博碩士論文 108223028 詳細資訊



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姓名 戴鈺端(Yu-Duan Dai)  查詢紙本館藏   畢業系所 化學學系
論文名稱 研究二價金屬離子對TDP-43致病蛋白片段在細胞間傳遞之影響
(The impact of divalent metal ions on cell-to-cell transmission of pathological TDP-43 fragments)
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摘要(中) 二價金屬離子參與很多重要的生理機制，近年研究顯示二價金屬離子的失調與神經退化性疾病成因有關。在臨床上，有研究中發現ALS病人腦脊髓液中含有較高的金屬離子濃度；在實驗中，也有文獻指出過量的二價金屬離子會促進ALS病理相關蛋白TDP-43的錯位(Mislocalization)及堆疊。另外，在其他神經退化性疾病的研究中發現二價金屬離子的刺激會促進致病蛋白的傳遞，但二價金屬離子刺激是否會促使TDP-43蛋白釋出則尚未被探討。由於致病蛋白的傳遞，常常被視為細胞的一種保護機制，因此在本篇研究中，我們想要探討二價金屬離子的刺激是否會促使TDP-43蛋白產生致病的蛋白片段(C-TDP-43)，以及這些片段是否會藉由細胞間的傳遞，影響其他受體細胞。本篇研究發現二價金屬離子(鋅、鎂、鉛)會造成神經細胞內TDP-43錯位，其中以鉛離子最為顯著。接著，我們觀察到金屬離子刺激細胞後所收集的培養基，也會造成受體細胞內TDP-43的錯位，顯示培養基中可能含有致病蛋白。我們成功的從培養基收集到不同大小的囊泡，發現到其中包含不少的外泌體(Exosome)，而這些外泌體裡含有一定的TDP-43致病蛋白片段。藉由奈米粒子追蹤分析儀，我們進一步發現到鋅和鉛離子會促進細胞釋出更多的外泌體，配合電顯的結果，我們發現鉛離子刺激出的外泌體尺寸較小。而這些經由金屬離子刺激細胞後所釋出的外泌體除了會造成受體細胞內TDP-43蛋白錯位，也會降低其在細胞核內的流動性，甚至會促使免疫細胞發生發炎反應，顯示含有TDP-43致病蛋白片段的外泌體可能是造成細胞出現疾病相關表現型(Phenotype)的原因。進一步探討外泌體裡發現在鎂和鉛離子的刺激下會增加TDP-43致病蛋白片段的含量，並且鋅和鉛離子的刺激會促使其有較高的聚集程度。總結來說，我們的研究顯示二價金屬離子(鋅、鎂、鉛)的刺激，不只會造成神經供體細胞內TDP-43蛋白錯位，還會增進細胞釋放出更多的外泌體，其中以鉛離子最為顯著。而在鉛離子的刺激下，藉由外泌體釋放出的TDP-43致病蛋白片段顯示出較多的含量及較高的聚集程度，我們推測這是造成受體細胞產生較嚴重的疾病相關表現型的原因。因此我們調查的二價金屬離子中，鉛離子可能是TDP-43致病蛋白片段在細胞間傳遞的重要影響因子。
摘要(英) Divalent metal ions are participated in many physiological mechanisms. Current literature has shown the cause of neurodegenerative diseases is correlated with disrupted metal homeostasis. In clinical study, it was found that the concentration of metal ions in cerebrospinal fluid (CSF) in amyotrophic lateral sclerosis (ALS) patients is higher. In addition, divalent metal ions induce pathological TDP-43 formation in vivo. Though it is shown divalent metal ions enhance the transmission of pathological proteins, however, there is no direct evidence showing TDP-43 following the similar scenario. Since the cell-to-cell transmission of pathological TDP-43 is considered as a defensive mechanism, we would like to investigate whether if the stimulation of divalent metal ion enhanced the formation of pathological TDP-43 fragments (C-TDP-43) and transmitted C-TDP-43 into recipient cells. In this study, we found three divalent metal ions (Zn2+, Mg2+, Pb2+) cause TDP-43 mislocalization in neuronal cells. By characterizing the vesicles in cultured medium, we found that pathological TDP-43 fragments were included in exosomes. Furthermore, we found Zn2+ and Pb2+ induced more exosomes by nanoparticle tracking analysis. With the help of transmission electron microscopy, we also notice Pb2+ induced smaller exosomes than all the other cases. In addition, metal ion-induced exosomes not only cause TDP-43 mislocalization in recipient cells, but also decrease TDP-43 fluidity in nucleus. Even more, these exosomes induced inflammation response in microglia cells. Finally, we have shown that the stimulation Mg2+ and Pb2+ enhanced the content of C-TDP-43 in exosomes, while Zn2+ and Pb2+ enhanced the compactness of C-TDP-43 inside exosomes. Conclusively, we demonstrated three divalent metal ions (Zn2+, Mg2+, Pb2+) were able to cause TDP-43 mislocalization and induce exosome secretion. Within these cases, Pb2+ particularly induced significant C-TDP-43 fragments accumulation and more cell-to-cell transmission. Therefore, Pb2+ could play a dominate role on cell-to-cell transmission of pathological TDP-43 fragments.
關鍵字(中) ★ 肌萎縮性脊髓側索硬化症
★ TDP-43蛋白
★ 外泌體
★ 細胞間傳遞
★ 二價金屬離子		 關鍵字(英) ★ Amyotrophic lateral sclerosis
★ TDP-43
★ Exosome
★ Cell-to-cell transmission
★ Divalent metal ion
論文目次 摘要 i
Abstract ii
致謝 iii
目錄 v
圖目錄 vii
表目錄 viii
縮寫表 ix
第一章 緒論 1
1-1 TDP-43蛋白與神經退化性疾病之關聯性 1
1-2 肌萎縮性脊髓側索硬化症(ALS)中TDP-43蛋白質病學 2
1-3 致病性蛋白之傳遞途徑 3
1-4 胞外囊泡在神經退化性疾病中扮演之角色 5
1-5 金屬離子對肌萎縮性脊髓側索硬化症(ALS)與TDP-43蛋白之影響 6
1-6 研究動機與目的 8
第二章 實驗器材與方法 9
2-1 實驗儀器與藥品 9
2-1-1 實驗儀器 9
2-1-2 實驗藥品 10
2-2 細胞培養與繼代 13
2-3 細胞中TDP-43蛋白表達及二價金屬離子處理 14
2-4 細胞毒性測試 15
2-5 免疫螢光染色 16
2-6 條件培養基收集方法 18
2-7 外泌體之分離方法 19
2-7-1 超高速離心分離法 19
2-7-2 試劑分離法 20
2-8 奈米粒子追蹤分析儀 21
2-9 穿透式電子顯微鏡 22
2-10 光漂白螢光恢復術 23
2-11 西方墨點法 24
2-12 螢光相關光譜顯微鏡 26
2-13 蛋白質快速純化液相層析儀 27
2-14 窄縫式點雜交法 28
第三章 實驗結果 29
3-1 二價金屬離子對神經供體細胞的毒性以及內源性TDP-43蛋白錯位之測定 29
3-1-1 二價金屬離子對神經供體細胞毒性測試 29
3-1-2 二價金屬離子對神經供體細胞中內源性TDP-43蛋白錯位之測定 30
3-2 經由二價金屬離子刺激後神經供體細胞所傳遞的物質對受體細胞造成的影響 32
3-2-1 二價金屬離子刺激後所收集之條件培養基對神經受體細胞中內源性TDP-43蛋白錯位之測定 32
3-2-2 利用西方墨點法鑑定內源性TDP-43蛋白之傳遞途徑 34
3-2-3 優化收集外泌體總量及外泌體中TDP-43致病蛋白片段含量之條件 35
3-2-4 二價金屬離子刺激下神經供體細胞所分泌之外泌體對於受體細胞的影響 37
3-3 鑑定二價金屬離子刺激下神經供體細胞中所分泌的外泌體之特性分析 47
3-3-1 利用奈米粒子追蹤分析儀測定外泌體之濃度與粒徑大小分佈 47
3-3-2 利用穿透式電子顯微鏡觀察外泌體之微觀影像及粒徑大小 49
3-3-3 利用西方墨點法鑑定收集之樣品為外泌體 51
3-4 二價金屬離子刺激下神經供體細胞所分泌之外泌體中過表達eGFP-TDP-43蛋白組成變化 52
3-4-1 利用西方墨點法鑑定外泌體中過表達eGFP-TDP-43蛋白之組成變化 52
3-4-2 利用螢光相關光譜顯微鏡鑑定外泌體中TDP-43致病蛋白片段聚集化程度 53
第四章 實驗討論 55
第五章 結論 57
第六章 未來展望 58
參考文獻 60
參考文獻 1. Guo, J. L.; Lee, V. M., Cell-to-cell transmission of pathogenic proteins in neurodegenerative diseases. Nature medicine 2014, 20 (2), 130-138.
2. Westergard, T.; Jensen, B. K.; Wen, X.; Cai, J.; Kropf, E.; Iacovitti, L.; Pasinelli, P.; Trotti, D., Cell-to-cell transmission of dipeptide repeat proteins linked to C9orf72-ALS/FTD. Cell reports 2016, 17 (3), 645-652.
3. McAlary, L.; Plotkin, S. S.; Yerbury, J. J.; Cashman, N. R., Prion-like propagation of protein misfolding and aggregation in amyotrophic lateral sclerosis. Frontiers in molecular neuroscience 2019, 12, 262.
4. Prasad, A.; Bharathi, V.; Sivalingam, V.; Girdhar, A.; Patel, B. K., Molecular mechanisms of TDP-43 misfolding and pathology in amyotrophic lateral sclerosis. Frontiers in molecular neuroscience 2019, 12, 25.
5. Takahashi, R. H.; Nagao, T.; Gouras, G. K., Plaque formation and the intraneuronal accumulation of β‐amyloid in Alzheimer′s disease. Pathology international 2017, 67 (4), 185-193.
6. Stefanis, L., α-Synuclein in Parkinson′s disease. Cold Spring Harbor perspectives in medicine 2012, 2 (2), a009399.
7. Mackenzie, I. R.; Rademakers, R.; Neumann, M., TDP-43 and FUS in amyotrophic lateral sclerosis and frontotemporal dementia. The Lancet Neurology 2010, 9 (10), 995-1007.
8. Nonaka, T.; Arai, T.; Buratti, E.; Baralle, F. E.; Akiyama, H.; Hasegawa, M., Phosphorylated and ubiquitinated TDP‐43 pathological inclusions in ALS and FTLD‐U are recapitulated in SH‐SY5Y cells. FEBS letters 2009, 583 (2), 394-400.
9. Sackmann, C.; Sackmann, V.; Hallbeck, M., TDP-43 is efficiently transferred between neuron-like cells in a manner enhanced by preservation of its N-terminus but independent of extracellular vesicles. Frontiers in neuroscience 2020, 14, 540.
10. Wegorzewska, I.; Baloh, R. H., TDP-43-based animal models of neurodegeneration: new insights into ALS pathology and pathophysiology. Neurodegenerative Diseases 2011, 8 (4), 262-274.
11. Guo, W.; Chen, Y.; Zhou, X.; Kar, A.; Ray, P.; Chen, X.; Rao, E. J.; Yang, M.; Ye, H.; Zhu, L., An ALS-associated mutation affecting TDP-43 enhances protein aggregation, fibril formation and neurotoxicity. Nature structural & molecular biology 2011, 18 (7), 822.
12. Fang, Y.-S.; Tsai, K.-J.; Chang, Y.-J.; Kao, P.; Woods, R.; Kuo, P.-H.; Wu, C.-C.; Liao, J.-Y.; Chou, S.-C.; Lin, V., Full-length TDP-43 forms toxic amyloid oligomers that are present in frontotemporal lobar dementia-TDP patients. Nature communications 2014, 5 (1), 1-13.
13. Harischandra, D. S.; Rokad, D.; Neal, M. L.; Ghaisas, S.; Manne, S.; Sarkar, S.; Panicker, N.; Zenitsky, G.; Jin, H.; Lewis, M., Manganese promotes the aggregation and prion-like cell-to-cell exosomal transmission of α-synuclein. Science signaling 2019, 12 (572).
14. Iguchi, Y.; Eid, L.; Parent, M.; Soucy, G.; Bareil, C.; Riku, Y.; Kawai, K.; Takagi, S.; Yoshida, M.; Katsuno, M., Exosome secretion is a key pathway for clearance of pathological TDP-43. Brain 2016, 139 (12), 3187-3201.
15. Menon, A. V.; Chang, J.; Kim, J., Mechanisms of divalent metal toxicity in affective disorders. Toxicology 2016, 339, 58-72.
16. Roos, P. M.; Vesterberg, O.; Syversen, T.; Flaten, T. P.; Nordberg, M., Metal concentrations in cerebrospinal fluid and blood plasma from patients with amyotrophic lateral sclerosis. Biological trace element research 2013, 151 (2), 159-170.
17. Hozumi, I.; Hasegawa, T.; Honda, A.; Ozawa, K.; Hayashi, Y.; Hashimoto, K.; Yamada, M.; Koumura, A.; Sakurai, T.; Kimura, A., Patterns of levels of biological metals in CSF differ among neurodegenerative diseases. Journal of the neurological sciences 2011, 303 (1-2), 95-99.
18. Caragounis, A.; Price, K. A.; Soon, C. P.; Filiz, G.; Masters, C. L.; Li, Q.-X.; Crouch, P. J.; White, A. R., Zinc induces depletion and aggregation of endogenous TDP-43. Free Radical Biology and Medicine 2010, 48 (9), 1152-1161.
19. Garnier, C.; Devred, F.; Byrne, D.; Puppo, R.; Roman, A. Y.; Malesinski, S.; Golovin, A. V.; Lebrun, R.; Ninkina, N. N.; Tsvetkov, P. O., Zinc binding to RNA recognition motif of TDP-43 induces the formation of amyloid-like aggregates. Scientific reports 2017, 7 (1), 1-10.
20. Toffa, D. H.; Magnerou, M. A.; Kassab, A.; Djibo, F. H.; Sow, A. D., Can magnesium reduce central neurodegeneration in Alzheimer′s disease? Basic evidences and research needs. Neurochemistry international 2019, 126, 195-202.
21. Dang, T.; Lim, N.; Grubman, A.; Li, Q.-X.; Volitakis, I.; White, A. R.; Crouch, P., Increased metal content in the TDP-43A315T transgenic mouse model of frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Frontiers in aging neuroscience 2014, 6, 15.
22. Tesauro, M.; Bruschi, M.; Filippini, T.; D′Alfonso, S.; Mazzini, L.; Corrado, L.; Consonni, M.; Vinceti, M.; Fusi, P.; Urani, C., Metal (loid) s role in the pathogenesis of amyotrophic lateral sclerosis: Environmental, epidemiological, and genetic data. Environmental Research 2021, 192, 110292.
23. Ash, P. E.; Dhawan, U.; Boudeau, S.; Lei, S.; Carlomagno, Y.; Knobel, M.; Al Mohanna, L. F.; Boomhower, S. R.; Newland, M. C.; Sherr, D. H., Heavy metal neurotoxicants induce ALS-linked TDP-43 pathology. Toxicological Sciences 2019, 167 (1), 105-115.
24. Turchinovich, A.; Drapkina, O.; Tonevitsky, A., Transcriptome of extracellular vesicles: state-of-the-art. Frontiers in immunology 2019, 10, 202.
25. Jo, M.; Lee, S.; Jeon, Y.-M.; Kim, S.; Kwon, Y.; Kim, H.-J., The role of TDP-43 propagation in neurodegenerative diseases: integrating insights from clinical and experimental studies. Experimental & Molecular Medicine 2020, 52 (10), 1652-1662.
26. Yang, D.; Zhang, W.; Zhang, H.; Zhang, F.; Chen, L.; Ma, L.; Larcher, L. M.; Chen, S.; Liu, N.; Zhao, Q., Progress, opportunity, and perspective on exosome isolation-efforts for efficient exosome-based theranostics. Theranostics 2020, 10 (8), 3684.
27. Wang, C.; Duan, Y.; Duan, G.; Ma, Z.; Zhang, K.; Deng, X.; Qian, B.; Gu, J.; Wang, Q.; Zhang, S., Stress induces cytoprotective TDP-43 nuclear bodies through lncRNA NEAT1-promoted phase separation. bioRxiv 2019, 802058.
28. Ashford, B. A.; Boche, D.; Cooper‐Knock, J.; Heath, P. R.; Simpson, J. E.; Highley, J. R., Microglia in motor neuron disease. Neuropathology and Applied Neurobiology 2021, 47 (2), 179-197.
29. Salvi, V.; Sozio, F.; Sozzani, S.; Del Prete, A., Role of atypical chemokine receptors in microglial activation and polarization. Frontiers in aging neuroscience 2017, 9, 148.
30. Hessvik, N. P.; Llorente, A., Current knowledge on exosome biogenesis and release. Cellular and Molecular Life Sciences 2018, 75 (2), 193-208.
31. Fändrich, M., Oligomeric intermediates in amyloid formation: structure determination and mechanisms of toxicity. Journal of molecular biology 2012, 421 (4-5), 427-440.
32. Thompson, A. G.; Gray, E.; Mäger, I.; Thézénas, M.-L.; Charles, P. D.; Talbot, K.; Fischer, R.; Kessler, B. M.; Wood, M.; Turner, M. R., CSF extracellular vesicle proteomics demonstrates altered protein homeostasis in amyotrophic lateral sclerosis. Clinical Proteomics 2020, 17 (1), 1-12.
33. Cañas, J. A.; Sastre, B.; Rodrigo-Muñoz, J. M.; Del Pozo, V., Exosomes: a new approach to asthma pathology. Clinica Chimica Acta 2019, 495, 139-147.
指導教授 黃人則 謝發坤(Jen-Tse Huang Fa-Kuen Shieh) 審核日期 2021-8-19
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