Effect And Its Mechanism Of α-synuclein Knockdown On Methamphetamine-induced Neurotoxicity

BACKGROUND AND OBJECTIVEMethamphetamine is a new type of dope and has the following characteristics including simple chemical synthesis and multi-pathway for uptake, which makes it abused and extended so quickly. Therefore, the toxicity and its mechanism of methamphetamine have become a hot world issue nowadays. METH possesses the traits of the nervous excitation and psychological dependence. The drug can induce the injury on heart, liver, kidney and other organs, but the most severe toxicity due to its neurotoxicity, especially to dopaminergic neuron, including DA depletion, decreased DAT and TH activity, loss of DA uptake sites and VMAT-2in the striatum. In the present, the mechanism of METH-induced neurotoxicity is not very clear. The possible mechanisms of METH induced neurotoxicity included dopaminergic defect and oxidative stress, cytotoxicity caused by glutamate and NO, mitochondrial dysfunction and neuron apoptosis.In our previous study, we have showed METH induced up-regulation of α-synuclein (α=SN) expression in the striatum, cortex and hippocamp of the rat brain, which was identified by western blot. Many studies suggest that abnormal accumulation and fibrosis of α-synuclein has an important role in the pathophysiology of Parkinson’s disease, Alzheimer’s disease and other neurodegeneration. And α-synuclein is identified as a drug target of Parkinson’s disease. Ajjimaporn demonstrates METH induced up-regulation of α-synuclein of SK-N-SH cell line. But the role of α-synuclein in the METH induced neurotoxicity is not reported and needed further research.RNA interference (RNAi) is an effective tool to explore the function of a gene, which can suppress gene expression on post-transcriptional level with high efficiency and specificity. Sapru MK reported successful silencing of the α-synuclein gene of SH-SY5Y by vector-based RNAi. Our study will employ the approach above and obtain stable cells which are transfected α-SN-shRNA, to explore the effect of α-SN knockdown on dopaminergic defect, oxidative stress, mitochondrial dysfunction and neuron apoptosis induced by METH. The study may be useful for exploring the theory of METH neurotoxicity mechanism and drug target of METH prevention and cure.METHODS1. The toxicity and α-SN expression of SH-SY5Y treated with various concentrations of METHSH-SY5Y cells were cultured in DMEM/F12medium containing10%fetal bovine serum,50units/mL penicillin G, and50mg/mL streptomycin sulfate. Cells were grown in a CO2incubator at37℃, with5%CO2and95%filtered air. We carry out the following studies when the cells grow to80%confluent. The SH-SY5Y cells of experiment group were treated by METH with the concentration of0.5,1.5,2.5,3.5,4.5mmol/L respectively for24h, and the control group was added with same volume of DMEM medium. The morphological changes were observed by inverted microscope, the viability of SH-SY5Y was assessed by CCK-8assay, the cell apoptosis rates were assessed by flow cytometry (FCM), the expression changes of α-SN gene and protein were detected by real time PCR and western bolt respectively.2. Construct SH-SY5Y cells whose α-synuclein expression is suppressed by RNAi and study the silencing effect of α-synuclein1) ShRNA expressing pLVTHM vectors were constructed according to the valid shRNA reported by previous research and two pairs of designed shRNA. AND they were identified by DNA sequencing. 2) These plasmid vectors were packaged with lentivirus and the recombinant lentiviral vectors were transfected into SH-SY5Y cells.3) The silencing effect on α-SN protein was investigated by western blot and the most effective shRNA was selected for stably transfected cells.4) The optimal shRNA were recombinated to pLKO.1vectors without GFP gene. The recombinants were transfected into SH-SY5Y cells and α-SN level was investigated by western blot3. Evaluate the effect of α-synuclein knockdown on neurotoxicity induced by METH in SH-SY5YThere are six big groups in our experiment, untransfected control group (CON), Empty vector control group (Empty vector), α-SN shRNA cell group (RNAi), CON+METH, Empty vector+METH, RNAi+METH. The treated METH concentration is3.5mmol/L.1) The viability and cell cycle of various group cells was detected by CCK-8assay and flow cytometry (FCM) respectively.2) The gene expression of TH, DAT and VMAT-2was assessed by real time PCR.3) The DA level was detected by ELISA.4) The activity of ROS, NOS and NO was measured by the method of enzyme chemistry with ROS fluorescence detection kit, NOS detection kit, NO detection kit respectively.5) The cell apoptosis rates were assessed by FCM and the morphological changes of apoptotic cells were observed by electron microscope.6) Mitochondrial Δψm was measured using two fluorometric probes in separate experiments, rhodamine123(Rh123) and Tetramethylrhodamine ethyl ester (TMRE) and analyzed by FCM and confocal microscope respectively.7) Calcein was employed to detect MPTP opening by confocal microscope.8) Intracellular calcium was stained and measured by confocal microscope.9) Western blotting assay was used to evaluate the release of cytochrome C. RESULTS1. Contrast to the control group (untreated by METH), the morphological changes of SH-SY5Y cells treated by METH of0.5-4.5mmol/L were featured by shrinkage of the cell bodies, brighter cell cytoplasm, disruption of the dendrite and disappearance of cell edges, as well as cells taking off the wall and floating, which can be found especially and obviously in the cells exposed to2.5,3.5and4.5mmol/L. There were significant differences between the1.5,2.5,3.5and4.5mmol/L METH groups and the control group on cell viability (P<0.001). Cell viability of SH-SY5Y treated by METH was decreased in dose dependent manner. The cell early apoptosis rates of1.5,2.5,3.5and4.5mmol/L METH groups were higher than control groups (P<0.001). The cell early apoptosis rates were gradually increased in dose dependent manner. There were significant differences between the1.5,2.5,3.5and4.5mmol/L METH groups and the control group on the level of α-SN RNA expression (P<0.001). And the α-SN protein expression of SH-SY5Y cells was increased in dose dependent manner.2. ShRNA with optimal silencing effect was selected. And recombinant α-SN-shRNA-pLVTHM lentiviral vectors were constructed successfully. The mRNA of transfected SH-SY5Y cells can inhibit gene expression of α-SN up to84%. AND recombinant α-SN-shRNA-pLKO.1lentiviral vectors uncontained GFP were constructed and transfected SH-SY5Y cells successfully。Western blot experiments also confirmed the α-SN protein of both kinds of transfected cells was significantly inhibited.3. Compared with various groups of cells untreated by METH, floating dead cells were observed in the groups treated by METH, cell viability was decreased (P≤0.001), mRNA of DAT, TH and VMAT-2was decreased significantly (P<0.001, P≤0.001,P≤0.001), intracellular DA level was decreased significantly ((P<0.001), the levels of ROS, NOS and NO were significantly increased (P<0.001, P≤0.001,P≤0.001). However, RNAi could reduce floating dead cells, increase cell viability (P≤0.001), prevent the loss of DAT, TH and VMAT-2level (P<0.001, P≤0.001,P≤0.001), attenuate the elevated ROS, NOS and NO level (P<0.001, P≤0.001,P≤0.001).4. Annexin V-FITC staining and flow cytometry analysis showed the early apoptosis rates of cells treated by3.5mmol/L METH were increased significantly (P<0.001), contrast to the control group (untreated by METH). Among the treated cells, the early apoptosis rates of RNAi groups were lower than control significantly (P<0.001). Compared to untreated cells, the ultrastructural changes of SH-SY5Y cells treated by of3.5mmol/L METH were as following:1)Necrosis morphological changes were featured by distension of cellular organelles such as endoplasmic reticulum and golgiosome, early karyolysis and nucleolus disappearance.2) Apoptosis morphological changes were featured by shrunken cell body, cell membrane rupture and condensed crescent shaped chromatins attaching to the nuclear membranes.3) Autophagosomes were observed which devoured mitochondria.4) Multiple intracellular bodies similar as Lewy bodies were composed of homogeneous substance arranged in concentric circles shape. However, no intracellular bodies similar as Lewy bodies were observed in RNAi groups treated by METH and organelle injuries of RNAi groups was lighter than control groups.5. The mitochondria indexes detected by fluorescence labeling combined with laser scanning confocal microscope showed:1) The intensity of intracellular red fluorescence issued by TMRE label was decreased in METH treated groups, showing decrease of mitochondrial membrane potential after cells were exposed to METH for2h. The fluorescence intensity in METH treated RNAi groups is higher than in METH treated another control groups, demonstrating the mitochondrial membrane potential decrease rate in METH treated RNAi groups is lower than in METH treated control groups.2) The intensity of intracellular green fluorescence issued by calcein label was decreased in METH treated groups, showing METH made mitochondrial permeable transition pores opened. The fluorescence intensity decrease rate in METH treated RNAi groups was lower than in METH treated control groups, indicating a-SN knockdown suppressed METH-induced mitochondrial permeable transition pores opening.3) The intensity of intracellular green fluorescence issued by Fluo-3label was increased in METH treated groups, manifesting METH treatment led to a rise in intracellular calcium levels. The increase rate of cytoplasmic free Ca2+concentration in METH treated RNAi groups is lower than in METH treated control groups. Western blot experiments confirmed cytoplasmic cytochrome C level was increased in METH treated groups, showing METH exposure resulted in release of mitochondrial cytochrome C. The increase rate of cytochrome C level in METH treated RNAi groups was lower than in METH treated control groups.CONCLUSION1. METH can induce morphological changes, loss of cell viability and increased expression of α-SN RNA and protein in SH-SY5Y cells. And the injuries and the expression of α-SN were gradually enhanced with the increase of METH concentration.2. Recombinant lentiviral α-SN-shRNA was constructed and significantly reduced α-SN gene expression of SH-SY5Y. The stable transfected SH-SY5Y cells with long term silencing of α-SN were achieved3.α-SN knockdown suppressed METH-induced depression of cell viability, decrease of RNA level of TH, DAT and VMAT-2, depletion of dopamine and increase of ROS、NOS and NO. α-SN knockdown may alleviate METH-induced cytotoxicity via inhibiting dopaminergic dysfuncation and oxidative stress injury.4. Exposure to METH induced mitochondrial dysfunction in the form of marked decrease in mitochondrial membrane potential, the opening of mitochondrial permeable transition pore, intracellular calcium overload and increase of mitochondrial cytochrome c release.α-SN knockdown suppressed METH-induced apoptosis and protected mitochondrial normal function.α-SN knockdown may protect SH-SY5Y against METH-induced injury via inhibiting cytochrome c release to cytoplasm and attenuating the depression of mitochondrial membrane potential, the opening of mitochondrial MPTP and the increase of intracellular calcium.