Overexposure to methamphetamine (METH), a psychoactive drug, induces a variety of adverse effects to the nervous system, including apoptosis of dopaminergic neurons. Insulin-like growth factor binding protein 5 (IGFBP5), a member of insulin-like growth factor (IGF) system, is a pro-apoptotic factor that plays important roles in neuronal apoptosis. To test the hypothesis that IGFBP5 can mediate METH-induced neuronal apoptosis, we examined IGFBP5 mRNA and protein expression changes in PC12 cells exposed to METH (3.0mM) for 24h and in the striatum of rats following 15mg/kg×8 intraperitoneal injections of METH at 12h interval. We also checked the effect on neuronal apoptosis after silencing IGFBP5 expression with TUNEL staining and flow cytometry; Western blot was used for detecting the expression of apoptotic markers active-caspase3 and PARP. To elucidate the mechanisms underlying IGFBP5-mediated neuronal apoptosis, we determined the release of cytochrome c (cyto c), an apoptogenic factor, from the mitochondria after METH treatment with or without IGFBP5 knockdown. Our results showed that IGFBP5 expression was increased significantly after METH exposure in PC12 cells and in the METH-treated rats' striatum. Further, METH-exposed PC12 cells exhibited higher apoptosis-positive cell number and activity of caspase3 and PARP compared with control cells, while these changes can be blocked by silencing IGFBP5 expression. In addition, a significant increase of cyto c release from mitochondria after METH exposure was observed and it was inhibited after silencing IGFBP5 expression in PC12 cells. These results indicate that IGFBP5 plays key roles in METH-induced neuronal apoptosis and may be a potential gene target for therapeutics in METH-caused neurotoxicity.
The over-expression of ?-synuclein is a major factor in the death of dopaminergic neurons in a methamphetamine-induced model of Parkinson's disease. In the present study, ?-synuclein knockdown rats were created by injecting ?-synuclein-shRNA lentivirus stereotaxically into the right striatum of experimental rats. At 2 weeks post-injection, the rats were injected intraperitoneally with methamphetamine to establish the model of Parkinson's disease. Expression of ?-synuclein mRNA and protein in the right striatum of the injected rats was significantly downregulated. Food intake and body weight were greater in ?-synuclein knockdown rats, and water intake and stereotyped behavior score were lower than in model rats. Striatal dopamine and tyrosine hydroxylase levels were significantly elevated in ?-synuclein knockdown rats. Moreover, superoxide dismutase activity was greater in ?-synuclein knockdown rat striatum, but the levels of reactive oxygen species, malondialdehyde, nitric oxide synthase and nitrogen monoxide were lower compared with model rats. We also found that ?-synuclein knockdown inhibited methamphetamine-induced neuronal apoptosis. These results suggest that ?-synuclein has the capacity to reverse methamphetamine-induced apoptosis of dopaminergic neurons in the rat striatum by inhibiting oxidative stress and improving dopaminergic system function.
The protein ?-synuclein (?-syn) is abundant in neurons and has been claimed to play critical roles in the pathophysiology of Parkinsons disease. Overexpression of ?-syn has been shown to be toxicity in methamphetamine (METH)-induced model in vivo and in vitro which has Parkinsons-like pathology. However, the exact mechanisms underlying toxicity of ?-syn mediated METH-induced neuron remain unknown. In the present study, human dopaminergic-like neuroblastoma SH-SY5Y cells were used as METH-induced model in vitro. Cell viability was found to be dramatically increased after silencing ?-syn expression followed by METH treatment compared with a-syn wild-type cells and the morphological damage to cells after METH treatment was abated through knockdown of ?-syn expression in this model. The expression levels of tyrosine hydroxylase (TH), dopamine transporter (DAT) and vesicular monoamine transporter 2(VMAT-2) were significantly decreased and the activity/levels of reactive oxygen species (ROS), nitric oxide synthase (NOS) and nitrogen (NO) were notably increased after METH treatment. However, the changes of these expression levels were reversed in cells transfected with ?-syn-shRNA. These results suggested that TH, DAT, VMAT-2, ROS and NOS maybe involved in ?-syn mediated METH-induced neuronal toxicity.
Methamphetamine is a type of psychoactive drug. It is well known that neurotoxicity caused by Methamphetamine(METH) can damage the nervous system and lead to apoptosis and cell loss of dopaminergic neurons. ROCK2 is a prominent target for gene therapy because its inhibition has proved to have a protective effect in various cell lines and pathophysiological conditions. Although several of the negative effects of METH on the dopaminergic system have been studied, the protective molecular mechanisms and the effective treatment of METH-induced apoptosis remain to be clarified. We hypothesized that ROCK2 is involved in METH-induced apoptosis. We tested our hypothesis using RT-PCR and western blotting to analyze whether silencing of ROCK2 with small interfering RNA (siROCK2) could reduce damage and apoptosis in PC12 cells after METH exposure. Increases in viability and cytomorphological changes were detected by MTT assay and bright field microscopy after pretreatment of METH-treated PC12 cells with 100 nM siROCK2. Apoptosis decreased significantly after ROCK2 silencing, as shown by Annexin V and TUNEL staining. The results show that ROCK2 is a possible gene target for therapeutics in METH-induced neurotoxicity in vitro, providing a foundation for future in vivo research.
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