Upregulation of clusterin/apolipoprotein J in lactacystin-treated SH-SY5Y cells

Clusterin (apolipoprotein J) is a highly conserved, multifunctional, vertebrate glycoprotein. Several isoforms of clusterin have been described including the predominant secreted isoform (sCLU) and several nuclear isoforms (nCLU) associated with cell death. sCLU has been shown to bind a variety of partly unfolded, stressed proteins including those associated with Lewy bodies (LBs) in patients with Parkinson's disease (PD). The development of familial and sporadic PD has been associated with the ubiquitin-proteasome system (UPS) dysfunction and aberrant protein degradation. This suggests that failure of the UPS to degrade abnormal proteins may underlie nigral degeneration and LB formation in PD. The effects of toxin-mediated proteasomal impairment on changes in gene expression and cell viability were studied in differentiated SH-SY5Y cells. Clusterin expression was increased in cells exposed for 24 hr to the proteasomal inhibitor lactacystin (10 microM) as determined by gene microarray analysis. RT-PCR showed that sCLU, not nCLU, was the major clusterin isoform expressed in both control and lactacystin-treated cells. Western blot analysis identified statistically significant increases in sCLU in total cell lysates after 24 hr of lactacystin exposure and showed that sCLU fractionates with the endoplasmic reticulum. Time-course studies demonstrated that maximal decreases in proteasome activity (4 hr) preceded maximal increases in clusterin expression (24 hr). Together these data suggest that proteasome impairment results in the upregulation of sCLU in SH-SY5Y cells, supporting the hypothesis that the association of clusterin with LBs in PD may be related to UPS failure.     

蛋白酶体抑制剂对神经细胞的双重作用

目的探索蛋白酶体抑制剂在不同浓度时对多巴胺能神经元的作用及原因。方法用6-羟多巴(6-OHDA)50uM处理的人SH-SY5Y细胞作为细胞受损伤的模型,加用不同浓度的蛋白酶体抑制剂lactacystin,镜下细胞计数和SRB法测定细胞活力,平行对照组测定蛋白酶体活性。用选择性MEKl/2抑制剂PD98059验证蛋白酶体抑制剂是否通过MAPK途径起作用。结果蛋白酶体抑制剂lactacystin在0.1、0.25、0.5uM浓度时提高细胞存活率,在2、5uM时降低细胞存活率,相应的蛋白酶体活性分别是对照组的83.43%、73.84%、66.14%、24.11%、12.36%,加用PD98059后,0.25、0.5uMlactacystin的保护作用被阻断。结论蛋白酶体抑制剂在低浓度时对多巴胺能神经元有保护作用,高浓度时对多巴胺能神经元有毒性作用,这种不同作用的原因可能与蛋白酶体抑制的程度有关。蛋白酶体抑制剂的保护作用可能通过MAPK途径起作用。     

Ubiquitin proteasome system in Parkinson's disease: A keeper or a witness?

ObjectiveThe aim of this work was to evaluate the role of ubiquitin–proteasome system (UPS) on mitochondrial-driven alpha-synuclein (aSN) clearance in in vitro, ex vivo and in vivo Parkinson's disease (PD) cellular models.MethodWe used SH-SY5Y ndufa2 knock-down (KD) cells, PD cybrids and peripheral blood mononuclear cells (PBMC) from patients meeting the diagnostic criteria for PD. We quantified aSN aggregation, proteasome activity and protein ubiquitination levels. In PBMC of PD patient population we evaluated the aSN levels in the plasma and the influence of several demographic characteristics in the above mentioned determinations.ResultsWe found that ubiquitin-independent proteasome activity was up-regulated in SH-SY5Y ndufa2 KD cells while a downregulation was observed in PD cybrids and PBMC. Moreover, we observed an increase in protein ubiquitination that correlates with a decrease in ubiquitin-dependent proteasome activity. Accordingly, proteasome inhibition prevented ubiquitin-dependent aSN clearance. Ubiquitin-independent proteasome activity was positively correlated with ubiquitination in PBMC.We also report a negative correlation of chymotrypsin-like activity with age in control and late-onset PD groups. Total ubiquitin content is positively correlated with aSN oligomer levels, which leads to an age-dependent increase of aSN ubiquitination in LOPD. Moreover, aSN levels are increased in the plasma of PD patients.InterpretationaSN oligomers are ubiquitinated and we identified a ubiquitin-dependent clearance insufficiency with the accumulation of both aSN and ubiquitin. However, SH-SY5Y ndufa2 KD cells showed a significant up-regulation of ubiquitin-independent proteasomal enzymatic activity that could mean a cell rescue attempt. Moreover, we identified that UPS function is age-dependent in PBMC.     

6-羟基多巴胺诱导SH-SY5Y细胞帕金森病模型中Peroxiredoxin6表达的研究

目的 应用蛋白质组学技术研究6-羟基多巴胺(6-OHDA)诱导SH-SY5Y细胞帕金森病(PD)模型中Peroxiredoxin6(Prx6)的表达变化.方法 在建立6-OHDA诱导SH-SY5Y细胞帕金森病模型的基础上,分别提取6-OHDA实验组和对照组孵育24h的细胞总蛋白,应用荧光差异双向凝胶电泳(DIGE)技术获得蛋白点的差异表达信息,运用基质辅助激光解吸/电离飞行时间质谱仪(MALDI-TOF MS)鉴定出差异蛋白质.结果 DIGE分析发现实验组有一个表达明显上调的差异蛋白质点(P<0.01),经质谱分析鉴定确认为Prx6.结论 6-OHDA诱导SH-SY5Y细胞的帕金森病模型中Prx6表达上调,提示PD中有氧化应激存在,Prx6上调及氧化应激可能与PD的发病机制有关.     

6-羟基多巴胺诱导PC12细胞帕金森模型中GRP78表达的研究

目的 探讨神经毒素6-羟基多巴胺（6-OHDA）诱导PC12细胞的帕金森（PD）模型中GRP78的表达.方法 在建立6-OHDA诱导PC12细胞帕金森模型的基础上,MTT法测细胞存活率和Hoechst33342染色检测细胞凋亡.分别提取6-OHDA处理组和对照组细胞总蛋白,应用荧光差异凝胶电泳（Differential Gel Electrophoresis,DIGE）技术获得蛋白点的差异表达信息,运用MALDI-TOF质谱鉴定出差异蛋白质.结果 实验组细胞存活率为60%±4.8%,与对照组比较显著下降（P＜0.05）.荧光染色可见细胞核呈固缩状或碎裂状的典型的凋亡形态学改变.DIGE分析发现6-OHDA组表达明显增高的一个蛋白质点,经质谱分析鉴定确认为GRP78.结论 6-OHDA能够诱导PC12细胞凋亡,凋亡过程中GRP78表达增高,提示GRP78增高可能与PD的发病机制有关.     

Activation of Wnt/β-catenin Pathway by Exogenous Wnt1 Protects SH-SY5Y Cells Against 6-Hydroxydopamine Toxicity

Wnt1, initially described as a modulator of embryonic development, has recently been discovered to exert cytoprotective effects in cellular models of several diseases, including Parkinson's disease (PD). We, therefore, examined the neuroprotective effects of exogenous Wnt1 on dopaminergic SH-SY5Y cells treated with 6-hydroxydopamine (6-OHDA). Here, we show that 10–500 μM 6-OHDA treatment decreased cell viability and increased lactate dehydrogenase (LDH) leakage. SH-SY5Y cells treated with 100 μM 6-OHDA for 24 h showed reduced Wnt/β-catenin activity, decreased mitochondrial transmembrane potential, elevated levels of reactive oxidative species (ROS) and phosphatidylserine (PS) extraversion, increased levels of Chop and Bip/GRP78 and reduced level of p-Akt (Ser473). In contrast, exogenous Wnt1 attenuated 6-OHDA-induced changes. These results suggest that activation of the Wnt/β-catenin pathway by exogenous Wnt1 protects against 6-OHDA-induced changes by restoring mitochondria and endoplasmic reticulum (ER) function.     

Dopamine induces TNFalpha and TNF-R1 expression in SH-SY5Y human neuroblastoma cells

Cytotoxic concentrations of dopamine (100-500 microM DA) induce expression of tumour necrosis factor receptor-1 (TNF-R1) and tumour necrosis factor-alpha (TNFalpha) in SH-SY5Y human neuroblastoma cells. TNFalpha expression is dose-dependent and can also be detected after 6-hydroxydopamine (6-OHDA) or 1-methyl-4-phenylpyridinium iodide (MPP) treatment. The expression of TNF-R1 is also dose-dependent, but was not observed in 6-OHDA or MPP-treatment. Cells not expressing TNF-R1 were insensitive to TNFalpha, whereas those treated with DA showed a further decrease in viability when subsequently treated with TNFalpha. Thus, DA treatment confers sensitivity to TNFalpha. The decrease of cell viability caused by DA was in part prevented by neutralizing TNFalpha with anti-TNFalpha. As TNF-R1 is increased in substantia nigra of Parkinsonian brains, we suggest that nonvesiculated DA might also play a role in inducing TNF-R1 expression and predispose the neuron to the action of cytokines released in a microglia-mediated inflammatory response.     

Inhibitory effects of pesticides on proteasome activity: implication in Parkinson's disease

Epidemiological studies have suggested a correlation of pesticides and Parkinson's disease (PD) while genetic and biochemical studies have implicated the ubiquitin-proteasome system (UPS) in the pathogenesis of PD. In the present studies, we tested the hypothesis that pesticide exposure increases the risk of developing PD by inhibiting the UPS. The effects of pesticides on proteasome activity were examined in SK-N-MC neuroblastoma cells overexpressing a GFP-conjugated proteasome degradation signal, GFP(u). Six out of 25 representative pesticides, including rotenone, ziram, diethyldithiocarbamate, endosulfan, benomyl, and dieldrin, showed inhibitory effects on proteasome activities at low concentrations (10 nM to 10 microM). Unlike proteasome inhibitors, they did not inhibit 20 S proteasome activities in cell lysates. Except for rotenone, the other five pesticides did not induce significantly cellular oxidative stress. The cytotoxic effects of these pesticides were closely correlated with proteasome inhibition. Our results suggest proteasome inhibition as a potential mechanism for the epidemiological association of pesticides and PD.     

The pattern of neuronal loss and survival may reflect differential expression of proteasome activators in Parkinson's disease

The basis of neuronal vulnerability, degeneration, and sparing in PD are unknown, but there is increasing evidence to suggest that the ubiquitin‐proteasome system (UPS) plays an important role in the pathogenesis of the disorder. In this study, we employed an immunocytochemical approach to determine if the differential expression of key UPS components in various brain regions and cells might underlie the pattern of neuronal degeneration and survival seen in PD. We showed that the ubiquitin activating enzyme (E1), ubiquitin conjugating enzyme (E2), and 26/20S proteasome α‐ and β‐subunits, are abundantly expressed in the substantia nigra pars compacta (SNc) and in cultured dopaminergic neurons. Although the proteasome activator PA700 is expressed in the medial SNc, levels are low in the lateral region, and expression of the other proteasome activator, PA28, is near absent in the entire SNc. PA28 (but not PA700) was found to be poorly expressed in noradrenergic neurons in the locus coeruleus (LC) compared with adjacent cells in the mesencephalic nucleus. PA700 and PA28 are also poorly expressed in dopaminergic neurons compared with other cell types in culture. Inhibition of proteasomal function, generation of misfolded proteins, induction of oxidative stress or impairment of mitochondrial complex I activity, caused a compensatory upregulation in PA700 and PA28 in a variety of cells but not in dopaminergic neurons in culture. These findings are consistent with the demonstration that, in sporadic PD, proteasomal activity and levels of PA700/PA28 are reduced in the SNc butare markedly upregulated in regions/cells that are spared from the neurodegenerative process. Thus, the differential distribution and activity of proteasome activations could play a significant role in the pathogenesis of PD. Synapse 64:241–250, 2010. © 2009 Wiley‐Liss, Inc.     

D-beta-hydroxybutyrate inhibits the apoptosis of PC12 cells induced by 6-OHDA in relation to up-regulating the ratio of Bcl-2/Bax mRNA

D-beta-hydroxybutyrate (DbetaHB) is a predominant member of ketone bodies produced by hepatocytes and, to a lesser extent, by astrocytes. It is an alternative source of energy in the brain when glucose supply is depleted such as during starvation. It has been reported that ketone bodies could protect dopaminergic culture. However, the biological function of DbetaHB in Parkinson disease (PD) is still unclear. In the present work, we investigated the role of DbetaHB in protecting rat pheochromocytoma (PC12) cells from apoptosis induced by 6-Hydroxydopamine (6-OHDA). DbetaHB rescued PC12 cells from apoptotic death induced by 6-OHDA by MTT assay, acridine orange (AO) staining, terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling (TUNEL) staining and the activity of caspase-3. DbetaHB prevented the decrease of cell viability and the increase of caspase-3 activity induced by 6-OHDA in a dose-dependent manner in PC12 cells. AO and TUNEL staining showed that DbetaHB prevented the apoptosis of PC12 cells induced by 6-OHDA. The ratio of Bcl-2/Bax at mRNA levels, which regulates the apoptosis of PC12 cells when exposed to 6-OHDA, increased when DbetaHB was preincubated. The data showed that DbetaHB inhibited the apoptosis of PC12 cells induced by 6-OHDA in relation to up-regulating the ratio of Bcl-2/Bax mRNA.     

Rapid activation of ERK by 6-hydroxydopamine promotes survival of dopaminergic cells

Isoforms of the mitogen-activated protein kinase ERK have been implicated in both cell survival and cell death. In the present study we explored their role in cell viability in response to oxidative stress. Using the dopaminergic MN9D cell line, we determined that cell death occurred in a concentration-dependent manner after exposure to 6-hydroxydopamine (6-OHDA). The toxicity of 6-OHDA was mediated through generation of reactive oxygen species and was accompanied by a large increase in phosphorylated ERK1/2 but no significant increase in phosphorylated ERK5. 6-OHDA produced a distinct temporal pattern of ERK1/2 activation, with phosphorylated ERK1/2 peaks occurring after 10-15 min (25-fold increase) and 6-24 hr (13-fold increase). Inhibition of the early phosphorylated ERK1/2 peak with U0126 increased the generation of reactive oxygen species by 6-OHDA as well as 6-OHDA-induced toxicity, whereas inhibition of the late peak did not affect 6-OHDA-induced cell death. The time course of phosphorylation of the prosurvival protein CREB mimicked the temporal profile of ERK1/2 activation after 6-OHDA, and blocking the early phospho-ERK1/2 peak also abolished CREB activation. In contrast, activation of caspase-3 by 6-OHDA was delayed, occurring after about 6 hr, and this activation was increased by inhibition of the first phosphorylated ERK1/2 peak. These results suggest that the rapid activation of ERK1/2 in dopaminergic cells by oxidative stress serves as a self-protective response, reducing the content of reactive oxygen species and caspase-3 activity and increasing downstream ERK1/2 substrates.     

黄芪甲苷对PC12细胞氧化应激损伤的保护作用

目的 探讨黄芪甲苷对PC12细胞氧化应激损伤的作用。方法 体外培养PC12细胞，6-羟基多巴胺（6-OHDA）作用PC12细胞导致氧化应激损伤。根据细胞作用方法随机分为4组:①对照组，采用完全培养基正常培养；②6-OHDA组，给予终浓度为100 μmol/L的6-OHDA处理24 h；③低、中、高剂量黄芪甲苷组，分别给予终浓度为25、50、100 μmol/L的黄芪甲苷预处理24 h，然后给予终浓度为100 μmol/L 6-OHDA处理24 h；④AG490组，以20 μmol/L AG490（JAK2/STAT3信号通路抑制剂）预处理16 h，加入终浓度为100 μmol/L黄芪甲苷处理24 h，然后再给予终浓度为100 μmol/L 6-OHDA处理24 h。CCK8法检测细胞生存率，流式细胞仪检测细胞凋亡率，酶联免疫吸附实验法检测细胞上清液超氧化物歧化酶（SOD）和丙二醛（MDA）水平，免疫印迹法检测细胞p-JAK2和p-STAT3蛋白表达。结果 6-OHDA作用后，PC12细胞存活率明显降低，细胞凋亡率明显升高，细胞培养液SOD水平明显降低、MDA水平明显升高，细胞p-JAK2和p-STAT3蛋白表达水平明显降低；黄芪甲苷预处理明显逆转6-OHDA的作用，而且呈剂量依赖性；AG490预处理明显逆转黄芪甲苷的作用。结论 6-OHDA作用PC12细胞，可导致氧化应激损伤促使细胞凋亡；黄芪甲苷预处理能激活JAK2/STAT3信号通路，抑制6-OHDA对PC12细胞的损伤，对PC12细胞起保护作用。     

Knocking Out DJ-1 Attenuates Astrocytes Neuroprotection Against 6-Hydroxydopamine Toxicity

Astrocytes are the most abundant glial cell type in the brain. Impairment in astrocyte functions can critically influence neuronal survival and leads to neurodegeneration. Parkinson’s disease (PD) is a common neurodegenerative disorder, characterized by motor dysfunction that results from progressive neuronal loss. Astrocytic dysfunction was demonstrated in human samples and in experimental models of PD. Mutations in DJ-1 (PARK7) leading to loss of functional protein cause familial PD and enhance sensitivity to oxidative insults. Recently, an increase in DJ-1’s expression was found in reactive astrocytes in various neurodegenerative disorders. Here we show that lack of DJ-1 attenuates astrocytes’ ability to support neuronal cells, thereby leading to accelerated neuronal damage. DJ-1 knockout mice demonstrated increased vulnerability in vivo to 6-hydroxydopamine (6-OHDA) hemiparkinsonian PD model. Astrocytes isolated from DJ-1 knockout mice showed an inferior ability to protect human neuroblastoma cells against 6-OHDA insult both by co-culture and through their conditioned media, as compared to wild-type astrocytes. DJ-1 knockout astrocytes showed blunted ability to increase the expression of cellular protective mechanisms against oxidative stress mediated via Nrf-2 and HO-1 in response to exposure to 6-OHDA. These experiments demonstrated that lack of DJ-1 impairs astrocyte-mediated neuroprotection.     

Baculovirus p35 gene greatly enhances PC12 cell's resistance against oxidative stress

Oxidative stress is thought to be a major contributor to the progress of the Parkinson's Disease (PD) because of the high vulnerability of dopaminergic cells against oxidative stress. The present work demonstrates that with the expression of the baculovirus p35 gene, PC12 cells could gain a high resistance against oxidative toxicants, hydrogen peroxide (H(2)O(2)) and 6-hydroxydopamine (6-OHDA). The DNA fragmentation analysis showed that PC12 cells underwent apoptosis after exposure to H(2)O(2) or 6-OHDA, while PP35 cells, a p35-expressing PC12 cell line, did not. Flow cytometric analysis showed that treatment with 150 microM H(2)O(2) or 120 microM 6-OHDA for 24 h caused 52.86% or 66.36% apoptotic cell, respectively, in PC 12 cells, but only 4.26% or 5.80% in PP35 cells. The cell viability measured by 3-(4,5-dimethylthiazal-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) assay indicated that H(2)O(2) and 6-OHDA induced a dose-dependent cell death on PC12 cells that were greatly remitted on PP35 cells. The viability of PP35 cells was even stronger than that of PC12 cells protected by glial cell line deprived neurotrophic factor (GDNF). The surviving PP35 cells remained normal cell morphology and showed positive with tyrosine hydroxylase (TH) immunocytochemical staining. These results indicate that baculovirus p35 gene possesses remarkable ability to rescue PC12 cells from death in experimental paradigms associated with oxidative stress.     

Proteasome inhibition suppresses Schwann cell dedifferentiation in vitro and in vivo

The ubiquitin‐proteasome system (UPS), lysosomes, and autophagy are essential protein degradation systems for the regulation of a variety of cellular physiological events including the cellular response to injury. It has recently been reported that the UPS and autophagy mediate the axonal degeneration caused by traumatic insults and the retrieval of nerve growth factors. In the peripheral nerves, axonal degeneration after injury is accompanied by myelin degradation, which is tightly related to the reactive changes of Schwann cells called dedifferentiation. In this study, we examined the role of the UPS, lysosomal proteases, and autophagy in the early phase of Wallerian degeneration of injured peripheral nerves. We found that nerve injury induced an increase in the ubiquitin conjugation and lysosomal‐associated membrane protein‐1 expression within 1 day without any biochemical evidence for autophagy activation. Using an ex vivo explant culture of the sciatic nerve, we observed that inhibiting proteasomes or lysosomal serine proteases prevented myelin degradation, whereas this was not observed when inhibiting autophagy. Interestingly, proteasome inhibition, but not leupeptin, prevented Schwann cells from inducing dedifferentiation markers such as p75 nerve growth factor receptor and glial fibrillary acidic protein in vitro and in vivo. In addition, proteasome inhibitors induced cell cycle arrest and cellular process formation in cultured Schwann cells. Taken together, these findings indicate that the UPS plays a role in the phenotype changes of Schwann cells in response to nerve injury. © 2009 Wiley‐Liss, Inc.     

Overexpression of amyloid precursor protein is associated with degeneration, decreased viability, and increased damage caused by neurotoxins (prostaglandins A1 and E2, hydrogen peroxide, and nitric oxide) in differentiated neuroblastoma cells

Inflammatory reactions are considered one of the important etiologic factors in the pathogenesis of Alzheimer's disease (AD). Prostaglandins such as PGE2 and PGA1 and free radicals are some of the agents released during inflammatory reactions, and they are neurotoxic. The mechanisms of their action are not well understood. Increased levels of beta-amyloid fragments (Abeta40 and Abeta42), generated through cleavage of amyloid precursor protein (APP), oxidative stress, and proteasome inhibition, are also associated with neurodegeneration in AD brains. Therefore, we investigated the effect of PGs and oxidative stress on the degeneration and viability of cyclic AMP-induced differentiated NB cells overexpressing wild-type APP (NBP2-PN46) under the control of the CMV promotor in comparison with differentiated vector (NBP2-PN1) or parent (NBP2) control cells. Results showed that differentiated NBP2-PN46 cells exhibited enhanced spontaneous degeneration and decreased viability in comparison with differentiated control cells, without changing the level of Abeta40 and Abeta42. PGA1 or PGE2 treatment of differentiated cells caused increased degeneration and reduced viability in all three cell lines. These effects of PGs are not due to alterations in the levels of vector-derived APP mRNA or human APP holoprotein, secreted levels of Abeta40 and Abeta42, or proteasome activity. H2O2 or SIN-1 (an NO donor) treatment did not change vector-derived APP mRNA levels, but H2O2 reduced the level of human APP protein more than SIN-1. Furthermore, SIN-1 increased the secreted level of Abeta40, but not of Abeta42, whereas H2O2 had no effect on the level of secreted Abeta fragments. Both H2O2 and SIN-1 inhibited proteasome activity in the intact cells. The failure of neurotoxins to alter APP mRNA levels could be due to the fact that they do not affect CMV promoter activity. These results suggest that the mechanisms of action of PGs on neurodegeneration are different from those of H2O2 and SIN-1 and that the mechanisms of neurotoxicity of H2O2 and SIN-1 are, at least in part, different from each other.     

Ucf-101 protects in vivo and in vitro models of PD against 6-hydroxydopamine toxicity by alleviating endoplasmic reticulum stress via the Wnt/β-catenin pathway

The accumulation of α-syn which induce endoplasmic reticulum stress (ERS) and mediate various signaling pathways involved in DA neuronal degeneration, and the apoptosis of dopamine (DA) neurons are pathological markers of Parkinson’s disease (PD). High-temperature requirement protein A2 (HtrA2) is synthesized in the endoplasmic reticulum, and the expression level of HtrA2 can be upregulated by drugs or by unfolded proteins. Ucf-101 is a specific inhibitor of HtrA2, and studies have shown that Ucf-101 reduced apoptosis in PC12 cells. Our study showed that PC12 cells treated with 60 μM 6-OHDA for 24 h had significantly decreased cell viability compared to that of controls. A low concentration (2.5 μM) of Ucf-101 decreased the apoptosis rate of the PD cell model, but a high concentration (≥10 μM) increased the apoptosis rate, compared to that of controls. 6-OHDA upregulated the expression of HtrA2, α-syn, CHOP, Grp78 and active caspase-3 and reduced the levels of TH and XIAP. Ucf-101 reduced the level of ERS and apoptosis both in vivo and in vitro. The ratio of p-GSK3β (Tyr216 to Ser9) increased in PD rats. However, Ucf-101 down-regulated the activation of GSK3β and activated the Wnt/β-catenin pathway that was caused by 6-OHDA. Ucf-101 activated the Wnt/β-catenin pathway and significantly attenuated 6-OHDA-induced neurotoxicity, which was related to the inhibition of ERS and the reduction of the apoptosis rate of PC12 cells and DA neurons in the midbrain of PD rats. Ucf-101 has certain neuroprotective effects.     

Divalent metal transporter 1 up-regulation is involved in the 6-hydroxydopamine-induced ferrous iron influx

The reasons underlying the high iron content found in the substantia nigra (SN) of Parkinson's disease (PD) are largely unknown. We suppose, based on our previous studies, that the newly discovered iron transporter divalent metal transporter 1 (DMT1) might be involved in this SN iron accumulation process. To investigate this, we first observed the cellular expression of DMT1 in rat SN, both with the iron response element (+IRE) and without the IRE (-IRE) forms. The results showed that both forms of DMT1 were expressed on neurons, astrocytes, and microglia but not on oligodendrocytes. We further observed the relationship between the increased iron influx and DMT1 expression in 6-hydroxydopamine (6-OHDA)-treated C6 cells. 6-OHDA (10 micromol/liter) caused a significant increase in ferrous iron influx, with the increased expression of DMT1+IRE, both in protein and in mRNA levels, whereas no change was observed for DMT1-IRE. To clarify further that the increased expression of DMT1 was not due to the increased intracellular iron content, C6 cells were overloaded with ferric ammonium citrate (100 microg/ml). Decreased expression of both forms of DMT1 was observed. Our data suggest that DMT1 is highly expressed in rat SN in a cell-specific manner. Increased DMT1+IRE expression is the mechanism behind ferrous iron influx induced by 6-OHDA treatment in C6 cells. This may give some evidence for the involvement of DMT1 in the iron accumulation in PD.