神经退化性疾病生物能量代谢和氧化应激研究进展

衰老是导致几种常见的神经系统退化性疾病的主要危险因素,包括帕金森氏病（Ｐａｒｋｉｎｓｏｎ’ｓ ｄｉｓｅａｓｅ ＰＤ）,肌萎缩性侧索硬化（Ａｍｙｏｔｒｏｐｈｉｃ ｌａｔｅｒａｌ ｓｃｌｅｒｏｓｉｓ,ＡＬＳ）,早老性痴呆（Ａｌｚｈｅｉｍｅｒ’ｓ ｄｉｓｅａｓｅ ＡＤ）和亨廷顿氏病（Ｈｕｎｔｉｎｇｔｏｎ’ｓ ｄｉｓｅａｓｅ ＨＤ）。最近研究表明,神经退化性疾病涉及到线粒体缺陷,氧化应激等因素。在脑和其它组织中,老化可导致线粒体功能的损伤和氧化损伤的增强。ＰＤ病人中,已发现线粒体复合酶体Ⅰ活性降低,氧化损伤增加和抗氧化系统活性的改变。在几例家族性ＡＬＳ病人中,也发现Ｃｕ、Ｚｎ超氧化物歧化酶（Ｃｕ,Ｚｎ ＳＯＤ）基因的突变,导致Ｃｕ、Ｚｎ超氧化物歧化酶活性减低;散发的ＡＬＳ病人氧化损伤增高。在ＨＤ病人中已发现能量代谢异常     

Molecular implications of the human glutathione transferase A-4 gene (hGSTA4) polymorphisms in neurodegenerative diseases

Several lines of evidence, including an increased level of lipid peroxidation and the depletion of antioxidant molecules like as glutathione (GSH), indicate that oxidative stress plays an important role in the pathogenesis of several neurodegenerative disorders, such as Parkinson's disease (PD) and Alzheimer's disease (AD). We previously observed a significant increased level of DNA oxidative damage in peripheral blood cells of PD patients, with respect to controls, moreover, the activity of glutathione transferases (GSTs) measured in circulating plasma was higher in controls than in PD patients, suggesting a lower enzymatic protection in PD individuals. Among human GSTs, glutathione transferase A4-4 displays a high catalitic activity towards 4-hydroxy-2-nonenal (HNE), a marker of lipid peroxidation whose levels have been found significantly increased in the substantia nigra of Parkinson's disease patients, in respect to controls. We performed this study to determine the presence of allelic variants of functional interest in the coding region of the hGSTA4 gene on 60 PD patients and 60 healthy controls. By the combined effort of polymerase chain reaction/single-strand conformation polymorphisms (PCR/SSCP) techniques, we observed a single nucleotide polymorphism (SNP) G351A leading to the silent mutation Gln117Gln. No significant difference was observed in the distribution of this polymorphism between PD individuals and controls, moreover, we did not observe any other polymorphism in the hGSTA4 gene in our population. Further studies are required to test the role played by both factors regulating the level of the expression of the hGSTA4 gene and any possible post-translational modification of the protein, in the protection against oxidative damage in neuronal cells.     

Lipid Peroxidation and Antioxidant Enzyme Activities in Vascular and Alzheimer Dementias

It has been reported that oxidative stress may play a role in the pathogenesis of dementia of the Alzheimer type (AD) and the cerebral ischemia which causes vascular dementia (VD). We measured malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) activities in blood samples from patients with AD and VD and in healthy non-demented controls (CTR) which similar ages to the patients, in order to evaluate the degree of oxidative stress in patients with AD and VD. A sample of 150 subjects consisting of 50 patients with AD; 50 patients with VD and 50 CTR, aged from 65 to 85 years on, was analyzed. Most of the changes observed were in SOD activity and MDA levels. Catalase activity were least affected. Significant differences were observed in SOD and GR activity between males and females in CRT and in patients with AD, but not in VD. We have found a decrease in antioxidant enzymes activities (SOD, CAT, GPx and GR) in patients with AD and VD and significant differences were observed between CRT and AD patients for ages from 65 to 74, 75 to 84 and from 85 years to 94 years in SOD activity and MDA levels (P < 0.001). MDA levels increase with age in VD, AD and CTR. No significant variation with respect to sex were detected, but significant variations in MDA levels were detected between CRT and patients with VD and AD (P < 0.001). We conclude that oxidative stress plays an important role in the brain damage for both AD and VD, being observed higher levels of oxidative stress for AD that for VD.     

Parkinson's disease: a genetic perspective

Parkinson's disease (PD) is a common neurodegenerative disorder in the aging population, affecting more than 1% over the age of 65 years. Certain rare forms of the disease are monogenic, representing 5-10% of PD patients, but there is increasing evidence that multiple genetic risk factors are important also for common forms of PD. To date, 13 genetic loci, PARK1-13, have been suggested for rare forms of PD such as autosomal dominant and autosomal recessive PD. At six of these loci, genes have been identified and reported by several groups to carry mutations that are linked to affected family members. Genes in which mutations have been linked to familial PD have also been shown to be candidate genes for idiopathic forms of PD, as those same genes may also carry other mutations that merely increase the risk. Four of the PARK genes, SNCA at PARK1, UCH-L1 at PARK5, PINK1 at PARK6 and LRRK2 at PARK8, have been implicated in sporadic PD. There are indeed multiple genetic risk factors that combine in different ways to increase or decrease risk, and several of these need to be identified in order to begin unwinding the causative pathways leading to the different forms of PD. In this review, we present the molecular genetics of PD that are understood today, to help explain the pathways leading to neurodegeneration.     

Oxidative stress in patients with nongenital warts

Comparison of oxidative stress status between subjects with or without warts is absent in the literature. In this study, we evaluated 31 consecutive patients with warts (15 female, 16 male) and 36 control cases with no evidence of disease to determine the effects of oxidative stress in patients with warts. The patients were classified according to the wart type, duration, number, and location of lesions. We measured the indicators of oxidative stress such as catalase (CAT), glucose-6-phosphate dehydrogenase (G6PD), superoxide dismutase (SOD), and malondialdehyde (MDA) in the venous blood by spectrophotometry. There was a statistically significant increase in levels of CAT, G6PD, SOD activities and MDA in the patients with warts compared to the control group (P< .05). However, we could not define a statistically significant correlation between these increased enzyme activities and MDA levels and the type, the duration, the number, and the location of lesions. We determined possible suppression of T cells during oxidative stress that might have a negative effect on the prognosis of the disease. Therefore, we propose an argument for the appropriateness to give priority to immunomodulatory treatment alternatives instead of destructive methods in patients with demonstrated oxidative stress.     

The involvement of nitric oxide in maneb- and paraquat-induced oxidative stress in rat polymorphonuclear leukocytes

Oxidative stress plays a crucial role in the manifestations of maneb (MB) and paraquat (PQ)-induced toxicity including MB+PQ-induced Parkinson's disease (PD). Polymorphonuclear leukocytes (PMNs) actively participate in the oxidative stress-mediated inflammation and organ toxicity. The present study was undertaken to investigate the MB- and/or PQ-induced alterations in the indices of oxidative stress in rat PMNs. Animals were treated with or without MB and/or PQ in an exposure time dependent manner. In some sets of experiments, the animals were pre-treated with NOS inhibitors N(G)-nitro-L-arginine methyl ester (L-NAME) and aminoguanidine (AG) along with respective controls. A significant increase in myeloperoxidase (MPO), superoxide dismutase (SOD), nitric oxide, iNOS expression and lipid peroxidation (LPO) was observed in PMNs of MB- and/or PQ-treated animals, while catalase and glutathione S-transferase (GST) activities were attenuated. L-NAME and AG significantly reduced the augmented nitrite content, iNOS expression and MPO activity to control level in MB and PQ exposed animals. Although the augmented LPO was also reduced significantly in L-NAME and AG treated rat PMNs, the level was still higher as compared with controls. Alterations induced in SOD and GST activities were not affected by NOS inhibitors. The results thus suggest that MB and/or PQ induce iNOS-mediated nitric oxide production, which in turn increases MPO activity and lipid peroxidation, thereby oxidative stress.     

Primary Skin Fibroblasts as a Model of Parkinson's Disease

Parkinson's disease is the second most frequent neurodegenerative disorder. While most cases occur sporadic mutations in a growing number of genes including Parkin (PARK2) and PINK1 (PARK6) have been associated with the disease. Different animal models and cell models like patient skin fibroblasts and recombinant cell lines can be used as model systems for Parkinson's disease. Skin fibroblasts present a system with defined mutations and the cumulative cellular damage of the patients. PINK1 and Parkin genes show relevant expression levels in human fibroblasts and since both genes participate in stress response pathways, we believe fibroblasts advantageous in order to assess, e.g. the effect of stressors. Furthermore, since a bioenergetic deficit underlies early stage Parkinson's disease, while atrophy underlies later stages, the use of primary cells seems preferable over the use of tumor cell lines. The new option to use fibroblast-derived induced pluripotent stem cells redifferentiated into dopaminergic neurons is an additional benefit. However, the use of fibroblast has also some drawbacks. We have investigated PARK6 fibroblasts and they mirror closely the respiratory alterations, the expression profiles, the mitochondrial dynamics pathology and the vulnerability to proteasomal stress that has been documented in other model systems. Fibroblasts from patients with PARK2, PARK6, idiopathic Parkinson's disease, Alzheimer's disease, and spinocerebellar ataxia type 2 demonstrated a distinct and unique mRNA expression pattern of key genes in neurodegeneration. Thus, primary skin fibroblasts are a useful Parkinson's disease model, able to serve as a complement to animal mutants, transformed cell lines and patient tissues.     

The mitochondrial protease HtrA2 is regulated by Parkinson's disease-associated kinase PINK1

In mice, targeted deletion of the serine protease HtrA2 (also known as Omi) causes mitochondrial dysfunction leading to a neurodegenerative disorder with parkinsonian features. In humans, point mutations in HtrA2 are a susceptibility factor for Parkinson's disease (PARK13 locus). Mutations in PINK1, a putative mitochondrial protein kinase, are associated with the PARK6 autosomal recessive locus for susceptibility to early-onset Parkinson's disease. Here we determine that HtrA2 interacts with PINK1 and that both are components of the same stress-sensing pathway. HtrA2 is phosphorylated on activation of the p38 pathway, occurring in a PINK1-dependent manner at a residue adjacent to a position found mutated in patients with Parkinson's disease. HtrA2 phosphorylation is decreased in brains of patients with Parkinson's disease carrying mutations in PINK1. We suggest that PINK1-dependent phosphorylation of HtrA2 might modulate its proteolytic activity, thereby contributing to an increased resistance of cells to mitochondrial stress.     

Peripheral markers of oxidative stress in Parkinson's disease. The role of L-DOPA.

Oxidative stress plays a central role in the pathogenesis of Parkinson's disease (PD). L-DOPA, the gold standard in PD therapy, may paradoxically contribute to the progression of the disease because of its pro-oxidant properties. The issue, however, is controversial. In this study, we evaluated peripheral markers of oxidative stress in normal subjects, untreated PD patients and PD patients treated only with L-DOPA. We also measured platelet and plasma levels of L-DOPA, 3-O-methyldopa (the long-lasting metabolite of the drug), and dopamine. We found that isolated platelets of treated PD patients form higher amounts of 2,3-dihydroxybenzoate, an index of hydroxyl radical generation, than platelets of controls or untreated patients. In treated patients, platelet levels of 2,3-dihydroxybenzoate were positively correlated with platelet levels of L-DOPA, 3-O-methyldopa, and with the score of disease severity. Disease severity was correlated with platelet and plasma levels of L-DOPA, as well as with the daily intake of the drug. No significant differences in platelet levels of cytosolic and mitochondrial isoforms of the antioxidant enzyme superoxide dismutase were found between PD patients, either treated or untreated, and controls. Our findings lend further support to the hypothesis that L-DOPA might promote free radical formation in PD patients.     

Increased level of DJ-1 in the cerebrospinal fluids of sporadic Parkinson's disease

DJ-1 is an antioxidant protein whose loss of function by gene mutations has been linked to familial Parkinson's disease (PD). The main objective of the present study was to determine if this molecule was also involved in the pathogenesis of sporadic PD. For this purpose, quantitative immunoblot assays were performed to evaluate DJ-1 in cerebrospinal fluids (CSF) collected from sporadic PD patients (n=40) and non-PD controls (n=38). The results showed that the CSF DJ-1 levels in PD were significantly higher than those in non-PD controls. Especially, upregulation of CSF DJ-1 in the early stage of PD (Yahr I-II) were distinct compared to those in the advanced stage of PD (Yahr III-IV) and non-PD controls (p<0.001 by ANOVA with post hoc Bonferroni's test), suggesting a protective role of DJ-1 against oxidative stress during the early stage. Thus, we propose that CSF DJ-1 could be a possible biomarker for early sporadic PD.     

Effect of MPTP and l-Deprenyl on Antioxidant Enzymes and Lipid Peroxidation Levels in Mouse Brain

Abstract: Excessive free radical formation or antioxidant enzyme deficiency can result in oxidative stress, a mechanism proposed in the toxicity of MPTP and in the etiology of Parkinson's disease (PD). However, it is unclear if altered antioxidant enzyme activity is sufficient to increase lipid peroxidation in PD. We therefore investigated if MPTP can alter the activity of the antioxidant enzymes, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GSH-PX) and the level of lipid peroxidation. l -Deprenyl, prior to MPTP administration, is used to inhibit MPP⁺ formation and its subsequent effect on antioxidant enzymes. MPTP induced a threefold increase in SOD activity in the striatum of C57BL/6 mice. No parallel increase in GSH-PX or CAT activities was observed, while striatal lipid peroxidation decreased. At the level of the substantia nigra (SN), even though increases in CAT activity and reduction in SOD and GSH-PX activities were detected, lipid peroxidation was not altered. Interestingly, l -deprenyl induced similar changes in antioxidant enzymes and lipid peroxidation levels, as did MPTP. Taken together, these results suggest that an alteration in SOD activity, without compensatory increases in CAT or GSH-PX activities, is not sufficient to induce lipid peroxidation.     

Proteomics analysis identifies PARK7 as an important player for renal cell resistance and survival under oxidative stress

Renal fibrosis is a process that is characterized by declining excretory renal function. The molecular mechanisms of fibrosis are not fully understood. Oxidative stress pathways were reported to be involved in renal tissue deterioration and fibrosis progression. In order to identify new molecular targets associated with oxidative stress and renal fibrosis, differential proteomics analysis was performed with established renal cell lines (TK173 and HK-2). The cells were treated with oxidative stress triggering factor H(2)O(2) and the proteome alterations were investigated. Two dimensional protein maps were generated and differentially expressed proteins were processed and identified using mass spectrometry analysis combined with data base search. Interestingly the increase of ROS in the renal cell lines upon H(2)O(2) treatment was accompanied by alteration of a large number of proteins, which could be classified in three categories: the first category grouped the proteins that have been described to be involved in fibrogenesis (e.g. ACTA2, VIN, VIM, DES, KRT, COL1A1, COL4A1), the second category, which was more interesting involved proteins of the oxidative stress pathway (PRDX1, PRDX2, PRDX6, SOD, PARK7, HYOU1), which were highly up-regulated under oxidative stress, and the third category represented proteins, which are involved in different other metabolic pathways. Among the oxidative stress proteins the up-regulation of PARK7 was accompanied by a shift in the pI as a result of oxidation. Knockdown of PARK7 using siRNA led to significant reduction in renal cell viability under oxidative stress. Under H(2)O(2) treatment the PARK7 knockdown cells showed up to 80% decrease in cell viability and an increase in apoptosis compared to the controls. These results highlight for the first time the important role of PARK7 in oxidative stress resistance in renal cells.     

Effect of centrophenoxine against rotenone-induced oxidative stress in an animal model of Parkinson's disease

Oxidative stress has been implicated in the etiology of Parkinson's disease (PD). The important biochemical features of PD, being profound deficit in dopamine (DA) content, reduced glutathione (GSH), and enhanced lipid peroxidation (LPO) in dopaminergic (DA-ergic) neurons resulting in oxidative stress, mitochondrial dysfunction and apoptosis. Rotenone-induced neurotoxicity is a well acknowledged preclinical model for studying PD in rodents as it produces selective DA-ergic neuronal degeneration. In our previous study, we have shown that chronic administration of rotenone to rats is able to produce motor dysfunction, which increases progressively with rotenone treatment and centrophenoxine (CPH) co-treatment is able to attenuate these motor defects. The present study was carried out to evaluate the antioxidant potential of CPH against rotenone-induced oxidative stress. Chronic administration of rotenone to SD rats resulted in marked oxidative damage in the midbrain region compared to other regions of the brain and CPH co-treatment successfully attenuated most of these changes. CPH significantly attenuated rotenone-induced depletion in DA, GSH and increase in LPO levels. In addition, the drug prevented the increase in nitric oxide (NO) and citrulline levels and also enhanced the activity of catalase and superoxide dismutase (SOD). Histological analysis carried out using hematoxylin and eosin staining has indicated severe damage to mid brain in comparison to cortex and cerebellum and this damage is attenuated by CPH co-treatment. Our results strongly indicate the possible therapeutic potential of centrophenoxine as an antioxidant in Parkinson's disease and other movement disorders where oxidative stress is a key player in the disease process.     

Progress in the pathogenesis and genetics of Parkinson's disease

Recent progresses in the pathogenesis of sporadic Parkinson's disease (PD) and genetics of familial PD are reviewed. There are common molecular events between sporadic and familial PD, particularly between sporadic PD and PARK1-linked PD due to alpha-synuclein (SNCA) mutations. In sporadic form, interaction of genetic predisposition and environmental factors is probably a primary event inducing mitochondrial dysfunction and oxidative damage resulting in oligomer and aggregate formations of alpha-synuclein. In PARK1-linked PD, mutant alpha-synuclein proteins initiate the disease process as they have increased tendency for self-aggregation. As highly phosphorylated aggregated proteins are deposited in nigral neurons in PD, dysfunctions of proteolytic systems, i.e. the ubiquitin-proteasome system and autophagy-lysosomal pathway, seem to be contributing to the final neurodegenerative process. Studies on the molecular mechanisms of nigral neuronal death in familial forms of PD will contribute further on the understanding of the pathogenesis of sporadic PD.     

The Role of Oxidative Stress in Amyotrophic Lateral Sclerosis and Parkinson’s Disease

We examined oxidative stress markers of 31 patients suffering from ALS, 24 patients suffering from PD and 30 healthy subjects were included. We determined the plasma levels of lipid peroxidation (malondialdehyde, MDA), of protein oxidative lesions (plasma glutathione, carbonyls and thiols) and the activity of antioxidant enzymes i.e. erythrocyte Cu,Zn-Superoxide dismutase (SOD), Glutathione peroxidase (GSH-Px) and catalase. MDA and thiols were significantly different in both neurodegenerative diseases versus control population. A trend for an enhancement of oxidized glutathione was noted in ALS patients. Univariate analysis showed that SOD activity was significantly decreased in ALS and GSH-Px activity was decreased in PD. After adjusting for demographic parameters and enzyme cofactors, we could emphasize a compensatory increase of SOD activity in PD. Different antioxidant systems were not involved in the same way in ALS and PD, suggesting that oxidative stress may be a cause rather than a consequence of the neuronal death.     

The effects of Maras powder (smokeless tobacco) on oxidative stress in users

Maras Powder (MP) is a special kind of smokeless tobacco widely used in the southeast region of Turkey especially in Kahramanmaras and Gaziantep and other southeastern cities. It is obtained from a tobacco species, Nicotiana rustica L and ash of oak or grapevine wood. Tobacco may increase oxidative stress, which is related to the products of the oxygen metabolism taking place in all cells. Cellular antioxidants, e.g. catalase (CAT), superoxide dismutase (SOD) and glucose 6-phosphate dehydrogenase (G6PD) protect the cell against oxidative damage. An imbalance between the ROS and antioxidants in favour of ROS is described as oxidative stress. In this study, we aimed to investigate the effect of MP on antioxidant enzyme levels and lipid peroxidation. We measured malondialdehyde (MDA), CAT, SOD and G6PD levels in blood of 68 MP users and 30 healthy controls who did not use MP. CAT, SOD and G6PD levels were lower in MP users than in the controls. On the other hand, lipid peroxidation levels (MDA), one of the best indicators of cytological damage, was increased in MP users compared with the controls. The present study showed that MP increases oxidative stress, which may cause many systemic disorders, including arteriosclerosis.     

DJ-1 is a redox-dependent molecular chaperone that inhibits alpha-synuclein aggregate formation

Parkinson's disease (PD) pathology is characterized by the degeneration of midbrain dopamine neurons (DNs) ultimately leading to a progressive movement disorder in patients. The etiology of DN loss in sporadic PD is unknown, although it is hypothesized that aberrant protein aggregation and cellular oxidative stress may promote DN degeneration. Homozygous mutations in DJ-1 were recently described in two families with autosomal recessive inherited PD (Bonifati et al. 2003). In a companion article (Martinat et al. 2004), we show that mutations in DJ-1 alter the cellular response to oxidative stress and proteasomal inhibition. Here we show that DJ-1 functions as a redox-sensitive molecular chaperone that is activated in an oxidative cytoplasmic environment. We further demonstrate that DJ-1 chaperone activity in vivo extends to alpha-synuclein, a protein implicated in PD pathogenesis.     

Isofurans,but not F2-isoprostanes,are increased in the substantia nigra of patients with Parkinson's disease and with dementia with Lewy body disease

F2-isoprostanes (F2-IsoPs) are well-established sensitive and specific markers of oxidative stress in vivo. Isofurans (IsoFs) are also products of lipid peroxidation, but in contrast to F2-IsoPs, their formation is favored when oxygen tension is increased in vitro or in vivo. Mitochondrial dysfunction in Parkinson's disease (PD) may not only lead to oxidative damage to brain tissue but also potentially result in increased intracellular oxygen tension, thereby influencing relative concentrations of F2-IsoPs and IsoFs. In this study, we attempted to compare the levels of F2-IsoPs and IsoFs esterified in phospholipids in the substantia nigra (SN) from patients with PD to those of age-matched controls as well as patients with other neurodegenerative diseases, including dementia with Lewy body disease (DLB), multiple system atrophy (MSA), and Alzheimer's disease (AD). The results demonstrated that IsoFs but not F2-IsoPs in the SN of patients with PD and DLB were significantly higher than those of controls. Levels of IsoFs and F2-IsoPs in the SN of patients with MSA and AD were indistinguishable from those of age-matched controls. This preferential increase in IsoFs in the SN of patients with PD or DLB not only indicates a unique mode of oxidant injury in these two diseases but also suggests different underlying mechanisms of dopaminergic neurodegeneration in PD and DLB from those of MSA.