Mechanism Of Protein Disulphideisomerase On Methamphetamine Induced Abnormal Expression Of α-synuclein

Backgroud:Methamphetamine (METH) also named metamfetamine, it has benzene ring structure and very similar with catecholamine neurotransmitter. METH is soluble in water and alcohol, its hydrochloride is transparent crystal, so commonly known as "ice" for its ice-like. METH as a new drug, due to it was easily synthesized and widely available, METH has become popular in the world and one of the most abused drugs, which poses a tremendous burden to the succumbed individuals. METH abuse also can cause all kinds of criminal, pose great harm to social. Therefore, the mechanism study of METH induced neurotoxicity and prevention has become one of the most important topics to the world.We have found a-synuclein (a-SN) expression in striatum and related brain areas increased significantly after treated METH, and confirmed it played an important role in METH induced oxidative stress, dopamine (DA) metabolic disorders and loss of mitochondrial function. Lots of evidence showed abnormal expression of a-SN associated with Parkinson’s disease (Parkinson, PD), Alzheimer’s disease (AD) and other neurodegenerative diseases. Medical research of image data and epidemiological data suggest that the ratio of PD increased significantly in METH users, and METH can result in the frontal cortex, hippocampus and striatum of brain neurons injury, similar to the AD and PD pathological change. It has paper showed a-SN gene is PD disease genes. In some family type of PD patients have a-SN mutation. And whether familial or scattered PD, a-SN is the main components of inclusion body of Lewy body, which is the characteristic of PD. Especially missense mutation (A30P A53Tå'Œ E46K) and core sequence (66VGGAVVTGV74) of a-SN is vital to PD. a-SN is a soluble and not folded protein in physiological status, and played an important role in regulating synaptic plasticity and neurotransmitter release. In pathological conditions, a-SN is an aggregate protein. In some neurodegenerative diseases, the aggregation a-SN not only can restrain the speed limit DA synthetic enzyme tyrosine hydroxylase expression, also can promote the cell oxidative stress and lead to neurotoxicity. In the same time, the oxidative stress can promote a-SN aggregation, caused vicious circle and strengthen neurodegenerative disease progression. So it’s very important to study the mechanism of METH induced a-SN aggregation. And it may provide important targets for treat METH induced neurotoxicity or other neurodegenerative disease.Protein disulfide isomerase is an endoplasmic reticulum retention protein. It participate the modification and fold of new synthetic secretory protein, catalytic thiol-disulfide bond exchange reaction, and formed disulfide bond. Disulfide bond plays an important role in the stability of protein three-dimensional structure.PDI has multiple domain structure, can not only catalytic mercapto-disulfide bond exchange, promote the formation of protein disulfide bond mismatch and disulfide bond rearrangement, also has the oxidation, reduction and heterogeneous functions. In addition, PDI also has a molecular chaperone activity, can restrain the aggregation of misfolded proteins, and stable the correct structure of the protein. In many neurodegenerative disease such as PD and AD PDI can inhibit accumulation of misfolded proteins and has neuroprotective effect. But in some degenerative diseases, PDI easy combined with NO and formed S-nitrosylating protein disulphideisomerase. PDI-SNO resulted PDI lost its function.As early as 2006, Uehara has reportedPDI and PDI-SNO have important role in abnormal protein aggregation associated degeneration disease. In PD and AD samples brain tissue, PDI is S-nitrosylated obviously. We have found the oxidative stress was enhanced obviously in vivo and in vitro after treated with METH in previous study. However, it still unclear in METH induced oxidative stress on PDI function and PDI on a-SN aggregation.This study intends to establish a METH poisoning cell model, and via detect the morphology, the expression of NO and NOS, a-SN aggregation and S-nitrosylating protein disulphideisomerase to confirm the METH induced neurotoxicity. We also prove the change of PDI and the effect of PDI on a-SN aggregation by add L-NNA and L-Arginine respectively. So as to further study the mechanism of METH induced abnormal expression of a-SN, providing theoretical basis for treatment of METH neurotoxicity.Objectives;To establish METH poisoning cell model, and add NOS inhibitor and NO donor, via detect the morphology, the expression of NO and NOS, a-SN aggregation and S-nitrosylating protein disulphideisomerase to study the effect of METH induced oxidative stress on PDI function and PDI on abnormal expression of a-SN. And then verify the previous results by RNA interference, further confirm the relations between METH, PDI and a-SN, for METH neurotoxic mechanism study provide new targets.Methods:1. The establishment of METH poisoning cell model(1) PC 12 cells were cultured in DMEM medium containing 10% fetal bovine serum in 96-well plates. Cells were grown in a CO2 incubator at 37℃ with 5% CO2. PC12 cells were treated by METH with the concentration of 0.5,1,2,3mmol/L respectively for 24h when cells grow to 70% confluent. The viability of PC 12 cells was assessed by CCK-8 assay.(2) PC 12 cells were cultured in ten 6cm dish, divided into two groups when cells grow to 70% confluent, one group pre-treated with 0.1 mmol/L L-NNA for 30min, then two group of dish treated by METH with the concentration of 0.5,1,2,3mmol/L respectively for 24h. Detect the expression of PDI in each group by Biotin-switch method and Western Blot.2. Study the mechanism of PDI on METH induced abnormal expression of a-SNPC 12 cells were cultured in 6-well plates,96-well plates and 10cm dish, and divided to 6 groups:empty control group (CON), empty NOS inhibitor group (CON+L-NNA), empty NO donor group (CON+L-Arginine), METH group (METH), METH+NOS inhibitor group (METH+L-NNA), METH+NO donor group (METH+ L-Arginine).(1) The morphological changes were observed by inverted microscope and the viability of cells was detected by CCK-8 assay.(2) The cell apoptosis rates were assessed by TUNEL and Hoechst.(3) The activity of NOS and NO was measured by the method of enzyme chemistry with NOS detection kit and NO detection kit respectively, the expression of NOS protein was measured by Western Blot.(4) The expression of PDI-SNO in each group was detected by Biotin-switch method and Western Blot.(5) The expression of a-SN was measured by Western Blot, the morphology of a-SN was measured by confocal microscopy.3. Theexpression of a-SN in PDI knockdown PC12 cells after treated with METH and L-NNA(1) Design and synthesis of 3 PDI siRNA to knockdown PC 12 PDI gene, detect the most effective siRNA sequences by Western Blot.(2) METH and L-NNA treated with PDI knockdown PC 12 cells, and then measured the expression of a-SN by Western Blot and immunofluorescence.Results:1. The establishment of METH poisoning cell model(1) Contrast to the control group, the viability of PC12 cells treated by METH was decreased in dose dependent manner and there were significant differences between the 1,2 and 3mmol/L METH group and the control group (p<0.05)(2) Compared with control group, the expression of PDI-SNOwas increased in dose dependent manner and there were significant differences between the 2 and 3mmol/L METH group and the control group (p<0.05). When add L-NNA to cells, L-NNA can suppress PDI-SNO expression significantly (p<0.05), especially in 3mmol/L METH concentration. So we select 3mmol/L METH in our study.2. Study the mechanism of PDI on METH induced abnormal expression of a-SN(1) Compared with control group, the morphological changes of PC 12 cells treated with METH were featured by shrinkage of the cell bodies, disruption of the dendrite and brighter cell cytoplasm. The cells treated with L-Arginine have the same change. But the cells treated with L-NNA have not significant change. When L-NNA and METH treat with cells together, L-NNA can effectively suppress the morphological changes of PC 12 cells induced by METH. The viability of PC 12 cells detected by CCK-8 has the same trend with its morphological changes.(2) The cell apoptosis rates assessed by TUNEL and Hoechst showed METH and L-Arginine increased cell apoptosis significantly (p<0.05), while L-NNA not. When L-NNA and METH treated cells together, L-NNA can decrease METH induced apoptosis (p<0.05). But when L-Arginine and METH treated cells together, L-Arginine can increase METH induced apoptosis (p<0.05)(3) The method of enzyme chemistry with NOS detection kit and NO detection kit showed the activity of NOS and NO in METH and L-Arginine increased observably compared with control(p<0.05). When add L-NNA with METH together, L-NNA can weaken the effect of METH (p<0.05). While the effect of L-Arginine in contrast to the L-NNA. The results of Western Blot are similar with the method of enzyme chemistry’s results.(4) The expression of a-SN measured by Western Blot showed METH can induce the expression of a-SN increase (p<0.05). And L-NNA can inhibit METH induced a-SN increase (p<0.05). Similarly, the morphology of a-SN measured by confocal microscopy showed METH induced a-SN aggregation significantly compared with control(p<0.05). L-NNA can weaken the effect of METH(p<0.05), while L-Arginine can strengthen the effect of METH (p<0.05)3. Theexpression of α-SN in PDI knockdown PC12 cells after treated with METH and L-NNA(1) We designed 3 PDI siRNA to knockdown PC 12 PDI gene; detect the effect of the 3 PDI siRNA by Western Blot. The result showed all the 3 PDI siRNA can inhibit the expression of PDI effetely, while the second siRNA effect is the most obvios.(2) We used the most useful siRNA to knockdown the PDI gene of PC 12 cells, and then treated cells with METH and L-NNA together, after 24h, Western Blot showed L-NNA cannot inhibit METH induced the expression and aggregation of a-SN after PDI gene knockdown.Conclusions1. PDI-SNO increased significantly after METH treat with cells, and the expression of PDI-SNO were gradually enhanced with the increase of METH concentration. The NOS inhibitor L-NNA can effetely decrease the expression of PDI-SNO, and when METH concentration is 3mmol/L, the effect is the most significant.2. METH can induce cells oxidative stress and a-SN aggregation, oxidative stress can result S-nitrosylate of protein disulphideisomerase which could not inhibit a-SN aggregation. The aggregation ofa-SN can result to cells apoptosis. The NOS inhibitor L-NNA can inhibit the process and decrease METH induced neurotoxicity. While the effect of NO donor L-Arginine is just the opposite. This effect showed the oxidative stress makes the PDI dysfunction, thus cannot via inhibit a-SN aggregation to protect nerve.3. After knockdown PDI gene, even though treated cells with L-NNA and METH together, a-SN aggregate significantly. It showed PDI is the most important for inhibit a-SN aggregation.