Insulin-like Growth Factor Binding Protein5(TGFBP5)Mediates Methamphetamine-induced Dopaminergic Neuron Apoptosis

Background and objectivesWith the growing number of new drugs, drug abuse has become a serious social problems facing the various countries around the world, and therefore propose a new drug prevention initiatives without delay. And our drug problem is also becoming serious increasingly. In order to raise capital, drug addicts participation in criminal cases, which destroys the social stability and become a serious burden. Moreover, drug addicts own bodily functions[1] and mental health[2] also suffered serious damage. Therefore, recent studies on the mechanism of injury of various drugs growing international concern.Methamphetamine (Methamphetamine, METH), commonly known as ice, belongs to the amphetamine-type stimulants(ATS), it is the chemical synthesis of a new drug. METH can cause a strong central excitatory effects, for example, stereotypical behavior, hallucinations, loss of appetite and other pharmacological effects and toxicological properties, and METH is easy to form drug dependence or addiction and even once. A large dose of METH may cause a direct result of acute poisoning and even death[3,4]. For long-term use of METH are concerned, it will not only cause damage on the mental and behavioral science, can also cause damage to changes in the central nervous system (CNS) structure and function. In recent years, animal models and clinical studies[5-7] have shown that occur with Parkinson’s disease (Parkinson’s disease, PD) and other neurodegenerative disorders like pathological changes of chronic METH users within the brain parenchyma, which may be the neural-toxicological effects of METH[8-11], for example, dopamine (Dopamine, DA) depletion and reduce the number of receptors[12,13]oxidative stress (Oxidative Stress, OS) damage[14], imbalance causes the body temperature regulation fever, inflammation of the injury, as well as activation of apoptosis-related pathways[15] eventually leading to related brain area and type of neuron damage and neurodegeneration occurs. But the current study is not enough and perfect to clarify the mechanism of METH neurotoxicity.The insulin-like growth factor (IGF) axis is a kind of peptides which induced cell growth, differentiation, and apoptosis, including IGFs (IGF-Ⅰ and IGF-Ⅱ), IGF receptors (IGF-Ⅰ R and IGF-Ⅱ R) and IGF binding proteins (IGFBPs,IGFBP1-7)[16]. The IGFBPs are able to enhance or inhibit the activity of IGFs in a cell-and tissue-specific manner. One of these proteins, IGFBP-5, also has an important role in controlling cell survival, differentiation and apoptosis[17]. Previous research demonstrated that IGFBP5plays an important role in rodent brain development. For example, Zhong et al[18] reported that adding IGFBP5antibody into culture media increased the survival of granule neurons, whereas adding IGFBP5decreased the survival of granule neurons. Therefore, they concluded that IGFBP5overexpression most likely exacerbates premature apoptosis of granule neurons. Marshman et al.[19]showed that addition of exogenous IGFBP5or IGFBP-3to murine mammary epithelial cells in vitro suppresses IGF-I-mediated survival, resulting in3fold greater rates of apoptosis. Butt et al[201showed that IGFBP5activated caspase-8 and caspase-9, causing apoptosis through bcl-2in the intrinsic apoptic pathway in MDA-MB-231breast cancer cells. These findings were consistent with a pro-apoptotic effect of IGFBP5.In the previous studies of METH, we found that rat striatum and other parts increased apoptosis of dopaminergic neurons after injection, and PC12cells (dopaminergic neuron cell line) with the same result of corresponding METH treatment. And our data shows that IGFBP5increased significantly after METH treated PC12cells within24hours. So we suspect that IGFBP5mediates METH-induced dopaminergic neuronal apoptosis. Through this study we are trying to prove that IGFBP5plays an important regulatory role in METH-induced dopaminergic neuronal apoptosis and provid a new ideas and effective drug targets for the treatment of METH addicts.Contents and Methods1. IGFBP5expression was increased by METH exposure in PC12cells and rat brain tissue.(1). To explore the molecular mechanisms underlying METH-induced neuronal apoptosis, microarray analysis was performed to examine the difference in the mRNA expression of all members of IGF axis between vehicle-and METH-treated PC12cells.(2). To further validate the data from microarray, QRT-PCR was performed to detect the expression of IGFBP4,5and7in PC12cells treated with or without METH.(3). To further clarify the role of IGFBP5in vivo, we built the model of Sprague-Dawley rats with METH intoxication, QRT-PCR was performed to detect the expression of IGFBP4,5and7in striatum of rat treated with or without METH. (4). To further determine the role of IGFBP5in the METH-exposed toxicity, we treated PC12cells with different concentration of METH for indicated times and then QPCR and western blot were performed to detect the IGFBP5expression at transcriptional and translational levels..2. Silencing of IGFBP5can protect METH-exposed apoptosis in PC12cells.(1). Three siRNA sequences were designed for targeting IGFBP5and synthesized by company. To verify the knockdown efficiency of siRNA for IGFBP5, we transfected the siRNA1-3and scrambled siRNA respectively into PC12cells followed by METH exposure, and QRT-PCR analysis was used.(2). To confirm IGFBP5protein expression in PC12cells after transfected the siRNA followed by METH exposure, western blot analysis was used.(3). To explore whether silencing of IGFBP5expression can reduced METH-exposed apoptosis in vitro, a TUNEL assay was performed to detect DNA damage in PC12cells..3. IGFBP5mediates neuronal apoptosis through regulating the release of cyto c and activation capase-3and PARP.(1). Previous study demonstrated that neuronal apoptosis induced by METH is accompanied by release of cyto c from mitochondria into the cytoplasm, cleavage of caspase-3and PARP is the substrate of activated caspase-3. We sought to determine whether IGFBP5mediated neuronal apoptosis via caspase-3pathway. Therefore we examined the caspase-3and PARP activation in PC12cells with IGFBP5silencing or not and treated with or without METH by western blot analysis.4. Statistical analysisData given in the text are expressed as mean±standard deviation(SD) of at least three independent replicates. Statistical analysis was performed using one-way ANOVA, factorial design ANOVA and independent-samples T Test with the scientific statistic software SPSS version13. The value of P<0.05was considered statistically significant.Results1. IGFBP5expression was increased by METH exposure in PC12cells and rat brain tissue.(1). To explore the molecular mechanisms underlying METH-induced neuronal apoptosis, microarray analysis was performed to examine the difference in the mRNA expression of all members of IGF axis between vehicle-and METH-treated PC12cells. We found that only IGFBP4、IGFBP5and IGFBP7expression were changed after METH treatment for24h in PC12cells.(2). To further validate the data from microarray, QRT-PCR was performed to detect the expression of IGFBP4,5and7in PC12cells treated with or without METH. Our data shown that METH exposure(3.0mM) for24h resulted in a7-fold increase of IGFBP5mRNA expression, but it had no effects on IGFBP4or IGFBP7in PC12cells.(3). To further clarify the role of IGFBP5in vivo, we built the model of Sprague-Dawley rats with METH intoxication, QRT-PCR was performed to detect the expression of IGFBP4,5and7in striatum of rat treated with or without METH. Our data shown that METH exposure(3.0mM) for24h resulted in a9-fold increase of IGFBP5mRNA expression, but it had no effects on IGFBP4or IGFBP7in striatum.(4). To further determine the role of IGFBP5in the METH-exposed toxicity, we treated PC12cells with different concentration of METH for indicated times(0,2,4,8,16,24h) and then QPCR and western blot were performed to detect the IGFBP5expression at transcriptional and translational levels. The results revealed that IGFBP5mRNA and protein expressions were dose-dependently and time-dependently increased.2. Silencing of IGFBP5can protect METH-exposed apoptosis in PC12cells.(1). Three siRNA sequences were designed for targeting IGFBP5and synthesized by company. To verify the knockdown efficiency of siRNA for IGFBP5, we transfected the siRNAl-3and scrambled siRNA respectively into PC12cells followed by METH exposure. QPCR analysis showed only two siRNA sequences(siRNA#2and siRNA#3) could effectively silenced the IGFBP5expression mRNA levels while siRNA#1has no effect on IGFBP5expression compared with control siRNA.(2). To confirm IGFBP5protein expression in PC12cells after transfected the siRNA followed by METH exposure, western blot analysis was used. Our data showed that METH induced IGFBP5expression while siRNA#2and siRNA#3can attenuate the IGFBP5induction by METH.(3). To explore whether silencing of IGFBP5expression can reduced METH-exposed apoptosis in vitro, a TUNEL assay was performed to detect DNA damage in PC12cells. The number of TUNEL-positive cells was increased by more than12-fold in PC12cells transfected with scrambled siRNA exposed to METH compared to control cells. The number of TUNEL-positive cells was decreased by60%and40%respectively in METH-exposed PC12cells transfected with siRNA#2and siRNA#3compared with cells transfected with scrambled siRNA.3. IGFBP5mediates neuronal apoptosis through regulating the release of cyto c and activation capase-3and PARP.(1). Previous study demonstrated that neuronal apoptosis induced by METH is accompanied by release of cyto c from mitochondria into the cytoplasm, cleavage of caspase-3and PARP is the substrate of activated caspase-3. We sought to determine whether IGFBP5mediated neuronal apoptosis via caspase-3pathway. Therefore we examined the caspase-3and PARP activation in PC12cells with IGFBP5silencing or not and treated with or without METH. western blot analysis showed that the concentration of cyto c in cellular subfractions was increased after METH treated in PC12cells, and silencing of IGFBP5expression blocked cyto c release from mitochondria into the cytoplasm. And cleaved caspase-3increased in PC12cells transfected with scrambled siRNA with METH treatment compared with the control.Conclusions1. IGFBP5expression was increased by METH exposure in PC12cells and rat brain tissue.2. Silencing of IGFBP5can protect METH-exposed apoptosis in PC12cells.3. IGFBP5mediates neuronal apoptosis through regulating the release of cyto c and activation capase-3and PARP.