Study On The Relationship Of Thioredoxin, Endoplasmic Reticulum Stress And Parkinson's Disease

Parkinson's disease (PD) is common neurodegenerative disorder in middle and aged population. Its etiology includes genetic and environmental factor. The clinical symptoms include resting tremor, bradykinesia, postural instability, rigidity and cognitive disorder. Pathological and biochemistry features are loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) of the brain, the accumulation of Lewy's body in the neurons, leading to reduce of dopamine secretion, low level of dopamined and decrease ratio of dopamine and acetylcholine. At present, the main treatment of PD is the replacement therapy of the L-DOPA, but its effect is not so good. The reason due to the molecular mechanism on PD is still unknown.The study on the mechanism of PD depends on the establishment of the cell model and the animal model of PD. 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) is commonly used in animals to produce an experiment model of PD. The 1-meythyl-4-phenylpyridinium ion (MPP+), the active metabolite of MPTP, is transported in dopaminergic terminals neuron and binds complex I NADH dehydrogenase in mitochondrial membrane-bound, inhibits its activity, causes ATP depletion, and leads to the increase of reactive oxygen species (ROS), finally results to the neurons death.Besides the mitochondrial damage and the production of ROS, the pathologic feature included the accumulation of Lewy's body results from the abnormal protein in cytoplasm. The metabolism of protein is associated with the endoplasmic reticulum. Thus, endoplasmic reticulum stress (ER stress) plays important roles in neuronal death of PD. Binding protein/glucose-regulated protein 78 (BiP/Grp78), C/EBP homologous protein (CHOP), caspase-12 and Irel/tumor necrosis factor receptor-associated factor 2 (TRAF2), apoptosis signal-regulating kinase 1 (ASK1) are associated with the ER stress. Furthermore, the decreased activity of phosphatidyl inositol 3 kinase (PI3K)/protein kinase B (PKB/Akt) and the increased activity of glycogen synthase kinase 3P (GSK-3β) is related to the ER stress.Thioredoxin (Trx) is a small molecular redox regulating protein. Trx has various important roles, including ROS scavenging, regulating the transcription factors, antiapoptosis and the neurotrophic function. Trx protects neuron from ER stress-mediated cytotoxicity induced by MPTP, and the molecular mechanism is still unclear. The further study on the molecular mechanism of Trx regulating ER stress will help to elucidate the pathological mechanism of PD and to apply targets of treatmenyt of PD.PD model established with MPTP/MPP+was used to explore the molecular mechanisms on ER stress of PD and on Trx protecting the the neuron from MPTP/MPP+ neurotoxicity.The major results were as follows:The rat pheochromocytoma cells (PC 12 cells) treated with the MPP+, and the viability of PC 12 cells was decreased in dose-dependent manner, and the LDH releasing was also increased. MPP+ induced the expression of BiP/Grp78, CHOP and the activation of caspase-12 in PC12 cells, the induction of BiP/Grp78 was prior to induction of CHOP and the activation of caspase-12. These results suggest that the ER stress was involved in the PC 12 cell apoptosis induced by MPP+. At the initial stages of ER stress, the increased expression of BiP/Grp78 reduced the ER stress, until the increased expression of CHOP and the activation of caspase-12.Nerve growth factor (NGF), the agonist of PI3K, and LiCl, the inhibitor of GSK-3βinhibited the expression of BiP/Grp78 and CHOP and the activation of caspase-12 induced by MPP+ in PC12 cells. The level of pAkt (ser 473) and pGSK-3β(ser 9) was significantly decreased after treatment with MPP+. This result suggests that the MPP+dephosphorylated the ser473 of Akt, and inhibited its kinase activity. In turn, dephosphorylated the ser9 of GSK-3β, and the activity of GSK-3βwas activated. Pretreatment with NGF inhibited the activation of GSK-3βinduced by MPP+. These results suggest that PI3K/Akt/GSK-3(3 is involved in the ER stress induced by MPP+ in PC 12 cells.After silencing the expression of Trx in PC12 cells using rat Trx siRNA, the expression of BiP and CHOP were not significantly affected, and the caspase-12 was activated much more. On the other hand, the Trx inducer geranylgeranylacetone (GGA) suppressed the ER stress by MPP+. The overexpression of Trx by GGA suppressed the expression of BiP/Grp78 and CHOP and the activation of caspase-12. These results suggest that Trx overexpression by GGA inhibited ER stress by MPP+and reduced the apoptosis induced by MPP+. Thus, Trx has the role in inhibiting the ER stress induced by MPP+in PC12 cells.Panaxatriol saponins (PTS) is one of the biologically active fractions of Panax notoginseng. It exerts many pharmacological effects to treat various diseases including anti oxidative stress, protecting brain, antithrombotic. Previous study has shown that PTS induced expression of Trx in vitro and in vivo suppressed the neurotocicity of MPP+In the behavioral experiment, the motor activity of mice of the MPTP group reduced significantly campared to the saline group. The pretreatment with PTS ameliorated MPTP-induced impairment of limb movements scored by traction tests. These results indicate that PTS has the ability to improve MPTP-induced functional impairment. The loss of dopaminergic neurons was assessed by expression of tyrosine hydroxylase (TH) which is the maker of dopamingenid neuron, in the the region of SNC. TH expression in the SNc was significantly decreased after injection of MPTP, however pretreatment with PTS rescued MPTP-induced down-regulation of TH expression. This indicates that MPTP-induced dopaminergic neurons loss is inhibited by PTS. After MPTP treatment, Trx expression was decreased, and the downregulation of Trx expression was restored by PTS pretreatment. PTS ameliorate the neuron damage induced by MPTP through the induction of Trx expression. MPTP administration increased the activity of caspase-9 and caspase-3, result in the neuron apoptosis. The pretreatment with PTS suppressed caspase-9 and caspase-3 activations caused by MPTP, while the decrease of pro-caspase-12 caused by MPTP was not inhibited by PTS. These data suggest that PTS prevents dopaminergic neurons againstMPTP induced neurotoxicity through suppressing mitochondria-mediated apoptosis pathway, but not ER-mediated pathway. Cyclooxygenase-2 (COX-2) expression is up-regulated in the SNc after MPTP treatment, and PTS suppressed MPTP-induced increase of COX-2. These results indicate that PTS ameliorated the move dysfunction induced by MPTP, prevented the loss of dopaminergic neurons in SNc. The main mechanism is that PTS induced the expression of Trx, inhibited the overexpression of COX-2, and suppressed the mitochondria-mediated apoptosis pathway.Our data suggested that the apoptosis mediated by ER stress palyed roles in the progress of PD, and the PI3K/Akt/GSK-3βsignal pathway was involved in ER stress induced by MPP+. This result clarified the ER stress-mediated neuron apoptosis induced by MPP+besides the mitochondria-mediated apoptosis pathway and further clarified the molecular mechanism on PD. GGA, an inducer of Trx, inhibited the ER stress induced by MPP+ in vitro. PI3K/Akt/GSK-3p involved in the ER stress is the potential therapy target of PD. In vivo, PTS, an inducer of Trx, inhibited the inflammation-or/and mitochondria-mediated neuron apoptosis through up-regulation of Trx. These results provided new theory foundation for finding the potential therapy target of PD. The Trx inducers, such as GGA, PTS, might be good therapeutic agent for PD. This study has the theory value and practical significance in the prevention and treatment of PD.