【作者】 刘林；

【导师】 王建秀；

【作者基本信息】 中南大学 ， 应用化学， 2011， 博士

【摘要】 神经退行性疾病是一类大脑和脊髓的神经元丧失的疾病。其中阿尔茨海默病(Alzheimer’s Disease, AD)和帕金森病(Parkinson’s Disease, PD)是全球发病率极高的老年神经退行性疾病,朊病毒病(Prion, PrP)是唯一具有传染性的神经退行性疾病。目前,有关诱导这类神经疾病发生的因素尚不明确。近年来的研究表明,除蛋白聚集之外,氧化应激效应被认为是导致神经退行性疾病的主要原因之一。由氧化还原活性的金属离子(如Cu(Ⅱ)、Fe(Ⅲ))催化产生的活性氧物质可以削弱线粒体的生理功能、破坏细胞膜等。基于上述考虑,本论文重点研究了氧化还原活性的金属离子在神经退行性疾病中所起的作用,探讨了金属离子与神经退行性疾病发病机理的关系,具体工作包括：一、β-淀粉样蛋白-Cu(Ⅱ)络合物(Aβ-Cu(Ⅱ))与AD的发生密切相关。本研究测定了Aβ-Cu(Ⅱ)氧化抗坏血酸(AA)的速率,提出了催化氧化的机制。所研究的Aβ包括Aβ(1-16)、Aβ(1-42)和Aβ(1-42)聚集体。催化AA氧化的次序依次为Aβ(1-16)-Cu(Ⅱ)>Aβ(1-42)-Cu(Ⅱ)>Aβ(1-42)聚集体-Cu(Ⅱ)>Aβ(1-42)-Cu(Ⅱ)聚集物。不同于游离的Cu(Ⅱ),Aβ-Cu(Ⅱ)络合物催化AA氧化的最终产物为H2O2。尽管Aβ-Cu(Ⅱ)络合物催化产生的H2O2的破坏能力远低于游离Cu(Ⅱ)催化产生的羟基自由基(OH·),但由于Aβ聚集体可以吸附在细胞膜表面,故Aβ-Cu(Ⅱ)催化产生的活性氧物质可能会对AD病人大脑的细胞膜造成破坏。二、多巴胺(DA)神经元的凋亡、铁离子的大量积累以及由α-突触核蛋白(alpha-synuclein,α-syn)聚集物所产生的路易小体是PD的三大病理学特征。α-syn可以与Cu(Ⅱ)形成α-syn-Cu(Ⅱ)络合物。本研究测定了α-syn-Cu(Ⅱ)络合物的氧化还原电位为0.018 V (vs. Ag/AgCl),该络合物可被生物体内常见还原性物种AA还原成α-syn-Cu(Ⅰ),而后者可被O2再氧化成α-syn-Cu(Ⅱ)。催化产生的H2O2可导致SY-HY5Y神经细胞的凋亡。因此,氧化应激效应可能是导致PD的重要原因之一,而α-syn-Cu(Ⅱ)在氧化应激过程中可能扮演了重要角色。三、Fe(Ⅲ)可以催化DA氧化生成黑色素。本研究表明三磷酸腺苷(ATP)可以与DA-Fe(Ⅲ)形成DA-Fe(Ⅲ)-ATP三元络合物,阻断Fe(Ⅲ)诱导DA氧化的途径,从而抑制了DA或DA-Fe(Ⅲ)的细胞毒性。正常情况下,ATP在细胞内的含量在1～10 mM之间,因此,DA与Fe(Ⅲ)在人体内完全可能以Ⅰ)A-Fe(Ⅲ)-ATP的形式存在。但在PD病人中,ATP的含量大大降低,因此,ATP的缺损可能是导致PD发病的重要原因之一。四、PrP蛋白可以络合Cu(Ⅱ),参与Cu(Ⅱ)的代谢过程。PrP蛋白的聚集是朊病毒的主要病理学特征。PrP的N-端有四个PHGGGWGQ(简称OP)单元,当[PrP]：[Cu(Ⅱ)]>1时,Cu(Ⅱ)与PrP中的四个PHGGGWGQ形成OP4-Cu(Ⅱ)络合物；当[PrP]：[Cu(Ⅱ)]<1时,Cu(Ⅱ)可以与PrP中的每个PHGGGWGQ形成OP4-Cu(Ⅱ)4络合物。本论文系统研究了OP4-Cu(Ⅱ)及OP4-Cu(Ⅱ)4络合物的氧化还原特性,计算了相应的OP4-Cu(Ⅰ)及OP4-Cu(Ⅰ)4络合物的络合系数并测试了其在氧气中的稳定性。结果表明OP4-Cu(Ⅱ)不能够催化AA的氧化,而OP4-Cu(Ⅱ)4可以催化AA氧化产生H2O2。阐述了PrP在铜离子代谢及信号传递等方面的作用原理。五、金属硫蛋白(MT)具有重金属解毒、清除自由基等作用。近年来,MT在神经系统中的生理作用引起了人们的广泛重视。我们研究了谷胱甘肽氧化还原电对诱导的MT的金属释放过程。采用超滤膜对谷胱甘肽氧化还原电对和MT的反应液进行分离,利用电化学和紫外可见光谱等手段检测了反应前后MT中金属离子的释放和巯基变化情况。该研究对阐述MT在环境毒理学方面的作用具有重要意义,为研究其它氧化还原电对(如α-syn-Cu(Ⅱ)、Aβ-Cu(Ⅱ))与MT的相互作用过程提供了一种简洁、高效的手段。更多还原

【Abstract】 Neurodegeneration is termed as the progressive loss of structures or functions of neurons, which eventually results in death of neurons. The main neurodegenerative diseases include Parkinson’s disease and Alzheimer’s disease. Prion disease is another neurodegenerative form with infectivity. Although many neurodegenerative diseases are manifested by the aggregation of the amyloidogenic proteins (e.g.,β-amyloid or Aβin Alzheimer’s disease (AD), alpha-synuclein (α-syn) in Parkinson’s disease (PD), and prion protein in Prion disease), oxidative stress has also been implicated in the pathogenesis of neurodegenerative diseases. Consequently, metal-induced oxidative stree processes have been an area under active pursuit. The implication of metal-induced oxidative stress is extremely broad, ranging from acceleration of the formation of reactive oxygen species (ROS) in the presence of redox-active metals such as copper and iron, mitochondria function impairment, and neuronal membrane damage through lipid peroxidation to depletion of vital intracellular species. Based on the above considerations, we have carried out the following studies:1. A forefront of the research on Alzheimer’s disease is the interaction of amyloid beta (Aβ) peptides with redox-active metal ions and the biological relevance of the Aβ-metal complexes to neuronal cell loss and homeostasis of essential metals and other cellular species. This work is concerned with the kinetic and mechanistic studies of the ascorbic acid (AA) oxidation by molecular oxygen, which is facilitated by Cu(Ⅱ) complexes with Aβ(1-16), Aβ(1-42), and aggregates of Aβ(1-42). The mechanism for the AA oxidation in which the oxidation states of the copper center in the Aβcomplexes alternate between +2 and +1 is proposed. The catalytic activity of Cu(Ⅱ) complex towards AA oxidation was found to decrease in the order of free Cu(Ⅱ)>Aβ(1-16)-Cu(Ⅱ)> Aβ(1-42)-Cu(Ⅱ)> Cu(Ⅱ) complexed by the Aβoligomer/fibril mixture> Cu(Ⅱ) in Aβfibrils. Unlike free Cu(Ⅱ), in the presence of AA, Aβ-Cu(Ⅱ) complexes facilitate the reduction of oxygen by producing H2O2 as a final product. Although Cu(Ⅱ) bound to oligomeric and fibrous Aβaggregates is less effective than free Cu(Ⅱ) and the monomeric Aβ-Cu(Ⅱ) complex in producing ROS, in vivo the Cu(Ⅱ)-containing Apoligomers and fibrils might be more biologically relevant given their strong association with cell membranes and the closer proximity of ROS to cell membranes.2. Parkinson’s disease, one of the leading neurodegenerative diseases, is characterized by the degeneration of dopaminergic neurons in the substantia nigra (SN), iron overload in SN and the presence of Lewy bodies of abnormal cytoplasmic inclusions that are mainly composed of alpha-synuclein (α-syn) in the dying neurons.α-syn is known to bind Cu(Ⅱ). The redox potential of theα-syn-Cu(Ⅱ) complex was determined to be 0.018 V (vs. Ag/AgCl). Furthermore, the Cu(Ⅱ) center can be readily reduced to Cu(Ⅰ), and possible reactions ofα-syn-Cu(Ⅱ) with cellular species (e.g., O2, ascorbic acid) have been investigated. In addition, the generated H2O2 was demonstrated to reduce the viability of the neuroblastoma SY-HY5Y cells. Our results thus suggest that oxidative stress is at least partially responsible for the loss of dopaminergic cells in PD brain and reveal the multifaceted role of theα-syn-Cu(Ⅱ) complex in oxidative stress associated with PD symptoms.3. Iron has been proven, in vitro, to catalyze the oxidation of DA by oxygen to generate neuromelanin. In this work, we investigated the formation of ternary complexes of DA-Fe(Ⅲ) with ATP. The results reveal that the ligation by ATP almost totally shut down the catalytic oxidation of DA via blockage of the oxygen access to the Fe(Ⅲ) center. In addition, ATP could decrease greatly the cell toxicity of DA or DA-Fe(Ⅲ) complex. In particular, ATP is the chemical energy for cell function and is abundant in cells (1～10 mM). It is possible that ATP-DA-Fe(Ⅲ) exists in vivo. In relevance to PD is the significant depletion of ATP in neuronal cell of PD afflicted brain. Therefore, the depletion of ATP might be a crucial cause for PD.4. A misfolded form of the prion protein (PrP) leads to the development of the prion diseases. The linkage between PrP and bioavailable Cu(Ⅱ) has been well established. PrP contains four highly conserved repeats of PHGGGWGQ octapeptide (OP) sequence within its N-terminal domain for Cu(Ⅱ) sequestration. In vitro studies have shown that when the concentration ratio between PrP and Cu(Ⅱ) is close to 1:1, three or four histidines in the OP4 domain coordinate the single Cu(Ⅱ) center (OP4-Cu(Ⅱ)). When the Cu(Ⅱ) concentration is four or more equivalents of Cu(Ⅱ), each of the four OPs binds one Cu(Ⅱ), giving rise to OP4-Cu(Ⅱ)4. We report here that the redox activity (cycling) of Cu(Ⅱ) is highly dependent on the mode in which Cu(Ⅱ) is coordinated within the PrP octarepeat domain. It is indicated that PrP possesses the unique ability to quench the copper redox activity in the form of OP4-Cu(Ⅱ) but promote the controlled H2O2 production in the form of OP4-Cu(Ⅱ)4. The results demonstrated herein are helpful for understanding the function of PrP in vivo.5. MTs paly an important role in regulation of essential metals, detoxification of heavy metals, and scavenging of free radicals. Recently, MTs have captured the attention of many researchists. In this study, modulation of metal release from MTs by the glutathione redox couple has been investigated. Upon separation of Zn2+ generated from the reaction mixture of MTs and glutathione redox couple (GSH/GSSG) with a centrifugal filter membrane, electrochemical technique was used in tandom with UV-vis spectrophotometry to characterize Zn2+ content and the concomitant conversion between free thiol groups and disulfide bonds. The new approach is demonstrated to be well suited for investigation of redox regulation of MT (e. g. MT/a-syn-Cu(Ⅱ) and MT/Aβ-Cu(Ⅱ)).更多还原