| Parkinson’s disease(PD) is a common neurodegenerative disorder characterized by a progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta(SNpc) and decreased level of dopamine in the putamen of the dorsolateral striatum. The molecular mechanism of PD has been intensively investigated; however, it remains largely unknown.It has been reported that oxidative stress and endoplasmic reticulum(ER) stress are involved in the pathogenesis of PD. Accumulation of unfolded proteins in the ER activates a transcriptional induction of path-proteins in the ER termed the unfolded protein response(UPR). ER stress elicits two major cellular-protecting responses. One is the attenuation of protein synthesis, and the other is the upregulation of genes encoding chaperones that facilitate the protein folding process. A majority of the UPR signaling pathways included:(1) inositol-requiring enzyme1(IRE1) activation that leads to X-box binding protein1(XBP1) mRNA splicing;(2) activating transcription factor6(ATF6) release from the ER membrane, followed by nuclear translocation and transcriptional upregulation of ER response genes; and (3) protein kinase RNA-like ER kinase(PERK) activation and phosphorylation of eukaryotic translation initiation factor2a(eIF2a), which leads to the inhibition of translation initiation. With continued ER stress, terminal UPR events occur, such as release of ER-sequestered calcium, subsequent activations of multiple proapoptotic pathways including tumor necrosis factor receptor associated factor2(TRAF2), apoptosis signal regulating kinase-1(ASK1), and c-jun N-terminal kinase(JNK) that ultimately lead to cell death.Thioredoxin-1(Trx-1) is a multifunctional protein, which the molecular weight is12kDa, having a conserved active site sequence:-Cys-Gly-Pro-Cys-. Accumulating studies have shown that Trx-1plays a cytoprotective role against cellular damage and stressful perturbations. Trx-1has been shown to scavenge singlet oxygen and hydroxyl radicals. Trx-1can protect cells from hydrogen peroxide, UV irradiation and focal ischemic brain damage. Our previous study showed that Trx-1expression was significantly suppressed by MPP+and overexpression or administration of Trx-1attenuated MPP+-induced neurotoxicity in PC12cells. Overexpression of Trx-1suppressed the activation of caspase12, the maker of ER-mediated apoptosis However, the mechanism remains unknown.1-Methy-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) and its active metabolite,1-methyl-4-phenylpyridinium ion(MPP+), are commonly used to make experimental models of PD in vivo or in vitro.In the present study, we detected the effect of MPP+on the expressions of Glucose Regulated Protein78(GRP78), C/EBP homologous protein(CHOP), procaspase-12, IRE1α, TRAF2and JNK in PC12cells. Furthermore, we examined the regulation of Trx-1on GRP78, CHOP, procaspase-12, IRE la, TRAF2and JNK. The main results were as follows:(1) MPP+treatment induced ER stress in PC12cells. PC12cells were treated with0.3mM MPP+for different time(0,2,6,12and24h or0,15,30,45and60min). We detected the effect of MPP+on the expressions of GRP78, IRE la, TRAF2, CHOP, procaspase-12and JNK by western blot. Our results showed that MPP+induced the expressions of GRP78, IRE1a, TRAF2and CHOP and the cleavage of procaspase-12in a time-dependent manner. JNK was activated by MPP+in very short time. To confirm whether procaspase-12, the executor of ER pathway-mediated apoptosis, is activated by JNK, PC12cells were pretreated with SP600125, a JNK inhibitor, for30min followed by MPP+treatment for24h. The result showed that the activation of procaspase-12was suppressed by SP600125.(2) Trx-1regulated MPP+-induced cytotoxicity. MPP+treatment significantly decreased the viability of PC12cells. MPP+decreased Trx-1expression in a time-dependent manner in PC12cells and MPTP decreased Trx-1expression in SNpc of WT C57BL/6mice. To clarify if Trx-1plays an important role in regulating the toxic effect of MPP+. Trx-1expression was downregulated by siRNA and upregulated by transfecting GFP-tagged human Trx-1. The effect of Trx-1on the toxicity by MPP+in PC12cells was examined by MTT assay. No evident cytotoxicity was observed in PC12cells transfected with Trx-1siRNA or GFP-tagged human Trx-1. Downregulation of Trx-1expression by siRNA increased the susceptibility of PC12cells to MPP+-induced toxicity. On the contrary, Trx-1overexpression partly inhibited MPP+-induced cell toxicity. These results suggest that Trx-1plays an important role in regulating MPP+-induced toxicity.(3) Trx-1siRNA aggravated MPP+-induced ER stress. After PC12cells were transfected with Trx-1siRNA for24h followed by treatment with MPP+for24h or15min, we investigated the effect of downregulation of Trx-1expression on MPP+-induced ER stress. The ER stress associated GRP78was increased after Trx-1siRNA treatment followed by MPP+treatment, suggesting that Trx-1siRNA aggravated ER stress induced by MPP+. We also tested the effect of Trx-1siRNA on expressions of IRE la, TRAF2, CHOP, JNK and procaspase-12. Accordantly, Trx-1siRNA further increased the expressions of IRE la, TRAF2and CHOP and induced activations of JNK and procaspase-12. These data suggest that Trx-1siRNA aggravates MPP+-induced ER stress via inductions of IRE1α, TRAF2, JNK, caspase-12and CHOP.(4) Trx-1overexpression attenuated MPP+-induced ER stress. The PC12cells were transfected with GFP-tagged human Trx-1and then we examined the effect of Trx-1 overexpression on MPP+-induced ER stress. Our result showed that Trx-1overexpression inhibited the increasing level of GRP78by MPP+Moreover, MPP+-induced the expressions of IRE la. TRAF2and CHOP and the activations of JNK and procaspase-12were inhibited by overexpression of Trx-1. These data suggest that Trx-1overexpression attenuated MPP+-induced ER stress by inhibiting the expressions of IRE1α, TRAF2and CHOP and the activations of procaspase-12and JNK.(5) Trx-1inhibited the MPTP-induced ER stress in mice. We further examined whether Trx-1could inhibit the toxicity of MPTP in mice. WT and hTrx-1transgenic mice of MPTP groups were treated with MPTP-HC1(20mg/kg. intraperitoneal injection, twice daily for7days). WT and hTrx-1transgenic mice of control groups were administered with equivoluminal saline. Locomotor deficit is an important hallmark of PD. The motor activity in the WT mice treated by MPTP reduced significantly compared to WT mice treated with saline, in contrast, motor activity in the hTrx-1overexpression transgenic mice treated by MPTP did not reduce. In present study, we also examined the effect of Trx-1on the impairment of limb movements caused by MPTP. Overexpression of Trx-1ameliorated MPTP-induced impairment of limb movements by traction tests. We further examined the expression of tyrosine hydroxylase (TH) in SNpc to assess the loss of DA neurons by immunohistochemistry and Western blot analysis. TH expression in the SNpc was significantly decreased in WT mice after treatment of MPTP. However, overexpression of Trx-1restored downregulation of TH expression induced by MPTP in hTrx-1transgenic mice. These results indicate that overexpression of Trx-1prevents DA neurons from the neurotoxicity by MPTP. Concordant with the results in PC12cells, overexpression of Trx-1significantly inhibited the increase of GRP78in the SNpc in hTrx-1transgenic mice, suggesting that overexpression of Trx-1attenuated MPTP-induced ER stress in hTrx-1transgenic mice. Moreover, MPTP-induced expressions of IRE1α, TRAF2and CHOP and activations of JNK and procaspase-12were suppressed in the SNpc in hTrx-1transgenic mice. These data indicate Trx-1suppresses MPTP-induced neurotoxicity through inhibiting the expressions of IRE1α, TRAF2and CHOP and the activations of procaspase-12and JNK.In the present study, we presented systematically the molecular mechanism on Trx-1suppressing endoplasmic reticulum stress by MPP+/MPTP in vitro and in vivo. Based on these findings, we concluded that Trx-1played a neuroprotective role in PD by suppressing ER stress through inhibiting the activations of IRE1α, TRAF2, CHOP, JNK and procaspase-12. |
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