铁与帕金森病

帕金森病又称“震颤麻痹”,是(中脑)黑质———纹状体系统中多巴胺神经元的退行性变,导致神经传递物质多巴胺的生成和可利用率降低,引起中枢神经系统锥体外系慢性、进行性功能紊乱。其症状特点是:可发生一系列自发性行动障碍,如强直和震颤、步态困难、运动迟缓、姿势反射丧失以     

基于质谱的蛋白质组学技术在神经退行性疾病生物标志物研究中的应用

蛋白质组学是在整体水平上研究细胞、组织或生物体蛋白质组成及变化规律的科学.与传统的生物学研究相比,蛋白质组学具有快速、灵敏、高通量的优点.神经退行性疾病是一类由神经系统内特定神经细胞的进程性病变或丢失而导致神经功能障碍的疾病,严重危害人类健康.近年来,基于质谱的蛋白质组学技术在神经退行性疾病的研究中得到了广泛应用.本文简要介绍了蛋白质组学在样品分离、多肽定量、质谱检测及生物标志物临床验证等方面的技术发展,并结合实例综述了基于质谱的蛋白质组学在神经退行性疾病生物标志物发现与验证中的研究进展.     

人参活性成分在防治神经退行性疾病中应用的研究进展

神经退行性疾病是一类由神经系统内特定神经细胞的进程性病变或丢失而导致的神经功能障碍疾病，随着全球人口的老龄化，其发病率呈明显上升趋势。目前，此类疾病的发病机制尚不明确，临床上缺乏有效的治疗措施。人参含有多种活性成分，具有十分广泛的药理功效，在治疗神经退行性疾病中表现出巨大应用潜力。本文总结归纳了人参在神经退行性疾病防治中的活性成分及检测方法；然后，概述了人参在防治神经退行性疾病中的具体药理作用；最后，对其相关机制和通路进行了总结和评述。目前已经发现的具有神经退行性疾病的预防治疗活性的化学成分种类多，但其更多的活性成分及临床应用研究仍有待进一步深入研究。     

小分子细胞自噬诱导剂用于治疗神经退行性疾病

大量突变和错误折叠的蛋白质在细胞内聚集是神经退行性疾病产生的基础,研究发现一些小分子可以通过引起细胞自噬而降解细胞内聚集的突变蛋白,为治疗神经退行性疾病提供了新的方法,本文对神经退行性疾病的发病机理,细胞自噬的机理以及对神经退行性疾病的作用进行了综述。     

肠道菌群及其代谢产物与神经退行性疾病关系研究进展

神经退行性疾病是一种发生于中枢神经系统,具有高度致残、致死性的疾病,主要发病人群为中老年群体,目前该类疾病的发病机制尚不清楚,没有有效的治疗策略。随着我国老龄化程度的加深,神经退行性疾病对居民身体健康造成严重威胁。肠道菌群作为寄生在胃肠道中的微生物,与人体呈互利共生的关系,对生命健康起到至关重要的作用,神经退行性疾病的发展伴随着肠道菌群及其相关代谢产物的改变。文章综述了肠道菌群及其代谢产物与神经退行性疾病相互影响的机制,并探讨通过肠道菌群治疗神经退行性疾病的潜在价值,以期为神经退行性疾病的治疗提供新的研究方向。     

基于金属的神经退行性疾病治疗策略*

本文综述了金属离子在神经退行性疾病中的重要作用以及针对该类疾病金属治疗药物的研究进展。以老年痴呆症和帕金森氏症为代表,结合本课题组的初步研究结果,讨论了金属离子在蛋白质聚集与氧化应激反应中的重要作用,暗示金属螯合策略应成为治疗该类疾病的首选策略,并介绍了数种已用于或即将用于临床实验的金属螯合制剂;还介绍了烷基化神经退行性疾病相关蛋白的金属结合位点,可以显著抑制该蛋白质聚集体的形成和活性氧的产生,这可能是继螯合策略后一种更有发展潜力的神经退行性疾病治疗方法。     

Raman光谱研究铜锌超氧化物歧化酶及其金属取代衍生物的二级结构

本文测定了铜锌超氧化物歧化酶（Cu2Zn2SOD)及其金属取代衍生物Cu2Ni2SOD的Raman光谱,对图谱进行了归属,并定量测定了两种SOD的二级结构,同时对结构与活性的关系进行了讨论。     

基于FKBP12结构的药物发现: L-1,4-噻嗪-3-羧酸衍生物的设计、合成及其神经营养活性

以神经亲免素FKBP12为靶点, 基于FKBP12, FK506与Calcineurin的复合物晶体结构, 设计、合成和筛选能够特异地靶向FKBP12的只具有促神经生长作用的功能而不影响免疫系统的新结构神经退行性疾病治疗药物. 结果显示化合物N308作为一种促神经生长和保护的候选药物具进一步开发的前景.     

慢性胃炎与微量元素

近来,越来越多的学者对微量元素与慢性胃炎的关系进行了探讨。发现患慢性胃炎时,头发、血清、胃液的锌含量均明显降低,机体易感性增加,从而导致幽门螺旋杆菌感染加重。另外,锌是超氧化物歧化酶(SOD)的辅因子,由于血锌降低,红细胞SOD活力降低,清除氧自由基能力随之降低,加重细胞膜上脂质过氧化损伤;锌又是细胞呼吸酶及DNA多聚酶的辅因子,可促进损伤的胃黏膜再生。缺锌时可能影响这些酶活性,进而影响黏膜修复。慢性胃炎与微量元素@颜世铭     

铜转运蛋白与癌症的研究进展

铜是所有生物体中必不可少的一类具有氧化还原性质的过渡金属,也是催化抗氧化、铁稳态、细胞呼吸和各种生化过程关键酶的辅助因子.但是,由于其潜在的毒副作用,细胞内游离的铜离子必须受到严格的调控.铜离子平衡的失调会导致包括癌症和神经退行性疾病的发生和发展.例如,铜离子的积累可能会导致氧化应激的增加以及与大分子非特异性地结合.因此,大多数细胞进化出复杂的铜调节和转运系统,以满足细胞对铜离子的需求,同时减少其潜在的毒性.本文详细介绍了铜转运蛋白Ctr1、铜伴侣蛋白CCS、Atox1、COX17对于维持细胞内铜离子平衡的重要意义,同时着重介绍了铜伴侣蛋白在肿瘤中的研究进展以及相关药物的发展,为铜紊乱疾病治疗新靶点的发现和药物的研发提供了理论基础.     

Fluorescence assay for monitoring Zn-deficient superoxide dismutase in vitro

A method has been developed for selective detection of the zinc-deficient form of Cu, Zn superoxide dismutase (SOD1) in vitro. Zinc-deficient SOD1 mutants have been implicated in the death of motor neurons leading in amyotrophic lateral sclerosis (ALS or Lou Gerhig's disease). Thus, this method may have applicability for detecting zinc-deficient SOD1 mutants in human ALS patients samples as well as in a transgenic mouse model of ALS and in cultured motor neurons. We determined previously that structural analogs of 1,10 phenanthroline, which react specifically with Cu(I), react with the active Cu(I) of SOD1 when zinc is absent, but not when zinc is also bound, as evidenced by the fact that the reaction is inhibited by pretreatment of the enzyme with zinc. We report herein that bathocuproine, or its water-soluble derivative bathocuproine disulfonate, react with zinc-deficient SOD1 to form a complex which fluoresces at 734 nm when excited at 482 nm. Fluorescent intensity is concentration dependent, thus we propose to use fluorescent confocal microscopy to measure intracellular levels of zinc-deficient SOD1 in situ.     

Sensory involvement in the SOD1-G93A mouse model of amyotrophic lateral sclerosis

A subset of patients of amyotrophic lateral sclerosis (ALS) present with mutation of Cu/Zn superoxide dismutase 1 (SOD1), and such mutants caused an ALS-like disorder when expressed in rodents. These findings implicated SOD1 in ALS pathogenesis and made the transgenic animals a widely used ALS model. However, previous studies of these animals have focused largely on motor neuron damage. We report herein that the spinal cords of mice expressing a human SOD1 mutant (hSOD1-G93A), besides showing typical destruction of motor neurons and axons, exhibit significant damage in the sensory system, including Wallerian-like degeneration in axons of dorsal root and dorsal funiculus, and mitochondrial damage in dorsal root ganglia neurons. Thus, hSOD1-G93A mutation causes both motor and sensory neuropathies, and as such the disease developed in the transgenic mice very closely resembles human ALS.     

2-DE and MALDI-TOF-MS for a comparative analysis of proteins expressed in different cellular models of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegenerative disorder characterized by the selective loss of motor neurons from the spinal cord and brain. About 10% of ALS cases are familial (FALS), and in 20% of these cases the disease has been linked to mutations in the Cu,Zn-SOD1 gene. Although the molecular mechanisms causing these forms of ALS are still unclear, evidence has been provided that motor neurons injuries associated with mutant superoxide dismutase (SOD1)-related FALS result from a toxic gain-in-fuction of the mutated enzyme. To understand better the role of these mutations in the pathophysiology of FALS we have compared the pattern of proteins expressed in human neuroblastoma SH-SY5Y cell line with those of cell lines transfected with plasmids expressing the wild-type human SOD1 and the H46R and G93A mutants. 2-DE coupled to MALDI-TOF-MS were the proteomic tools used for identification of differentially expressed proteins. These included cytoskeletal proteins, proteins that regulate energetic metabolism and intracellular redox conditions, and the ubiquitin proteasome system. The proteomic approach allowed to expand the knowledge on the pattern of proteins, with altered expression, which we should focus on, for a better understanding of the possible mechanism involved in mutated-SOD1 toxicity. The cellular models considered in this work have also evidenced biochemical characteristics common to other SOD1-mutated cellular lines connected to the pathogenesis of ALS.     

Coupling of native IEF and extended X-ray absorption fine structure to characterize zinc-binding sites from pI isoforms of SOD1 and A4V pathogenic mutant

Extended X-ray absorption fine structure (EXAFS) has already provided high-resolution structures of metal-binding sites in a wide variety of metalloproteins. Usually, EXAFS is performed on purified metalloproteins either in solution or crystallized form but purification steps are prone to modify the metallation state of the protein. We developed a protocol to couple EXAFS analysis to metalloprotein separation using native gel electrophoresis. This coupling opens a large field of applications as metalloproteins can be characterized in their native state avoiding purification steps. Using native isoelectric focusing, the method enables the EXAFS analysis of metalloprotein pI isoforms. We applied this methodology to SOD1, wild-type, and Ala4Val mutant (A4V), a mutation found in amyotrophic lateral sclerosis (ALS) because decreased Zn affinity to SOD1 mutants is suggested to be involved in the pathogenesis of this neurodegenerative disease. We observed similar coordination structures for Zn in wild-type and mutant proteins, in all measured pI isoforms, demonstrating the feasibility of EXAFS on electrophoresis gels and suggesting that the Zn-binding site is not structurally modified in A4V SOD1 mutant.     

Intracellular amyloid beta interacts with SOD1 and impairs the enzymatic activity of SOD1: implications for the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease caused by the degeneration of motor neurons. Mutations in Cu/Zn superoxide dismutase (SOD1), including G93A, were reportedly linked to familial ALS. SOD1 is a key antioxidant enzyme, and is also one of the major targets for oxidative damage in the brains of patients suffering from Alzheimer''s disease (AD). Several lines of evidence suggest that intracellular amyloid beta (Aβ) is associated with the pathogenesis of AD. In this report we demonstrate that intracellular Aβ directly interacts with SOD1, and that this interaction decreases the enzymatic activity of the enzyme. We observed Aβ-SOD1 aggregates in the perinuclear region of H4 cells, and mapped the SOD1 binding region to Aβ amino acids 26-42. Interestingly, intracellular Aβ binds to the SOD1 G93A mutant with greater affinity than to wild-type SOD1. This resulted in considerably less mutant enzymatic activity. Our study implicates a potential role for Aβ in the development of ALS by interacting with the SOD1 G93A mutant.     

Optimizing separation efficiency of 2-DE procedures for visualization of different superoxide dismutase forms in a cellular model of amyotrophic lateral sclerosis

Neurodegenerative diseases such as Alzheimer disease (AD) and Parkinson disease (PD) have been associated with increased production of reactive oxygen species. In AD and PD patients, superoxide dismutase (SOD1) was also indicated as a major target of oxidative damage. In particular, in brain tissue of these patients, different SOD1 isoforms have been identified, although their functional role still remains to be elucidated. In the light of the possibility that different SOD1 entities could be expressed also in other neurodegenerative disorders, as a sort of unifying event with AD and PD, we have investigated amyotrophic lateral sclerosis (ALS) using human neuroblastoma SH-SY5Y cells with mutated SOD1 gene H46R as cellular model. 2-DE using a narrow-range IPG 4-7 strips in the first dimension and linear 15% SDS-PAGE in the second allowed to separate different SOD1 spots. MALDI-TOF MS and CapLC-MS/MS have been used for their complete identification. This is the first report in which the presence of SOD1 (iso) forms in a cellular model of ALS has been evidenced.     

Reduced net charge and heterogeneity of pI isoforms in familial amyotrophic lateral sclerosis mutants of copper/zinc superoxide dismutase

Familial cases of amyotrophic lateral sclerosis (fALS) are related to mutations of copper/zinc superoxide dismutase 1 (SOD1). Aggregation of SOD1 plays a central role in the pathogenesis of fALS and altered metallation of SOD1 mutants could be involved in this process. Using IEF gel electrophoresis under non‐denaturating conditions and particle induced X‐ray emission (PIXE) analysis, we studied the pI distribution and metallation status of fALS SOD1 mutants (A4V, G93A, D125H) compared to human wild‐type (hWT). SOD1 fALS mutants are characterized by a variable number of isoforms and higher pI compared to hWT, reflecting a reduced net charge that might explain their greater propensity to precipitation and aggregation. Cu/Zn ratios were slightly different for the predominant expressed isoforms of A4V, G93A, and D125H mutants compared to hWT. Differences in metallation were observed within each genotype, the more basic isoforms exhibiting lower Cu/Zn ratios. Moreover, we revealed the existence of a pool of fALS mutants SOD1 pI isoforms, slightly expressed (<10%), with a low Cu/Zn ratio and high pI values. Overall, IEF‐PIXE results suggest that the toxicity of SOD1 mutants should be studied at the pI isoform level with a particular attention to the species with the lowest charges.     

A Novel Anti-Inflammatory d-Peptide Inhibits Disease Phenotype Progression in an ALS Mouse Model

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease characterised by selective neuronal death in the brain stem and spinal cord. The cause is unknown, but an increasing amount of evidence has firmly certified that neuroinflammation plays a key role in ALS pathogenesis. Neuroinflammation is a pathological hallmark of several neurodegenerative disorders and has been implicated as driver of disease progression. Here, we describe a treatment study demonstrating the therapeutic potential of a tandem version of the well-known all-d-peptide RD2 (RD2RD2) in a transgenic mouse model of ALS (SOD1*G93A). Mice were treated intraperitoneally for four weeks with RD2RD2 vs. placebo. SOD1*G93A mice were tested longitudinally during treatment in various behavioural and motor coordination tests. Brain and spinal cord samples were investigated immunohistochemically for gliosis and neurodegeneration. RD2RD2 treatment in SOD1*G93A mice resulted not only in a reduction of activated astrocytes and microglia in both the brain stem and lumbar spinal cord, but also in a rescue of neurons in the motor cortex. RD2RD2 treatment was able to slow progression of the disease phenotype, especially the motor deficits, to an extent that during the four weeks treatment duration, no significant progression was observed in any of the motor experiments. Based on the presented results, we conclude that RD2RD2 is a potential therapeutic candidate against ALS.     

Characterization of superoxide dismutase 1 (SOD‐1) by electrospray ionization‐ion mobility mass spectrometry

In this paper, we report nano‐electrospray ionization‐ion mobility mass spectrometry (nano‐ESI‐IM‐MS) characterization of bovine superoxide dismutase (SOD‐1) and human SOD‐1 purified from erythrocytes. SOD‐1 aggregates are characteristic of amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disease in humans that could be triggered by dissociation of the native dimeric enzyme (Cu₂,Zn₂‐dimer SOD‐1). In contrast to ESI‐MS, nano‐ESI‐IM‐MS allowed an extra dimension for ion separation, yielding three‐way mass spectra (drift time, mass‐to‐charge ratio and intensity). Drift time provided valuable structural information related to ion size, which proved useful to differentiate between the dimeric and monomeric forms of SOD‐1 under non denaturing conditions. In order to obtain detailed structural information, including the most relevant post‐translational modifications, we evaluated several parameters of the IM method, such as sample composition (10 mM ammonium acetate, pH 7) and activation voltages (trap collision energy and cone voltage). Neutral pH and a careful selection of the most appropriate activation voltages were necessary to minimize dimer dissociation, although human enzyme resulted less prone to dissociation. Under optimum conditions, a comparison between monomer‐to‐dimer abundance ratios of two small sets of blood samples from healthy control and ALS patients demonstrated the presence of a higher relative abundance of Cu,Zn‐monomer SOD‐1 in patient samples. Copyright © 2013 John Wiley & Sons, Ltd.     

Parsing disease‐relevant protein modifications from epiphenomena: perspective on the structural basis of SOD1‐mediated ALS

Conformational change and modification of proteins are involved in many cellular functions. However, they can also have adverse effects that are implicated in numerous diseases. How structural change promotes disease is generally not well‐understood. This perspective illustrates how mass spectrometry (MS), followed by toxicological and epidemiological validation, can discover disease‐relevant structural changes and therapeutic strategies. We (with our collaborators) set out to characterize the structural and toxic consequences of disease‐associated mutations and post‐translational modifications (PTMs) of the cytosolic antioxidant protein Cu/Zn‐superoxide dismutase (SOD1). Previous genetic studies discovered >180 different mutations in the SOD1 gene that caused familial (inherited) amyotrophic lateral sclerosis (fALS). Using hydrogen–deuterium exchange with mass spectrometry, we determined that diverse disease‐associated SOD1 mutations cause a common structural defect – perturbation of the SOD1 electrostatic loop. X‐ray crystallographic studies had demonstrated that this leads to protein aggregation through a specific interaction between the electrostatic loop and an exposed beta‐barrel edge strand. Using epidemiology methods, we then determined that decreased SOD1 stability and increased protein aggregation are powerful risk factors for fALS progression, with a combined hazard ratio > 300 (for comparison, a lifetime of smoking is associated with a hazard ratio of ~15 for lung cancer). The resulting structural model of fALS etiology supported the hypothesis that some sporadic ALS (sALS, ~80% of ALS is not associated with a gene defect) could be caused by post‐translational protein modification of wild‐type SOD1. We developed immunocapture antibodies and high sensitivity top‐down MS methods and characterized PTMs of wild‐type SOD1 using human tissue samples. Using global hydrogen–deuterium exchange, X‐ray crystallography and neurotoxicology, we then characterized toxic and protective subsets of SOD1 PTMs. To cap this perspective, we present proof‐of‐concept that post‐translational modification can cause disease. We show that numerous mutations (N➔D; Q➔E), which result in the same chemical structure as the PTM deamidation, cause multiple diseases. Copyright © 2017 John Wiley & Sons, Ltd.