铁过载对神经元Clstn1和APP的调控作用研究

发布时间：2018-04-23 14:51

本文选题：铁过载 + 神经退行性疾病　；参考：《中国科学院大学(中国科学院上海药物研究所)》2017年博士论文

【摘要】：铁离子是维持正常生命活动所必须的微量元素,参与了机体多项基础生理过程。随着年龄增长,铁离子逐渐沉积于脑中与运动和认知相关的区域。越来越多的证据显示,铁代谢紊乱参与多种神经退行性疾病的发生发展。传统观点认为,铁离子主要通过氧化应激或铁响应元件调节神经元生理和病理功能,然而目前关于铁过载所致神经功能改变的其他调控机制研究仍然相当缺乏。在本研究中,我们首先建立原代神经元铁过载模型,确证铁过载可引起神经元活性氧增加、ATP生成减少以及突起网络损伤。随后,采用非标记质谱定量分析铁过载神经元在蛋白水平的变化,从中寻找变化显著且与神经系统功能及疾病密切相关的候选蛋白进行深入研究,以探索铁过载对相关蛋白的调控机制及对其功能水平的影响。质谱共检出336个差异表达蛋白。根据蛋白功能进行分类发现,其中82个差异蛋白参与神经系统发育和功能维持;按照疾病相关性进行分类表明,其中112个差异蛋白为神经疾病相关蛋白。通过蛋白质组学手段证实,包括铁蛋白、转铁蛋白受体1和顺乌头酸酶1等七个铁代谢相关蛋白受到铁过载调控。本论文进一步从变化最显著的前十个差异表达蛋白中选择了与神经退行性疾病密切相关的两个蛋白——钙同线蛋白1(Calsyntenin 1,Clstn1)和淀粉样前体蛋白(Amyloid precursor protein,APP)进行了深入研究。本论文首先研究了铁过载对神经元Clstn1的调控作用。Clstn1被报道参与了神经系统发育和学习记忆形成,在多种神经退行性疾病中均检测到该蛋白全长或其剪切片段的改变,提示Clstn1可能参与相关疾病的发生发展,然而迄今尚未有研究报道铁过载与Clstn1的关联性。本研究的质谱结果显示,Clstn1蛋白在所有差异蛋白中变化幅度位居第二,铁过载处理组Clstn1蛋白水平为对照组的20.502倍,该结果通过蛋白质免疫印迹得到进一步证实。本研究对神经元Clstn1的转录、修饰、分泌以及剪切进行了系统研究,并发现铁过载对Clstn1的转录水平、糖基化水平和剪切水平均无显著影响。后续研究证实,铁过载显著抑制Clstn1蛋白N端剪切片段sAlcα的分泌,表现为细胞培养液中sAlcα水平显著降低而细胞裂解液中sAlcα水平大幅增加,并且发现铁过载处理组增加的sAlcα主要滞留于细胞膜上。本研究进一步通过表达纯化重组sAlcα蛋白证实该片段可结合于原代神经元表面,且外源添加salcα有效逆转铁过载所致神经元突起损伤。上述研究首次证实铁过载影响神经元clstn1剪切片段salcα的分泌和细胞膜分布;此外,本研究提示分泌型的salcα具有神经保护作用,而铁过载导致的salcα分泌减少可能与铁过载诱发的神经元损伤相关。本论文其次研究了铁过载对神经元app的调控作用。app的突变是阿尔茨海默症发病的重要遗传因素之一,以往的研究报道提示铁过载上调app表达。本论文的质谱结果显示,铁过载显著上调神经元app蛋白水平。后续蛋白质免疫印迹的结果表明,铁过载并不影响神经元app总蛋白水平,并提示组学发现的app表达差异主要是剪切形式app变化的结果。app主要通过两条途径进行剪切。正常情况下,大部分app通过非淀粉样途径剪切,先由α-分泌酶剪切得到sappα和羧基端片段(c-terminalfragment,ctf)ctfα,ctfα进一步由γ-分泌酶剪切得到p3和app胞内结构域(appintracellulardomain,aicd);其次,app亦可通过淀粉样途径先由β-分泌酶剪切得到sappβ和ctfβ,后者进一步被γ-分泌酶剪切得到aβ和aicd。本研究表明,铁过载促进app非淀粉样剪切,具体表现为铁处理组ctfα显著增加,但sappα分泌受阻,表现为sappα分泌减少而细胞分布增加;同时,铁过载抑制神经元app淀粉样剪切,表现为ctfβ水平明显降低,sappβ和aβ分泌亦显著减少。进一步研究证实铁过载处理的神经元β-分泌酶(β-siteamyloidprecursorproteincleavingenzyme1,bace1)活性显著降低,而其蛋白水平无明显改变。分子水平的机制研究显示,三价铁剂柠檬酸铁铵可直接抑制bace1酶活,也可通过促进sappα与bace1的相互作用间接抑制淀粉样剪切。上述研究在原有报道的基础上进一步系统研究了铁过载对神经元app非淀粉样剪切和淀粉样剪切的调控,并首次证实铁过载对神经元app剪切产物细胞内外分布的影响。鉴于分泌形式sappα和aβ的重要生理和病理功能,铁过载所引起的sappα和aβ分泌异常有可能是铁过载所致神经损伤的机制之一。综上,本论文在蛋白质组学的基础上,探索了铁过载对原代神经元clstn1和app的调控作用。本研究首次揭示铁过载与神经元重要功能蛋白clstn1的关系,阐明了铁过载对神经元app代谢尤其是对其剪切产物细胞分布的调控作用,拓展了对铁过载相关神经损伤的认识,为铁代谢紊乱参与中枢神经系统疾病发生发展的这一理论提供支持,并为开发新型神经系统铁代谢紊乱干预药物提供线索。
[Abstract]:Iron ions are the trace elements necessary for the maintenance of normal life activities and participate in a number of basic physiological processes in the body. As age increases, iron ions are gradually deposited in the brain related areas associated with movement and cognition. More and more evidence shows that iron metabolic disorders are involved in the development of a variety of neurodegenerative diseases. Ions are mainly regulated by oxidative stress or iron response element to regulate the physiological and pathological functions of neurons. However, the research on other regulatory mechanisms of neural function changes caused by iron overload is still quite lacking. In this study, we first established the primary neuronal iron overload model, confirming that iron overload can cause the increase of reactive oxygen species in neurons, ATP Then, the changes in protein levels of iron overload neurons were quantitatively analyzed by non labelled mass spectrometry, and the candidate proteins with significant changes and closely related to the function of the nervous system and the disease were investigated in order to explore the regulation mechanism of iron overload and the level of its function. 336 differentially expressed proteins were detected by mass spectrometry. According to the protein function classification, 82 of them were involved in the development and function maintenance of the nervous system. According to the disease correlation, 112 of the difference proteins were neural disease related proteins. The protein was confirmed by proteomic methods, including ferritin and transferrin. Seven iron metabolism related proteins, such as receptor 1 and CIS cis 1, were regulated by iron overload. In this paper, two proteins closely related to neurodegenerative diseases were selected from the first ten differentially expressed proteins of the most significant changes - calcium co protein 1 (Calsyntenin 1, Clstn1) and amyloid precursor protein (Amyloid precursor prote). In, APP), this paper first studied the role of iron overload in the regulation of neuronal Clstn1..Clstn1 was reported to be involved in the development of the nervous system and the formation of learning and memory. In many neurodegenerative diseases, the whole length of the protein or the changes of its shear fragments were detected, suggesting that Clstn1 may be involved in the development of related diseases. However, the association between iron overload and Clstn1 has not been reported so far. The results of mass spectrometry in this study show that the variation of Clstn1 protein in all differential proteins is second, and the level of Clstn1 protein in the iron overload treatment group is 20.502 times that of the control group. This result is further confirmed by the immunofimting of protein. This study is a study on the neuron Cl. The transcriptional, modification, secretion and shearing of STN1 were systematically studied, and it was found that iron overload had no significant effect on Clstn1 transcriptional level, glycosylation level and shear level. Subsequent studies showed that iron overload significantly inhibited the secretion of sAlc alpha in the N terminal fragment of Clstn1 protein, which showed a significant decrease in the level of sAlc alpha in cell culture fluid. The level of sAlc alpha in the lysate increased significantly, and it was found that the increased sAlc alpha in the iron overload treatment group was mainly retained on the cell membrane. This study further confirmed that the fragment could be combined with the surface of the primary neuron by expressing the recombinant sAlc alpha protein, and the exogenous addition of SALC alpha was effective in reversing the neurite damage caused by iron overload. It is confirmed that iron overload affects the secretion of SALC alpha and the distribution of cell membrane in the clstn1 shear fragment of neurons. In addition, this study suggests that the secretory SALC alpha has a neuroprotective effect, and the decrease of SALC alpha secretion induced by iron overload may be related to the neuronal damage induced by iron overload. Secondly, the regulation of iron overload on the neuronal app is studied in this paper. The mutation of.App is one of the important genetic factors of Alzheimer's disease. Previous studies have suggested that iron overload is up to up APP expression. The results of mass spectrometry in this paper show that iron overload significantly up-regulated the level of APP protein in neurons. The difference in expression of app was mainly attributed to the changes in the shear form app..app was cut mainly through two pathways. Under normal conditions, most of app was cut by non amyloid pathway, Sapp alpha and carboxyl end fragments (c-terminalfragment, CTF) CTF alpha was obtained by alpha secretase shear, and CTF alpha was further cut by gamma secretase to P3. And app intracellular domain (appintracellulardomain, AICD); secondly, app can also be cut to Sapp beta and CTF beta by beta secretase in the amyloid pathway. The latter is further cut by gamma secretase by a beta and aicd. based studies. The results show that iron overload promotes app non amyloid shear, which is manifested as a significant increase in CTF alpha in the iron treatment group, but Sapp alpha secretion. Hindered, the expression of Sapp alpha secretion decreased and the cell distribution increased; at the same time, iron overload inhibited app amyloid shear, showing a significant decrease in CTF beta level and a significant decrease in Sapp beta and a beta secretion. Further studies have demonstrated that the activity of beta secretase (beta -siteamyloidprecursorproteincleavingenzyme1, BACE1) in iron overload treated neurons is significant. The molecular level of the molecular level studies showed that trivalent iron citrate can inhibit BACE1 enzyme activity directly and indirectly inhibit amyloid shear by promoting the interaction of Sapp alpha with BACE1. The effects of iron overload on the intracellular and external distribution of APP shear products were first demonstrated by the regulation of powder like shear and amyloid shear. In view of the important physiological and pathological functions of the secretory form of Sapp A and a beta, the abnormal secretion of Sapp A and a beta caused by iron overload may be one of the mechanisms of nerve damage induced by iron overload. On the basis of proteomics, the regulation of iron overload on primary neurons clstn1 and app was explored. The relationship between iron overload and neuron important functional protein clstn1 was revealed for the first time, and the regulation of iron overload on the metabolism of neuron APP metabolism, especially the distribution of its shear products, was elucidated, and the nerve loss related to iron overload was extended. The understanding of the injury provides support for the theory of iron metabolic disorders participating in the development of central nervous system diseases, and provides clues for the development of new types of nervous system iron metabolic disorders.

【学位授予单位】：中国科学院大学(中国科学院上海药物研究所)
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R741

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