研究生: |
洪一汝 I-Ju Hung |
---|---|
論文名稱: |
於漸凍症病人的單核淋巴球細胞質中誘導出TDP-43蛋白質聚集體 Induction of Cytoplasmic TDP-43 Puncta in the Peripheral Blood Mononuclear Cell of ALS Patients |
指導教授: |
黃人則
Jen-Tse Huang 何明樺 Ming-Hua Ho |
口試委員: |
黃人則
Jen-Tse Huang 何明樺 Ming-Hua Ho 陳儀莊 Yi-juang Chern |
學位類別: |
碩士 Master |
系所名稱: |
工程學院 - 化學工程系 Department of Chemical Engineering |
論文出版年: | 2019 |
畢業學年度: | 107 |
語文別: | 中文 |
論文頁數: | 81 |
中文關鍵詞: | 漸凍症 、單核淋巴球 、TDP-43蛋白質 、細胞質聚集體 |
外文關鍵詞: | ALS, PBMC, TDP-43, cytoplasmic puncta |
相關次數: | 點閱:199 下載:0 |
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肌萎縮性脊髓側索硬化症 (Amyotrophic Lateral Sclerosis, ALS) ,又名漸凍症,為一種漸進性神經退化性疾病,患者中樞神經系統的運動神經細胞會快速進行性凋亡,在兩三年內逐漸出現神經肌肉萎縮、四肢癱瘓、言語吞嚥困難,最終導致呼吸衰竭死亡,然而漸凍症自早期發病到臨床確診,卻需耗費至少一年以上的時間。此外,研究證實在漸凍症患者之病理切片中,在其病變神經細胞中均有內含TAR DNA-binding protein (TDP-43) 蛋白質的異常堆積物,因此TDP-43蛋白質於細胞質之堆積物被視為其中一個漸凍症病理特徵,近年研究中更是發現在患者血液之單核淋巴球中,有極高比例之TDP-43蛋白質錯位,或許未來可將此現象運用於漸凍症之疾病檢測上。
本研究欲發展一個不具侵入性且簡單快速之檢測方法作為醫師臨床診斷漸凍症之輔助性工具,並建立專一辨識疾病之生物指標 (TDP-43蛋白質錯位及TDP-43蛋白細胞質聚集體)。首先,我們以人類淋巴母細胞株建立漸凍症細胞模擬模型並且測試光控胜肽 (JHR1及ADP1) 在此模擬模型中誘導TDP-43蛋白質錯位的能力,並藉此來增強病理特徵,實驗結果也顯示以上之光控胜肽均能成功誘導TDP-43蛋白質錯位。之後,我們於漸凍症患者之單核淋巴球中加入光控胜肽並利用以上的生物指標進行分析。實驗結果顯示以患者功能評定量表分數(ALSFRS-R) 進行分群後,加入ADP1後能有效提升TDP-43蛋白細胞質聚集體百分比。除此之外,在臨床因素關聯性分析中我們發現,加入ADP1及JHR1後,患者之單核淋巴球TDP-43蛋白錯位百分比與病程呈現負相關;加入ADP1後,患者之單核淋巴球TDP-43蛋白細胞質聚集體百分比則與ALSFRS-R呈現正相關。然而,加入ADP1或是JHR1,不論是TDP-43蛋白質錯位或是細胞質聚集體,均與患者年齡、性別均無關聯性。本研究發現結合ADP1及TDP-43蛋白細胞質聚集體有潛力可應用於單核淋巴球作為一個輔助診斷漸凍症的生物指標,希望可以幫助病友提早進入治療療程,減輕其生理及心理上的煎熬。
Amyotrophic lateral sclerosis (ALS), also known as neurodegenerative motor neuron disease, is characterized by the degeneration of both upper and lower motor neurons, which leads to muscle weakness and eventual paralysis. However, there is a delay of 9–15 months from the onset of patients’ symptoms to confirmation of the diagnosis due to the complexity and heterogeneous nature of ALS. The cytoplasmic accumulation of TAR DNA-binding protein (TDP-43) has been identified as one of the hallmarks of ALS. Moreover, studies have discovered TDP-43 mislocalized from nucleus to cytoplasm in the peripheral blood mononuclear cell (PBMC) of ALS patients, shedding light on the possibilities to apply this feature for ALS detection.
In this study, we attempt to develop a simple and noninvasive method using two pathological features, TDP-43 mislocalization and TDP-43 cytoplasmic puncta, as detection biomarkers for the detection of ALS. We have successfully established TDP-43 mislocalization model in normal Lymphoblastoid cell line (normal LCL) and applied 2 photocontrollable probes, JHR1 and ADP1, to test their ability in enhancing the pathological features in normal LCL. Results showed both probes were able to induce TDP-43 mislocalization in cell. Therefore, we further applied JHR1 and ADP1 in ALS PBMC samples and evaluated their ability to enhance TDP-43 mislocalization and TDP-43 cytoplasmic puncta. Our results showed treatment of ADP1 increased the formation of TDP-43 cytoplasmic puncta. In addition, we found significant positive correlation between TDP-43 cytoplasmic puncta and ALSFRS-R score under ADP1 treatment. Also, ADP1 treatment also lead to significant negative correlation between TDP-43 cytoplasmic puncta and disease duration. TDP-43 mislocalization or TDP-43 cytoplasmic puncta were not correlated with either age or gender. To conclude, we found combining ADP1 and TDP-43 cytoplasmic puncta as a biomarker in ALS PBMC may serve as a useful platform in ALS detection. We truly hope this platform may assist clinicians and alleviate the sufferings in physiologically and psychologically of the patients in the future.
1. Brown, R. H.; Al-Chalabi, A., Amyotrophic Lateral Sclerosis. N Engl J Med 2017, 377 (2), 162-172.
2. Kiernan, M. C.; Vucic, S.; Cheah, B. C.; Turner, M. R.; Eisen, A.; Hardiman, O.; Burrell, J. R.; Zoing, M. C., Amyotrophic lateral sclerosis. The Lancet 2011, 377 (9769), 942-955.
3. Chiò, A., ISIS Survey: an international study on the diagnostic process and its implications in amyotrophic lateral sclerosis. Journal of Neurology 1999, 246 (15), s001-s005.
4. Hardiman, O.; van den Berg, L. H.; Kiernan, M. C., Clinical diagnosis and management of amyotrophic lateral sclerosis. Nat Rev Neurol 2011, 7 (11), 639-49.
5. Zarei, S.; Carr, K.; Reiley, L.; Diaz, K.; Guerra, O.; Altamirano, P. F.; Pagani, W.; Lodin, D.; Orozco, G.; Chinea, A., A comprehensive review of amyotrophic lateral sclerosis. Surg Neurol Int 2015, 6, 171.
6. Daube, J. R., Electrodiagnostic studies in amyotrophic lateral sclerosis and other motor neuron disorders. Muscle & Nerve 2000, 23 (10), 1488-1502.
7. Traynor, B. J.; Codd, M. B.; Corr, B.; Forde, C.; Frost, E.; Hardiman, O., Amyotrophic Lateral Sclerosis Mimic Syndromes. Archives of Neurology 2000, 57 (1).
8. Turner, M. R.; Kiernan, M. C.; Leigh, P. N.; Talbot, K., Biomarkers in amyotrophic lateral sclerosis. The Lancet Neurology 2009, 8 (1), 94-109.
9. Miller, R. G.; Munsat, T. L.; Swash, M.; Brooks, B. R., Consensus guidelines for the design and implementation of clinical trials in ALS. Journal of the Neurological Sciences 1999, 169 (1-2), 2-12.
10. Byrne, S.; Walsh, C.; Lynch, C.; Bede, P.; Elamin, M.; Kenna, K.; McLaughlin, R.; Hardiman, O., Rate of familial amyotrophic lateral sclerosis: a systematic review and meta-analysis. J Neurol Neurosurg Psychiatry 2011, 82 (6), 623-627.
11. M.D, J. H. P.; M.D, B. s. K.; M.D, Y. j. B.; M.D, H. r. J.; M.D, S.-H. K.; PhD; M.D, T. H. K.; PhD; M.D, J. W. S.; PhD; M.D, D. H. S.; PhD; M.D, S. S. P.; PhD; M.D, H. J. Y.; PhD; M.D, H. J. K.; PhD, Amyotrophic Lateral Sclerosis Identified by Failure to Wean From Mechanical Ventilation. Journal of the Korean Geriatrics Society 2012, 16 (3), 162-166.
12. Shin, J.-Y.; Lee, K.-W., Diagnosis and management of amyotrophic lateral sclerosis. Journal of the Korean Medical Association 2015, 58 (2).
13. Cedarbaum, J. M.; Stambler, N.; Malta, E.; Fuller, C.; Hilt, D.; Thurmond, B.; Nakanishi, A., The ALSFRS-R: a revised ALS functional rating scale that incorporates assessments of respiratory function. Journal of the Neurological Sciences 1999, 169 (1-2), 13-21.
14. Franchignoni, F.; Mora, G.; Giordano, A.; Volanti, P.; Chio, A., Evidence of multidimensionality in the ALSFRS-R Scale: a critical appraisal on its measurement properties using Rasch analysis. J Neurol Neurosurg Psychiatry 2013, 84 (12), 1340-1345.
15. Castrillo-Viguera, C.; Grasso, D. L.; Simpson, E.; Shefner, J.; Cudkowicz, M. E., Clinical significance in the change of decline in ALSFRS-R. Amyotroph Lateral Scler 2010, 11 (1-2), 178-180.
16. Neumann, M.; Sampathu, D. M.; Kwong, L. K.; Truax, A. C.; Micsenyi, M. C.; Chou, T. T.; Bruce, J.; Schuck, T.; Grossman, M.; Clark, C. M.; McCluskey, L. F.; Miller, B. L.; Masliah, E.; Mackenzie, I. R.; Feldman, H.; Feiden, W.; Kretzschmar, H. A.; Trojanowski, J. Q.; Lee, V. M., Ubiquitinated TDP-43 in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Science 2006, 314 (5796), 130-133.
17. Prasad, A.; Bharathi, V.; Sivalingam, V.; Girdhar, A.; Patel, B. K., Molecular Mechanisms of TDP-43 Misfolding and Pathology in Amyotrophic Lateral Sclerosis. Front Mol Neurosci 2019, 12, 25.
18. Feneberg, E.; Gray, E.; Ansorge, O.; Talbot, K.; Turner, M. R., Towards a TDP-43-Based Biomarker for ALS and FTLD. Mol Neurobiol 2018, 55 (10), 7789-7801.
19. Buratti, E., Multiple roles of TDP-43 in gene expression, splicing regulation, and human disease. Frontiers in Bioscience 2008, 13 (13).
20. Kuo, P. H.; Doudeva, L. G.; Wang, Y. T.; Shen, C. K.; Yuan, H. S., Structural insights into TDP-43 in nucleic-acid binding and domain interactions. Nucleic Acids Res 2009, 37 (6), 1799-1808.
21. Feneberg, E.; Steinacker, P.; Lehnert, S.; Schneider, A.; Walther, P.; Thal, D. R.; Linsenmeier, M.; Ludolph, A. C.; Otto, M., Limited role of free TDP-43 as a diagnostic tool in neurodegenerative diseases. Amyotroph Lateral Scler Frontotemporal Degener 2014, 15 (5-6), 351-356.
22. De Marco, G.; Lomartire, A.; Calvo, A.; Risso, A.; De Luca, E.; Mostert, M.; Mandrioli, J.; Caponnetto, C.; Borghero, G.; Manera, U.; Canosa, A.; Moglia, C.; Restagno, G.; Fini, N.; Tarella, C.; Giordana, M. T.; Rinaudo, M. T.; Chio, A., Monocytes of patients with amyotrophic lateral sclerosis linked to gene mutations display altered TDP-43 subcellular distribution. Neuropathol Appl Neurobiol 2017, 43 (2), 133-153.
23. De Marco, G.; Lupino, E.; Calvo, A.; Moglia, C.; Buccinna, B.; Grifoni, S.; Ramondetti, C.; Lomartire, A.; Rinaudo, M. T.; Piccinini, M.; Giordana, M. T.; Chio, A., Cytoplasmic accumulation of TDP-43 in circulating lymphomonocytes of ALS patients with and without TARDBP mutations. Acta Neuropathol 2011, 121 (5), 611-622.
24. Wang, I. F.; Chang, H. Y.; Hou, S. C.; Liou, G. G.; Way, T. D.; James Shen, C. K., The self-interaction of native TDP-43 C terminus inhibits its degradation and contributes to early proteinopathies. Nat Commun 2012, 3, 766.
25. Fuentealba, R. A.; Udan, M.; Bell, S.; Wegorzewska, I.; Shao, J.; Diamond, M. I.; Weihl, C. C.; Baloh, R. H., Interaction with polyglutamine aggregates reveals a Q/N-rich domain in TDP-43. J Biol Chem 2010, 285 (34), 26304-26314.
26. Jarrett, J. T.; Lansbury, P. T., Seeding “one-dimensional crystallization” of amyloid: A pathogenic mechanism in Alzheimer's disease and scrapie? Cell 1993, 73 (6), 1055-1058.
27. Butterfield, S.; Hejjaoui, M.; Fauvet, B.; Awad, L.; Lashuel, H. A., Chemical strategies for controlling protein folding and elucidating the molecular mechanisms of amyloid formation and toxicity. J Mol Biol 2012, 421 (2-3), 204-236.
28. Mayer, G.; Heckel, A., Biologically active molecules with a "light switch". Angew Chem Int Ed Engl 2006, 45 (30), 4900-4921.
29. Kataoka, H.; Tahara, H.; Watanabe, T.; Sugawara, M.; Ide, T.; Goto, M.; Furuichi, Y.; Sugimoto, M., Immortalization of immunologically committed Epstein-Barr virus-transformed human B-lymphoblastoid cell lines accompanied by a strong telomerase activity. Differentiation 1998, 62 (4), 203-211.
30. Sie, L.; Loong, S.; Tan, E. K., Utility of lymphoblastoid cell lines. J Neurosci Res 2009, 87 (9), 1953-1959.
31. Sugimoto, M.; Tahara, H.; Ide, T.; Furuichi, Y., Steps involved in immortalization and tumorigenesis in human B-lymphoblastoid cell lines transformed by Epstein-Barr virus. Cancer Res 2004, 64 (10), 3361-3364.
32. Goldberg, A. L., Development of proteasome inhibitors as research tools and cancer drugs. J Cell Biol 2012, 199 (4), 583-588.
33. Wente, M.; Eibl, G.; Reber, H.; Friess, H.; Büchler, M.; Hines, O., The proteasome inhibitor MG132 induces apoptosis in human pancreatic cancer cells. Oncology Reports 2005.
34. Scotter, E. L.; Vance, C.; Nishimura, A. L.; Lee, Y. B.; Chen, H. J.; Urwin, H.; Sardone, V.; Mitchell, J. C.; Rogelj, B.; Rubinsztein, D. C.; Shaw, C. E., Differential roles of the ubiquitin proteasome system and autophagy in the clearance of soluble and aggregated TDP-43 species. J Cell Sci 2014, 127 (Pt 6), 1263-1278.
35. He, R. Y.; Chao, S. H.; Tsai, Y. J.; Lee, C. C.; Yu, C. Y.; Gao, H. D.; Huang, Y. A.; Hwang, E.; Lee, H. M.; Huang, J. J., Photocontrollable Probe Spatiotemporally Induces Neurotoxic Fibrillar Aggregates and Impairs Nucleocytoplasmic Trafficking. ACS Nano 2017, 11 (7), 6795-6807.
36. Mittag, D.; Proietto, A. I.; Loudovaris, T.; Mannering, S. I.; Vremec, D.; Shortman, K.; Wu, L.; Harrison, L. C., Human dendritic cell subsets from spleen and blood are similar in phenotype and function but modified by donor health status. J Immunol 2011, 186 (11), 6207-6217.