytes. However, the mechanisms underlying METH-induced apoptosis in cardiomyocytes remain to be elucidated.DDIT4 (DNA damage-inducible transcript 4, also known as REDD1 and Dig2) is a protein with a molecular weight of 35 kD that is ubiquitously expressed in various human tissues. DDIT4 expression is low in physiological condition, but is induced in a series of cellular stress, such as hypoxia, DNA damage, and energy deprivation. It has been reported that DDIT4 can induce hypoxia-triggered apoptosis of differentiated PC 12 cells through activation of the caspase-3/PARP pathway. mTOR (mammalian target of rapamycin) is a highly-conserved modulator of many biological functions, including cell growth, metabolism, etc. DDIT4 is a negative regulator of mTOR signaling pathway. DDIT4 participates in mTOR signaling pathway by inhibiting the phosphorylation of mTOR, leading to up-regulated autophagy. It has been shown that exposure to another neurotoxicant alcohol increases DDIT4 expression in heart and DDIT4mediates the activation of mTOR when the heart undergoes ischemia/reperfusion injury. However, the role of DDIT4in the neurotoxicity and cardiotoxicity of METH has not been reported.The present study aimed to investigate the role of DDIT4 in METH-induced neurotoxicity and cardiotoxicity by the following two parts. In the first part, we determined changes of DDIT4 expression and the level of autophagy in PC12 cells, SH-SY5Y cells, and in the hippocampus, prefrontal cortex and striatum of rats exposed to METH. We also determined the effects of blocking DDIT4 expression with siRNA on METH-induced autophagy in PC12 cells and SH-SY5Y cells using Annexin V and GFP-LC3 immunofluorescence. We also explored whether DDIT4-mTOR pathway is involved in METH-induced dopaminergic neurotoxicity. Furthermore, we observed the protein expression changes of the autophagy and apoptotic markers, Beclin-1,LC3-Ⅱ, caspase-3 and PARP, after silencing the DDIT4 expression in rat striatum by injecting LV-shDDIT4 lentivrus using a stereotaxic positioning system. In the end, to demonstrate role of DDIT4 in METH-induced neurotoxicity. In the second part, we measured DDIT4 protein levels in cardiomyocytes and in heart tissues of METH-treated rats. We also evaluated the effects on METH-caused autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in cardiomyocytes. In the end, to demonstrate role of DDIT4 in METH-induced cardiotoxicity and provide a theoretical basis for the research of METH-induced toxicity mechanism and gene therapy.Aims:The present study aimed to investigate the role of DDIT4 in autophagy and apoptosis induced by METH. To this end, we examined the expression of DDIT4 in PC 12 cells, SH-SY5Y cells, cardiomyocytes and rats exposed to vehicle or METH. We also explored whether DDIT4-mTOR pathway is involved in METH-induced autophagy and apoptosis. In addition, we evaluated the effects on METH-caused autophagy and apoptosis after silencing DDIT4 expression with synthetic siRNA with or without pretreatment of a mTOR inhibitor rapamycin in vitro and in vivo. In the end, to demonstrate role of DDIT4 in METH-induced autophagy and apoptosis and provide a theoretical basis for the research of METH-induced toxicity mechanism and gene therapy.Methods:PART 11. Examined DDIT4 protein expression in PC 12 and SH-SY5Y after treated byMETH and METH-induced autophagy in PC 12 cells after blockade of DDIT4expression.(1) Examined the values of LC25 and LC50 of METH in PC 12 cell line and SH-SY5Y cell line.(2) Detected DDIT4 mRNA and protein levels by Western-blot,Q-PCR in METH-treated PC12 cell.(3) Detected Beclin-1 and LC3-Ⅱ protein expression by Western-blot after METH treat PC12 cell and SH-SY5Y cell.(4) Using Western-blot,GFP-LC3 transfection and electron microscopy to measure METH-induced autophagy in PC 12 cells and SH-SY5Y cells after blocking DDIT4 expression.(5) Measure METH-induced autophagy in PC 12 cells and SH-SY5Y cells after Rap or 3-MA exposured.(6) Using Western-blot and Annexin V to measure METH-induced apoptosis in PC 12 cells and SH-SY5 Y cells after blocking DDIT4 expression.2. Examined DDIT4 protein expression in rats exposed to METH and observed the protein expression of the autophagy and apoptotic markers after silencing the DDIT4 expression in rat striatum by injecting LV-shDDIT4 lentivrus using a stereotaxic positioning system.(1) Detected DDIT4 and autophagy markers protein expression by Western-blot in METH-treated rats.(2) Measure METH-induced autophagy and apoptosis in rat striatum after blocking DDIT4 expression. PART 21. Examined DDIT4 protein expression in cardiomyocytes after treated by METH and METH-induced autophagy and apoptosis in cardiomyocytes after blockade of DDIT4 expression.(1) Detected DDIT4 protein expression in METH-treated cardiomyocytes.(2) Using Western-blot and GFP-LC3 transfection to measure METH-induced autophagy in cardiomyocytes after blocking DDIT4 expression.(3) Using Western-blot and TUNEL staining to measure METH-induced apoptosis in cardiomyocytes after blocking DDIT4 expression.(4) Measure METH-induced autophagy and apoptosis in cardiomyocytes after Rap exposured.2. Examined DDIT4 protein expression in rats exposed to METH and observed the protein expression of the autophagy and apoptotic markers in vivo(1) Detected DDIT4 and autophagy markers protein expression by Western-blot in METH-treated rats.(2) Using Western-blot and TUNEL staining to measure METH-induced apoptosis in heart tissues of male Sprague-Dawley (SD) rats.Results:1. Effects of DDIT4 in methamphetamine-induced autophagy and apoptosis in dopaminergic neurons(1) METH induced autophagy in dopaminergic neurons. The results showed that 24h METH exposure increased Beclin-1 and LC3-Ⅱ expression in a dose- and time-dependent manner. For example,24h exposure to 1.0mM METH increased Beclin-1 and LC3-II protein expression by ～1.5-fold; this increase reached ～3.5-fold when METH exposure level was 3.0mM. Additionally, after 2h exposure to 3.0mM METH, Beclin-1 and LC3-II protein expression was ～1.5-fold higher in the METH-treated PC 12 cells than control cells, and this effect became the greatest at 24h.(2) METH increased DDIT4 mRNA and protein expression in dopaminergic neurons. After 24h exposure, DDIT4 mRNA level and protein expression were significantly increased by 4.24-fold and 4.20-fold, respectively, in the METH-treated (3.0mM) cells. Similarly, DDIT4 protein expression was 2.30-fold higher in the METH-treated (2.0mM) SH-SY5Y cells than in the control cells after 24h exposure.(3) METH increased DDIT4 expression and induced autophagy in vivo. The results showed that DDIT4 protein level in hippocampus was 2.65-fold and 4.38-fold higher in the subacute and chronic exposure groups, respectively, than in the control. Subacute and chronic METH exposures also increased DDIT4 protein expression in the prefrontal cortex (1.63-and 2.1-fold, respectively) and striatum (3.33- and 2.54-fold, respectively). Beclin-1 and LC3-Ⅱ protein expression was increased by ～2-fold in subacute group, and ～3.00-fold in chronic group compared to controls in the hippocampus. Likewise, Beclin-1 and LC3-Ⅱ protein expression was increased by ～2.40-fold in subacute group, and ～3.5-fold in chronic exposure group compared to controls in the prefrontal cortex. In the striatum, the increase of Beclin-1 and LC3-Ⅱ protein expression was ～4.6-fold and ～4.0-fold in the subacute and chronic groups, respectively.(4) Synthetic siDDIT4 knockdown DDIT4 mRNA and protein levels in dopaminergic neurons. We transfected the effective siDDIT4 (100nM) or siNC (100nM) into PC 12 cells for 48h followed by 24h METH (3mM) exposure. siDDIT4 significantly reduced DDIT4 mRNA and protein levels. METH exposure increased DDIT4 mRNA and protein expression by～2.5-fold in siNC group; this effect was significantly attenuated to 48.7% and 41.0% by co-exposure of siDDIT4. Similarly, we transfected siNC (100nM) or siDDIT4 (100nM) into SH-SY5Y cells, and the results showed that 2.0mM METH exposure increased DDIT4 protein expression by 2.09-fold; this effect was ameliorated to 40.2% by co-exposure of siDDIT4.(5) Silencing of DDIT4 expression affected METH-induced autophagy in PC 12 cells.(6) METH decreased p-mTOR expression and silencing of DDIT4 increased p-mTOR expression in PC 12 cells.(7) Rapamycin promoted METH-induced autophagyin DDIT4 silenced dopaminergic neurons, while 3-MA attenuated it.(8) Silencing of DDIT4 expression affected METH-induced apoptosis in dopaminergic neurons.(9) Silencing of DDIT4 expression reduced METH-induced caspase-3 and PARP activation in vitro.(10) Silencing of DDIT4 expression reduced METH-induced autophagy and apoptosis in vivo.2. Role of DDIT4 in methamphetamine-induced autophagy and apoptosis in cardiomyocytes(1) METH increased DDIT4 protein expression in cardiomyocytes.(2) METH induced autophagy and apoptosis in cardiomyocytes.(3) Synthetic siDDIT4 knockdown DDIT4 protein expression in cardiomyocytes.(4) Silencing of DDIT4 protected cardiomyocytes from METH-induced autophagy and apoptosis.(5) Rapamycin promoted METH-induced autophagy and apoptosis in DDIT4 knockdown cardiomyocytes.Conclusion:PART 1(1) METH increased DDIT4 expression and induced autophagy and apoptosis in dopaminergic neurons;(2) Silencing of DDIT4 expression reduced METH-induced autophagy and apoptosis in PC 12 cell and SH-SY5Y cell;(3) DDIT4 mediates methamphetamine-induced autophagy through mTOR signaling pathway in dopaminergic neurons;(4) METH induced autophagy in dopaminergic neurons,elevated Beclin-1 expression induced by METH can form a complex with Bcl2, an anti-apoptotic protein, leading to apoptosis. PART 2(1) DDIT4 expression in cardiomyocytes is increased after METH exposure in vivo and in vitro;(2) DDIT4can mediate METH-induced autophagy and apoptosis in cardiomyocytes;(3) DDIT4 silencing can activate the phosphorylation of mTOR and protect cardiomyocytes against METH-causedautophagy and apoptosis;(4) The effect of DDIT4 silencing in METH-induced autophagy and apoptosis could be reversed after exposure to the mTOR inhibitor rapymycin. These findings indicate that DDIT4 plays acritical role in METH-induced autophagy and apoptosis in cardiomyocytes.In summary, DDIT4 plays acritical role in METH-induced neurotoxicity and cardiotoxicity.