本文選題：鐵過載 + 神經(jīng)退行性疾病��； 參考：《中國科學(xué)院大學(xué)(中國科學(xué)院上海藥物研究所)》2017年博士論文

【摘要】：鐵離子是維持正常生命活動所必須的微量元素,參與了機體多項基礎(chǔ)生理過程。隨著年齡增長,鐵離子逐漸沉積于腦中與運動和認知相關(guān)的區(qū)域。越來越多的證據(jù)顯示,鐵代謝紊亂參與多種神經(jīng)退行性疾病的發(fā)生發(fā)展。傳統(tǒng)觀點認為,鐵離子主要通過氧化應(yīng)激或鐵響應(yīng)元件調(diào)節(jié)神經(jīng)元生理和病理功能,然而目前關(guān)于鐵過載所致神經(jīng)功能改變的其他調(diào)控機制研究仍然相當缺乏。在本研究中,我們首先建立原代神經(jīng)元鐵過載模型,確證鐵過載可引起神經(jīng)元活性氧增加、ATP生成減少以及突起網(wǎng)絡(luò)損傷。隨后,采用非標記質(zhì)譜定量分析鐵過載神經(jīng)元在蛋白水平的變化,從中尋找變化顯著且與神經(jīng)系統(tǒng)功能及疾病密切相關(guān)的候選蛋白進行深入研究,以探索鐵過載對相關(guān)蛋白的調(diào)控機制及對其功能水平的影響。質(zhì)譜共檢出336個差異表達蛋白。根據(jù)蛋白功能進行分類發(fā)現(xiàn),其中82個差異蛋白參與神經(jīng)系統(tǒng)發(fā)育和功能維持;按照疾病相關(guān)性進行分類表明,其中112個差異蛋白為神經(jīng)疾病相關(guān)蛋白。通過蛋白質(zhì)組學(xué)手段證實,包括鐵蛋白、轉(zhuǎn)鐵蛋白受體1和順烏頭酸酶1等七個鐵代謝相關(guān)蛋白受到鐵過載調(diào)控。本論文進一步從變化最顯著的前十個差異表達蛋白中選擇了與神經(jīng)退行性疾病密切相關(guān)的兩個蛋白——鈣同線蛋白1(Calsyntenin 1,Clstn1)和淀粉樣前體蛋白(Amyloid precursor protein,APP)進行了深入研究。本論文首先研究了鐵過載對神經(jīng)元Clstn1的調(diào)控作用。Clstn1被報道參與了神經(jīng)系統(tǒng)發(fā)育和學(xué)習記憶形成,在多種神經(jīng)退行性疾病中均檢測到該蛋白全長或其剪切片段的改變,提示Clstn1可能參與相關(guān)疾病的發(fā)生發(fā)展,然而迄今尚未有研究報道鐵過載與Clstn1的關(guān)聯(lián)性。本研究的質(zhì)譜結(jié)果顯示,Clstn1蛋白在所有差異蛋白中變化幅度位居第二,鐵過載處理組Clstn1蛋白水平為對照組的20.502倍,該結(jié)果通過蛋白質(zhì)免疫印跡得到進一步證實。本研究對神經(jīng)元Clstn1的轉(zhuǎn)錄、修飾、分泌以及剪切進行了系統(tǒng)研究,并發(fā)現(xiàn)鐵過載對Clstn1的轉(zhuǎn)錄水平、糖基化水平和剪切水平均無顯著影響。后續(xù)研究證實,鐵過載顯著抑制Clstn1蛋白N端剪切片段sAlcα的分泌,表現(xiàn)為細胞培養(yǎng)液中sAlcα水平顯著降低而細胞裂解液中sAlcα水平大幅增加,并且發(fā)現(xiàn)鐵過載處理組增加的sAlcα主要滯留于細胞膜上。本研究進一步通過表達純化重組sAlcα蛋白證實該片段可結(jié)合于原代神經(jīng)元表面,且外源添加salcα有效逆轉(zhuǎn)鐵過載所致神經(jīng)元突起損傷。上述研究首次證實鐵過載影響神經(jīng)元clstn1剪切片段salcα的分泌和細胞膜分布;此外,本研究提示分泌型的salcα具有神經(jīng)保護作用,而鐵過載導(dǎo)致的salcα分泌減少可能與鐵過載誘發(fā)的神經(jīng)元損傷相關(guān)。本論文其次研究了鐵過載對神經(jīng)元app的調(diào)控作用。app的突變是阿爾茨海默癥發(fā)病的重要遺傳因素之一,以往的研究報道提示鐵過載上調(diào)app表達。本論文的質(zhì)譜結(jié)果顯示,鐵過載顯著上調(diào)神經(jīng)元app蛋白水平。后續(xù)蛋白質(zhì)免疫印跡的結(jié)果表明,鐵過載并不影響神經(jīng)元app總蛋白水平,并提示組學(xué)發(fā)現(xiàn)的app表達差異主要是剪切形式app變化的結(jié)果。app主要通過兩條途徑進行剪切。正常情況下,大部分app通過非淀粉樣途徑剪切,先由α-分泌酶剪切得到sappα和羧基端片段(c-terminalfragment,ctf)ctfα,ctfα進一步由γ-分泌酶剪切得到p3和app胞內(nèi)結(jié)構(gòu)域(appintracellulardomain,aicd);其次,app亦可通過淀粉樣途徑先由β-分泌酶剪切得到sappβ和ctfβ,后者進一步被γ-分泌酶剪切得到aβ和aicd。本研究表明,鐵過載促進app非淀粉樣剪切,具體表現(xiàn)為鐵處理組ctfα顯著增加,但sappα分泌受阻,表現(xiàn)為sappα分泌減少而細胞分布增加;同時,鐵過載抑制神經(jīng)元app淀粉樣剪切,表現(xiàn)為ctfβ水平明顯降低,sappβ和aβ分泌亦顯著減少。進一步研究證實鐵過載處理的神經(jīng)元β-分泌酶(β-siteamyloidprecursorproteincleavingenzyme1,bace1)活性顯著降低,而其蛋白水平無明顯改變。分子水平的機制研究顯示,三價鐵劑檸檬酸鐵銨可直接抑制bace1酶活,也可通過促進sappα與bace1的相互作用間接抑制淀粉樣剪切。上述研究在原有報道的基礎(chǔ)上進一步系統(tǒng)研究了鐵過載對神經(jīng)元app非淀粉樣剪切和淀粉樣剪切的調(diào)控,并首次證實鐵過載對神經(jīng)元app剪切產(chǎn)物細胞內(nèi)外分布的影響。鑒于分泌形式sappα和aβ的重要生理和病理功能,鐵過載所引起的sappα和aβ分泌異常有可能是鐵過載所致神經(jīng)損傷的機制之一。綜上,本論文在蛋白質(zhì)組學(xué)的基礎(chǔ)上,探索了鐵過載對原代神經(jīng)元clstn1和app的調(diào)控作用。本研究首次揭示鐵過載與神經(jīng)元重要功能蛋白clstn1的關(guān)系,闡明了鐵過載對神經(jīng)元app代謝尤其是對其剪切產(chǎn)物細胞分布的調(diào)控作用,拓展了對鐵過載相關(guān)神經(jīng)損傷的認識,為鐵代謝紊亂參與中樞神經(jīng)系統(tǒng)疾病發(fā)生發(fā)展的這一理論提供支持,并為開發(fā)新型神經(jīng)系統(tǒng)鐵代謝紊亂干預(yù)藥物提供線索。
[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.

【學(xué)位授予單位】：中國科學(xué)院大學(xué)(中國科學(xué)院上海藥物研究所)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2017
【分類號】：R741

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