Mechanism of 6-hydroxydopamine neurotoxicity: the role of NADPH oxidase and microglial activation in 6-hydroxydopamine-induced degeneration of dopaminergic neurons

Cell death induced by 6-hydroxydopamine (6-OHDA) is thought to be caused by reactive oxygen species (ROS) derived from 6-OHDA autooxidation and by a possible direct effect of 6-OHDA on the mitochondrial respiratory chain. However, the process has not been totally clarified. In rat primary mesencephalic cultures, we observed a significant increase in dopaminergic (DA) cell loss 24 h after administration of 6-OHDA (40 micromol/L) and a significant increase in NADPH subunit expression, microglial activation and superoxide anion/superoxide-derived ROS in DA cells that were decreased by the NADPH inhibitor apocynin. Low doses of 6-OHDA (10 micromol/L) did not induce a significant loss of DA cells or a significant increase in NADPH subunit expression, microglial activation or superoxide-derived ROS. However, treatment with the NADPH complex activator angiotensin II caused a significant increase in all the latter. Forty-eight hours after intrastriatal 6-OHDA injection in rats, there was still no loss of DA neurons although there was an increase in NADPH subunit expression and NADPH oxidase activity. The results suggest that in addition to the autooxidation-derived ROS and the inhibition of the mitochondrial respiratory chain, early microglial activation and NADPH oxidase-derived ROS act synergistically with 6-OHDA and constitute a relevant and early component of the 6-OHDA-induced cell death.     

Relationship between microglial activation and dopaminergic neuronal loss in the substantia nigra: a time course study in a 6-hydroxydopamine model of Parkinson's disease

Cellular interactions between activated microglia and degenerating neurons in in vivo models of Parkinson's disease are not well defined. This time course study assesses the dynamics of morphological and immunophenotypic properties of activated microglia in a 6-hydroxydopamine (6-OHDA) model of Parkinson's disease. Neurodegeneration in the substantia nigra pars compacta (SNc) was induced by unilateral injection of 6-OHDA into the medial forebrain bundle. Activated microglia, identified using monoclonal antibodies: clone of antibody that detects major histocompatibility complex (MHC) class II antigens (OX6) for MHC class II, clone of antibody that detects cell surface antigen-cluster of differentiation 11b – anti-complement receptor 3, a marker for complement receptor 3 and CD 68 for phagocytic activity. Activation of microglia in the lesioned SNc was rapid with cells possessing amoeboid or ramified morphology appeared on day 1, whilst antibody clone that detects macrophage-myeloid associated antigen immunoreactivity was observed at day 3 post-lesion when there was no apparent loss of tyrosine hydroxylase (TH)+ve dopaminergic (DA) SNc neurons. Thereafter, OX6 and antibody clone that detects macrophage-myeloid associated antigen activated microglia selectively adhered to degenerating axons, dendrites and apoptotic (caspase 3+ve) DA neurons in the SNc were observed at day 7. This was followed by progressive loss of TH+ve SNc neurons, with the peak of TH+ve cell loss (51%) being observed at day 9. This study suggests that activation of microglia precedes DA neuronal cell loss and neurons undergoing degeneration may be phagocytosed prematurely by phagocytic microglia.     

Drosophila Cu,Zn superoxide dismutase gene confers resistance to paraquat in Escherichia coli

Superoxide dismutase (SOD) is known to protect organisms from reactive oxygen metabolites. We tested the hypothesis that the Drosophila Cu,Zn SOD is capable of protecting Escherichia coli from oxidative damage caused by the herbicide paraquat. The Cu,Zn Sod gene of Drosophila sechellia was subcloned into pET-20b(+) expression vector. Transformation of E. coli with the constructed vector resulted in an overexpression of this eukaryotic superoxide dismutase, as evidenced by dramatically increased levels of the Cu,Zn SOD polypeptide in bacterial cytosolic extracts. As well, the E. coli transformants showed resistance to paraquat-mediated inhibition of growth and survival. Paraquat is known to promote formation of the superoxide radical anion inside cells and thus the data have been interpreted as indicating that the cloned superoxide dismutase provides protection in E. coli against damage attributable to free radicals.     

Prion-like activity of Cu/Zn superoxide dismutase

Neurodegenerative diseases belong to a larger group of protein misfolding disorders, known as proteinopathies. There is increasing experimental evidence implicating prion-like mechanisms in many common neurodegenerative disorders, including Alzheimer disease, Parkinson disease, the tauopathies, and amyotrophic lateral sclerosis (ALS), all of which feature the aberrant misfolding and aggregation of specific proteins. The prion paradigm provides a mechanism by which a mutant or wild-type protein can dominate pathogenesis through the initiation of self-propagating protein misfolding. ALS, a lethal disease characterized by progressive degeneration of motor neurons is understood as a classical proteinopathy; the disease is typified by the formation of inclusions consisting of aggregated protein within and around motor neurons that can contribute to neurotoxicity. It is well established that misfolded/oxidized SOD1 protein is highly toxic to motor neurons and plays a prominent role in the pathology of ALS. Recent work has identified propagated protein misfolding properties in both mutant and wild-type SOD1, which may provide the molecular basis for the clinically observed contiguous spread of the disease through the neuroaxis. In this review we examine the current state of knowledge regarding the prion-like properties of SOD1 and comment on its proposed mechanisms of intercellular transmission.     

Mitochondrial membrane depolarization and the selective death of dopaminergic neurons by rotenone: protective effect of coenzyme Q10

Chronic exposure to the pesticide rotenone induces a selective degeneration of nigrostriatal dopaminergic neurons and reproduces the features of Parkinson's disease in experimental animals. This action is thought to be relevant to its inhibition of the mitochondrial complex I, but the precise mechanism of this suppression in selective neuronal death is still elusive. Here we investigate the mechanism of dopaminergic neuronal death mediated by rotenone in primary rat mesencephalic neurons. Low concentrations of rotenone (5-10 nM) induce the selective death of dopaminergic neurons without significant toxic effects on other mesencephalic cells. This cell death was coincident with apoptotic events including capsase-3 activation, DNA fragmentation, and mitochondrial membrane depolarization. Pretreatment with coenzyme Q10, the electron transporter in the mitochondrial respiratory chain, remarkably reduced apoptosis as well as the mitochondrial depolarization induced by rotenone, but other free radical scavengers such as N-acetylcysteine, glutathione, and vitamin C did not. Furthermore, the selective neurotoxicity of rotenone was mimicked by the mitochondrial protonophore carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), a cyanide analog that effectively collapses a mitochondrial membrane potential. These data suggest that mitochondrial depolarization may play a crucial role in rotenone-induced selective apoptosis in rat primary dopaminergic neurons.     

No correlation between aggregates of Cu/Zn superoxide dismutase and cell death in familial amyotrophic lateral sclerosis

Aggregates of Cu/Zn superoxide dismutase (SOD) have been demonstrated in familial amyotrophic lateral sclerosis (FALS) and other neurodegenerative diseases; however, their role in disease pathogenesis is unclear. In this study, we investigated the presence of SOD aggregates in nerve growth factor (NGF)-differentiated PC12 cells and cell viability following: (i) transduction with replication-deficient recombinant adenoviruses (AdVs) expressing wild-type SOD (SODWT) or mutant SOD (SODMT, V148G or A4V); (ii) transfection of yellow fluorescent protein-tagged SODWT (SODWT-YFP) or SODMT (SODA4V-YFP, SODV148G-YFP). SOD aggregates were more prominent in cells following transduction of AdSODMT than AdSODWT and following treatment with H2O2, suggesting that mutant SOD leads to oxidation of cellular components. In addition, cells expressing SODMT-YFP yielded SOD aggregates that were significantly larger and more frequent than SOD aggregates in cells expressing SODWT-YFP. Proteasome inhibitors, but not cathepsin B inhibitors, increased aggregate formation but did not increase cell death. In addition, treatments that increased cell viability did not significantly decrease SOD aggregates. Taken together, our data demonstrate that there is no association between SOD aggregates and cell death in FALS.     

转铜/锌超氧化物歧化酶和抗坏血酸过氧化物酶基因马铃薯的耐氧化和耐盐性研究

高盐等逆境可以加剧植物体内活性氧的产生,进而引起植物细胞死亡。为开发抗逆境作物,以置于氧化诱导型启动子下定位于叶绿体的转铜/锌超氧化物歧化酶（Cu/ZnSOD）和抗坏血酸过氧化物酶基因(APX)马铃薯为材料,研究了其对MV和 NaCl所引起的氧化胁迫的耐受性。结果表明, MV胁迫下,转基因马铃薯叶片膜的相对电导率明显低于对照; NaCl胁迫下,其叶绿素含量高于对照。 在含NaCl 的培养基上,转基因幼苗生根率明显大于对照。另外,NaCl胁迫下转基因马铃薯叶片的SOD和APX酶活性显著高于对照,与其耐盐性的提高相一致。这些研究表明,转入Cu/ZnSOD和APX基因的马铃薯清除活性氧的能力增强,抗逆性得到提高。本实验采用氧化诱导型启动子调控下的SOD和APX两个基因协同作用,使外源基因只有在逆境胁迫时才特异性表达,增强转基因植株的抗逆效果,为培育抗逆经济作物开阔了思路。     

Heavy metal-mediated activation of the rat Cu/Zn superoxide dismutase gene via a metal-responsive element

The Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced in the course of biological oxidations. When placed under the control of the rat SOD1 gene promoter and transfected into human HepG2 hepatoma cells, the activity of a chloramphenicol acetyltransferase reporter gene was found to increase three- to four-fold in the presence of heavy metals (cadmium, zinc and copper). Functional analysis of mutant derivatives of the SOD1 gene promoter and the use of a heterologous promoter system confirmed that the induction of the SOD1 gene by metal ions requires a metal-responsive element (MRE) located between positions −273 and −267 (GCGCGCA). It was also shown by gel mobility shift assays that an MRE binding protein is induced by the exposure of the human liver cell line HepG2 to heavy metals. These results suggest that the MRE participates in the induction of the SOD1 gene by heavy metals. Received: 5 February 1999 / Accepted: 21 May 1999     

Fibroblast growth factor 9 prevents MPP+-induced death of dopaminergic neurons and is involved in melatonin neuroprotection in vivo and in vitro

Oxidative stress and down-regulated trophic factors are involved in the pathogenesis of nigrostriatal dopamine(DA)rgic neurodegeneration in Parkinson's disease. Fibroblast growth factor 9 (FGF9) is a survival factor for various cell types; however, the effect of FGF9 on DA neurons has not been studied. The antioxidant melatonin protects DA neurons against neurotoxicity. We used MPP⁺ to induce neuron death in vivo and in vitro and investigated the involvement of FGF9 in MPP⁺ intoxication and melatonin protection. We found that MPP⁺ in a dose- and time-dependent manner inhibited FGF9 mRNA and protein expression, and caused death in primary cortical neurons. Treating neurons in the substantia nigra and mesencephalic cell cultures with FGF9 protein inhibited the MPP⁺-induced cell death of DA neurons. Melatonin co-treatment attenuated MPP⁺-induced FGF9 down-regulation and DA neuronal apoptosis in vivo and in vitro . Co-treating DA neurons with melatonin and FGF9-neutralizing antibody prevented the protective effect of melatonin. In the absence of MPP⁺, the treatment of FGF9-neutralizing antibody-induced DA neuronal apoptosis whereas FGF9 protein reduced it indicating that endogenous FGF9 is a survival factor for DA neurons. We conclude that MPP⁺ down-regulates FGF9 expression to cause DA neuron death and that the prevention of FGF9 down-regulation is involved in melatonin-provided neuroprotection.     

Superoxide dismutase activity in organotypic midbrain-striatum co-cultures is associated with resistance of dopaminergic neurons to excitotoxicity

We have previously demonstrated that dopaminergic neurons in midbrain-striatum slice co-cultures are more resistant to NMDA cytotoxicity than the same neuronal population in single midbrain slice cultures. Here, we show that dopaminergic neurons in midbrain-striatum co-cultures also exhibit resistance to the cytotoxicity of nitric oxide donors, 2,2'-(hydroxynitrosohydrazono)bis-ethanamine (NOC-18) and 3-morpholinosydnonimine (SIN-1). The cytotoxicity of NMDA (30 microM) in single cultures was significantly attenuated by the nitric oxide synthase (NOS) inhibitor N(omega)-nitro-L-arginine (100 microM), whereas the toxicity in co-cultures was not. The levels of tyrosine residue nitration of tyrosine hydroxylase, a hallmark of the occurence of peroxynitrite anion in dopaminergic neurons, were lower in co-cultures than those in single cultures. Single cultures and co-cultures did not show appreciable differences in the number or distribution of NOS-containing neurons as assessed by NADPH diaphorase histochemistry. On the other hand, midbrain slices cultured with striatal slices showed higher levels of superoxide dismutase (SOD) activity as well as increased protein levels of Cu,Zn-SOD, than midbrain slices cultured alone. These results suggested that the generation of NO is involved in NMDA cytotoxicity on dopaminergic neurons, and that increased activity of SOD in co-cultures renders dopaminergic neurons resistant to NMDA cytotoxicity by preventing the formation of peroxynitrite.     

Initiation of Neuronal Damage by Complex I Deficiency and Oxidative Stress in Parkinson's Disease

Oxidative stress and partial deficiencies of mitochondrial complex I appear to be key factors in the pathogenesis of Parkinson's disease. They are interconnected; complex I inhibition results in an enhanced production of reactive oxygen species (ROS), which in turn will inhibit complex I. Partial inhibition of complex I in nerve terminals is sufficient for in situ mitochondria to generate more ROS. H2O2 plays a major role in inhibiting complex I as well as a key metabolic enzyme, alpha-ketoglutarate dehydrogenase. The vicious cycle resulting from partial inhibition of complex I and/or an inherently higher ROS production in dopaminergic neurons leads over time to excessive oxidative stress and ATP deficit that eventually will result in cell death in the nigro-striatal pathway.     

Regeneration of dopaminergic neurons after 6-hydroxydopamine-induced lesion in planarian brain

Planarians have robust regenerative ability dependent on X-ray-sensitive pluripotent stem cells, called neoblasts. Here, we report that planarians can regenerate dopaminergic neurons after selective degeneration of these neurons caused by treatment with a dopaminergic neurotoxin (6-hydroxydopamine; 6-OHDA). This suggests that planarians have a system to sense the degeneration of dopaminergic neurons and to recruit stem cells to produce dopaminergic neurons to recover brain morphology and function. We confirmed that X-ray-irradiated planarians do not regenerate brain dopaminergic neurons after 6-OHDA-induced lesioning, suggesting that newly generated dopaminergic neurons are indeed derived from pluripotent stem cells. However, we found that the majority of regenerated dopaminergic neurons were 5-bromo-2'-deoxyuridine-negative cells. Therefore, we carefully analyzed when proliferating stem cells became committed to become dopaminergic neurons during regeneration by a combination of 5-bromo-2'-deoxyuridine pulse-chase experiments, immunostaining/in situ hybridization, and 5-fluorouracil treatment. The results strongly suggested that G(2) -phase stem cells become committed to dopaminergic neurons in the mesenchymal space around the brain, after migration from the trunk region following S-phase. These new findings obtained from planarian regeneration provide hints about how to conduct cell-transplantation therapy for future regenerative medicine.     

Transcriptional activation of the human Cu/Zn superoxide dismutase gene by 2,3,7,8-tetrachlorodibenzo-p-dioxin through the xenobiotic-responsive element

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced during biological oxidations and environmental stress. Here we have investigated the effect of the most toxic dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), on the promoter of the Cu/Zn superoxide dismutase (SOD1) gene in HepG2 and HeLa cells using the chloramphenicol acetyltransferase gene as a reporter. The SOD1 promoter was activated 4- to 5-fold by TCDD treatment, in a concentration-dependent manner. In addition, the level of SOD1 mRNA and the enzymatic activity of the SOD1 protein were also enhanced on exposure of the cells to TCDD. Functional analysis of the regulatory region of the SOD1 gene by deletion and point mutation, and the use of a heterologous promoter system, showed that the SOD1 gene was transactivated by TCDD via the xenobiotic-responsive element (XRE). Gel mobility shift assays also confirmed the induction and the inducible binding of a receptor-ligand complex to XRE. Yeast cells that overexpress hSOD1 appeared to be more resistant to TCDD than the wild type. These results demonstrate that SOD1 is induced by TCDD via the XRE. The induced SOD1 may accelerate the neutralization of the superoxide anion and thus reduce the oxidative damage associated with dioxin toxicity.     

Xenobiotic-responsive element for the transcriptional activation of the rat Cu/Zn superoxide dismutase gene

Cu/Zn superoxide dismutase (SOD1) catalyzes the dismutation of superoxide radicals produced from biological oxidation and environmental stresses. A number of xenobiotics are toxic because they generate free radicals, such as superoxide and hydroxyl radicals, through a redox cycle. The xenobiotic responsive element (XRE) was located between the nt -268 and -262 region of the 5'-flanking sequence of the SOD1 gene. Functional analyses of this element by deletion, mutations, and heterologous promoter systems confirmed that the expression of the SOD1 gene was induced by a xenobiotic through the XRE. Gel mobility shift assays showed the xenobiotic inducible binding of the receptor-ligand complex to XRE. The cytoplasmic fraction from nontreated HepG2 cells also contains the factor as a cryptic form and prominently reveals its DNA-binding activity by incubation with betaNF in vitro. These results suggest that the XRE participates in the induction of the rat SOD1 gene by xenobiotics.     

Neurokinin-3 peptide instead of neurokinin-1 synergistically exacerbates kainic acid-inducing degeneration of neurons in the substantia nigra of mice

Neurokinin peptides neurokinin-1 (NK1), neurokinin-3 (NK3), and related receptors are abundantly distributed in the substantia nigra (SN) and evidenced by their possible roles in the Parkinson's disease. Differential intervention roles of NK3 on kainic acid (KA)-induced neuronal injury in the SN of mice were thus in vitro and in vivo studied by Fluoro-Jade C (FJC) staining, immunohistochemistry to tyrosine hydroxylase (TH) or phospho-NMDA receptor, and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. It revealed that (i) in contrast to protective effect of NK1 agonist septide that reduced FJC-positive degenerative neurons and lesion volume insulted by KA, NK3 agonist senktide significantly increased FJC-positive ones and lesion volume, and this effect was sufficiently reversed by NK3 antagonist SB218795; (ii) similarly, senktide reduced TH-positive neurons and this effect was antagonized by SB218795, but septide increased TH-positive ones; (iii) mechanistic observation showed differential influences of NK1 and NK3 agonists on phosphorylated-NMDA receptor subunit 1 (phospho-NMDAR1) and glial fibrillary acidic protein-expressing astrocytes, i.e. senktide enhanced of NMDA receptor phosphorylation and astrocyte activity, while septide reduced NMDA receptor phosphorylation and astrocytic response; (iv) cell culture further confirmed the exacerbating effect of NK3 agonist on KA-induced lesion of nigral cells or dopaminergic neurons, in which administration of senktide alone did not show significant cell toxicity. This study presents new evidence that neurokinin NK3 instead of NK1 synergistically exacerbate excitotoxic neuronal degeneration in the SN in a dose-dependent manner and possibly through modulation of NMDA receptor phosphorylation and astrocyte activity, suggesting their potential significance in novel pharmaceutical therapy against Parkinson's disease.     

A comprehensive study on long‐term injury to nigral dopaminergic neurons following intracerebroventricular injection of lipopolysaccharide in rats

Parkinson's disease (PD) is characterized by selective and progressive degeneration of dopaminergic neurons in the substantia nigra (SN). Lipopolysaccharide (LPS) can induce chronic inflammation and has been widely used to study the pathogenesis of PD. In this study, a single intracerebroventricular injection of LPS was used to induce neurotoxic effects on dopaminergic neurons in Sprague–Dawley rats. The long‐term neurotoxic effects of LPS were evaluated at different time points. Microglia were activated in the hippocampus and striatum at 4 weeks, and in the SN at 24 weeks. Astrocytes were activated in the hippocampus and nigrostriatal system at 2 and 24 weeks. The expression of brain‐derived neurotrophic factor in the SN increased at 4 weeks and decreased after 12 weeks, and tyrosine hydroxylase‐positive neurons in the SN were shown to have an atrophic appearance, with cell loss evident after 24 weeks. Phospho‐α‐synuclein expression, a reflection of parkinsonian pathogenesis, increased at 12 weeks, and peaked at 24 weeks. Abnormal motor behavior appeared at 16 weeks and lasted up to 48 weeks. These results indicate that microglia are activated for several months after a single, low dose injection of LPS, which eventually results in progressive and selective damage to dopaminergic neurons in the SN.