透過 western blotting 觀察 insoluble form 的 polyQ 蛋白在細胞中聚 集，在加入不同濃度的藥物處理後細胞，是否因細胞自噬增加而降低。
以誘導表現的 polyQ 穩定細胞株中處理 compound C 48 小時之後，收 取蛋白質作 western blotting 分析。由 stacking gel interface 可以看到 79Q-EGFP 高分子有明顯聚集，但隨藥物處理濃度的增加，被清除 insoluble form 的聚集下降 50%，然而 soluble form 的 polyQ 卻會因 為藥物處理而增加 70%(Figure4A)。因此推測 autophagy 除了降解 insoluble form polyQ 外，另外 soluble form polyQ 也會增加。
5、藥物處理可以清除堆積的多麩醯胺酸
利用誘導四天 polyQ-EGFP 的細胞，以 5、10 和 20 μM 不同濃度處理 48 小時後利用螢光顯微鏡觀察(Figure5A)，計算每 500 個有 79Q-EGFP 表現的細胞具有聚集的細胞數目。並與 DMSO 的控制組為基準，觀 察加藥組的聚集比例上是否減少。發現 compound C 能隨濃度增加而 增加消除聚集的數量，並且有顯著差異其中在5μM 處理後 polyQ 聚
23
集下降接近 50% (Figure5B)。
6.利用細胞自噬抑制劑證明藥物透過促進細胞自噬去清除 polyQ 聚集 PolyQ 聚集的清除有可能是藉由其他路徑達成，例如:UPS 系統。
因此利用細胞自噬抑制劑阻斷 compound C 的能力，因抑制劑使藥物 所帶來的清除能力下降，確定清除 polyQ 聚集的能力是來至於增強 autophagy 的能力。使用誘導四天 polyQ-EGFP 的細胞，以 20 μM 細 胞自噬抑制劑—3-MA 及 0.5 nM Bafilomycin A1 作用 24 小時後移除，
再加入20 μM 藥物處理 48 小時後利用螢光顯微鏡觀察量(Figure6A)，
比較只有藥物處理和同時在以抑制劑處理再進行藥物處理兩組之間 的差異，發現抑制劑處理後藥物效力下降，polyQ-EGFP 的堆積物上 升，量化後也具統計意義(Figure6B)。表示藥物透過誘導細胞自噬而 達到減少 polyQ-EGFP 的堆積物的效果。
7.藥物對細胞存活率的影響
細胞存活率對藥物是個很重要的，我們利用 PI 染色方法來確認藥 物沒有毒性使細胞造成凋亡。在细胞加入 PI 染劑，PI 不能穿透完整 细胞膜，而壞死细胞由於失去膜的完整性，PI 可進入细胞内與 DNA 结合，依據此特點，使用 PI 染色可鑑定死细胞。將細胞誘導後並經 藥物處理後利用流式細胞儀程式(cell Quest Pro)分析，染劑無法穿透
24
細胞膜，代表藥物處理在不同長度 polyQ-EGFP 的細胞中能有效幫助 細胞存活。
8.藥物減少細胞核內 polyQ 聚集
以 5、10 和 20 μM 不同濃度處理 48 小時後，透過螢光顯微鏡觀 察 Q79 細胞內 polyQ-EGFP 分布情況，發現 insoluble form 的 polyQ 蛋白在細胞質與細胞核有顯著下降(Figure8A)。另外透過 western blotting 也發現 insoluble form 的 polyQ 蛋白在細胞核的表現有顯著下 降(Figure8B)。
陸、討論
本研究希望透過藥物誘導細胞自噬減少擴增PolyQ 蛋白的堆積。
實驗以flow cytometry 進行第一步的篩選，以多種化合物處理Q36、
Q61及Q79 細胞株後，觀察lysotracker的紅色螢光(42)。利用compound A 作為本實驗的控制組，由lysotracker紅色螢光結果顯示，compound C處理後，細胞中的lysosome確實增加和compound A有相似結果 (Figure1)。研究結果顯示藥物誘導的細胞自噬LC3-II會明顯增加，我 們在Western blot 與螢光顯微鏡觀察compound C會增加LC3-II表現量 達50%(Figure2,3)。前文提及細胞自噬形成初期Vps34會與Beclin-1結 合参與自噬小體雙層膜的形成，本實驗結果Beclin-1的表現量與控制 組相比增加2.5倍，結果在Western blot中發現compound C會增加
25
Beclin-1的表現量與控制組compound A 結果相似(Figure2)。
在某些神經變性疾病中有研究顯示細胞會透過細胞自噬清除 polyQ聚集，然而透過螢光顯微鏡觀察加入compound C有明顯使polyQ 聚集減少(Figure5)。要進一步確認我們的藥物與細胞自噬的關係，加 入compound C與細胞自噬的抑制劑，數據顯示當細胞加入compound C與細胞自噬的抑制劑處理後，清除polyQ聚集能力有下降趨勢，可 以確認我們藥物compound C是高度敏感的自噬調節劑(Figure.6)。藥 物是否具毒性是一個很重要的問題，實驗利用流式細胞儀分析可以發 現compound C處理下不會導致細胞死亡(Figure7)，代表compound C 有效幫助細胞存活。有研究指出當細胞核累積大量的多麩醯胺酸重複 序列蛋白導致轉錄失活(43)，因此想探討compound C是否可清除細胞 核累積的多麩醯胺酸重複序列蛋白，首先，透過螢光顯微鏡觀察，當 加入compound C 確認polyQ聚集在細胞核的程度有dose-dependent 下降並沒有顯著差異，進一步透過 western blot 確認，藥物使細胞核 的insoluble form的polyQ蛋白有下降(Figure8)。有研究指出細胞內含有 Xpo1基因蛋白可將細胞核內insoluble form的polyQ蛋白游離到細胞質 (43)。因此推測我們的藥物可以促進Xpo1基因將細胞核內insoluble form的polyQ蛋白清除，堆積在細胞質的insoluble form的polyQ蛋白可 透過細胞自噬清除。未來可以進一步利用western blot 確認藥物是透
26
過mTOR dependent或mTOR independent 路徑去促進細胞自噬。
27
柒、參考文獻
1. Orr, H. T., and Zoghbi, H. Y. (2007) Trinucleotide repeat disorders. Annual review of neuroscience 30, 575-621
2. Huang, C. C., Faber, P. W., Persichetti, F., Mittal, V., Vonsattel, J. P., MacDonald, M. E., and Gusella, J. F. (1998) Amyloid formation by mutant huntingtin: threshold, progressivity and recruitment of normal polyglutamine proteins. Somatic cell and molecular genetics 24, 217-233
3. Semaka, A., Creighton, S., Warby, S., and Hayden, M. R. (2006) Predictive testing for Huntington disease: interpretation and significance of intermediate alleles. Clinical genetics 70, 283-294
4. Langbehn, D. R., Hayden, M. R., and Paulsen, J. S. (2010) CAG-repeat length and the age of onset in Huntington disease (HD): a review and validation study of statistical approaches. American journal of medical genetics. Part B, Neuropsychiatric genetics : the official publication of the International Society of Psychiatric Genetics 153b, 397-408
5. DiFiglia, M., Sapp, E., Chase, K. O., Davies, S. W., Bates, G. P., Vonsattel, J.
P., and Aronin, N. (1997) Aggregation of huntingtin in neuronal intranuclear inclusions and dystrophic neurites in brain. Science (New York, N.Y.) 277, 1990-1993
6. Wellington, C. L., Brinkman, R. R., O'Kusky, J. R., and Hayden, M. R. (1997) Toward understanding the molecular pathology of Huntington's disease. Brain pathology (Zurich, Switzerland) 7, 979-1002
7. Scherzinger, E., Lurz, R., Turmaine, M., Mangiarini, L., Hollenbach, B., Hasenbank, R., Bates, G. P., Davies, S. W., Lehrach, H., and Wanker, E. E.
(1997) Huntingtin-encoded polyglutamine expansions form amyloid-like protein aggregates in vitro and in vivo. Cell 90, 549-558
8. Shao, J., and Diamond, M. I. (2007) Polyglutamine diseases: emerging concepts in pathogenesis and therapy. Human molecular genetics 16 Spec No.
2, R115-123
9. Cortes, C. J., and La Spada, A. R. (2014) The many faces of autophagy dysfunction in Huntington's disease: from mechanism to therapy. Drug discovery today
10. Imai, Y., Soda, M., Murakami, T., Shoji, M., Abe, K., and Takahashi, R. (2003) A product of the human gene adjacent to parkin is a component of Lewy bodies and suppresses Pael receptor-induced cell death. The Journal of biological chemistry 278, 51901-51910
11. Mattson, M. P., LaFerla, F. M., Chan, S. L., Leissring, M. A., Shepel, P. N.,
28
and Geiger, J. D. (2000) Calcium signaling in the ER: its role in neuronal plasticity and neurodegenerative disorders. Trends in neurosciences 23, 222-229
13. Emili, A., Greenblatt, J., and Ingles, C. J. (1994) Species-specific interaction of the glutamine-rich activation domains of Sp1 with the TATA box-binding protein. Molecular and cellular biology 14, 1582-1593
14. Dunah, A. W., Jeong, H., Griffin, A., Kim, Y. M., Standaert, D. G., Hersch, S.
M., Mouradian, M. M., Young, A. B., Tanese, N., and Krainc, D. (2002) Sp1 and TAFII130 transcriptional activity disrupted in early Huntington's disease.
Science (New York, N.Y.) 296, 2238-2243
15. Chen-Plotkin, A. S., Sadri-Vakili, G., Yohrling, G. J., Braveman, M. W., Benn, C. L., Glajch, K. E., DiRocco, D. P., Farrell, L. A., Krainc, D., Gines, S., MacDonald, M. E., and Cha, J. H. (2006) Decreased association of the transcription factor Sp1 with genes downregulated in Huntington's disease.
Neurobiology of disease 22, 233-241
16. Nucifora, F. C., Jr., Sasaki, M., Peters, M. F., Huang, H., Cooper, J. K., Yamada, M., Takahashi, H., Tsuji, S., Troncoso, J., Dawson, V. L., Dawson, T.
M., and Ross, C. A. (2001) Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity. Science (New York, N.Y.) 291, 2423-2428
17. Sugars, K. L., Brown, R., Cook, L. J., Swartz, J., and Rubinsztein, D. C. (2004) Decreased cAMP response element-mediated transcription: an early event in exon 1 and full-length cell models of Huntington's disease that contributes to polyglutamine pathogenesis. The Journal of biological chemistry 279, 4988-4999
18. Cui, L., Jeong, H., Borovecki, F., Parkhurst, C. N., Tanese, N., and Krainc, D.
(2006) Transcriptional repression of PGC-1alpha by mutant huntingtin leads to mitochondrial dysfunction and neurodegeneration. Cell 127, 59-69
19. McConoughey, S. J., Basso, M., Niatsetskaya, Z. V., Sleiman, S. F., Smirnova, N. A., Langley, B. C., Mahishi, L., Cooper, A. J., Antonyak, M. A., Cerione, R.
A., Li, B., Starkov, A., Chaturvedi, R. K., Beal, M. F., Coppola, G., Geschwind, D. H., Ryu, H., Xia, L., Iismaa, S. E., Pallos, J., Pasternack, R., Hils, M., Fan, J., Raymond, L. A., Marsh, J. L., Thompson, L. M., and Ratan,
29
R. R. (2010) Inhibition of transglutaminase 2 mitigates transcriptional dysregulation in models of Huntington disease. EMBO molecular medicine 2, 349-370
20. Lee, J., Kim, C. H., Simon, D. K., Aminova, L. R., Andreyev, A. Y., Kushnareva, Y. E., Murphy, A. N., Lonze, B. E., Kim, K. S., Ginty, D. D., Ferrante, R. J., Ryu, H., and Ratan, R. R. (2005) Mitochondrial cyclic AMP response element-binding protein (CREB) mediates mitochondrial gene expression and neuronal survival. The Journal of biological chemistry 280, 40398-40401
21. Shirendeb, U., Reddy, A. P., Manczak, M., Calkins, M. J., Mao, P., Tagle, D.
A., and Reddy, P. H. (2011) Abnormal mitochondrial dynamics, mitochondrial loss and mutant huntingtin oligomers in Huntington's disease: implications for selective neuronal damage. Human molecular genetics 20, 1438-1455
22. Song, W., Chen, J., Petrilli, A., Liot, G., Klinglmayr, E., Zhou, Y., Poquiz, P., Tjong, J., Pouladi, M. A., Hayden, M. R., Masliah, E., Ellisman, M., Rouiller, I., Schwarzenbacher, R., Bossy, B., Perkins, G., and Bossy-Wetzel, E. (2011) Mutant huntingtin binds the mitochondrial fission GTPase dynamin-related protein-1 and increases its enzymatic activity. Nature medicine 17, 377-382 23. Kim, J., Moody, J. P., Edgerly, C. K., Bordiuk, O. L., Cormier, K., Smith, K.,
Beal, M. F., and Ferrante, R. J. (2010) Mitochondrial loss, dysfunction and altered dynamics in Huntington's disease. Human molecular genetics 19, 3919-3935
24. Browne, S. E., Bowling, A. C., MacGarvey, U., Baik, M. J., Berger, S. C., Muqit, M. M., Bird, E. D., and Beal, M. F. (1997) Oxidative damage and metabolic dysfunction in Huntington's disease: selective vulnerability of the basal ganglia. Annals of neurology 41, 646-653
25. Pandey, M., Varghese, M., Sindhu, K. M., Sreetama, S., Navneet, A. K., Mohanakumar, K. P., and Usha, R. (2008) Mitochondrial NAD+-linked State 3 respiration and complex-I activity are compromised in the cerebral cortex of 3-nitropropionic acid-induced rat model of Huntington's disease. Journal of neurochemistry 104, 420-434
26. Seong, I. S., Ivanova, E., Lee, J. M., Choo, Y. S., Fossale, E., Anderson, M., Gusella, J. F., Laramie, J. M., Myers, R. H., Lesort, M., and MacDonald, M. E.
(2005) HD CAG repeat implicates a dominant property of huntingtin in mitochondrial energy metabolism. Human molecular genetics 14, 2871-2880 27. Watson, L. M., Scholefield, J., Greenberg, L. J., and Wood, M. J. (2012)
Polyglutamine disease: from pathogenesis to therapy. South African medical journal = Suid-Afrikaanse tydskrif vir geneeskunde 102, 481-484
30
28. Renna, M., Jimenez-Sanchez, M., Sarkar, S., and Rubinsztein, D. C. (2010) Chemical inducers of autophagy that enhance the clearance of mutant proteins in neurodegenerative diseases. The Journal of biological chemistry 285, 11061-11067
29. Zhang, L., Yu, J., Pan, H., Hu, P., Hao, Y., Cai, W., Zhu, H., Yu, A. D., Xie, X., Ma, D., and Yuan, J. (2007) Small molecule regulators of autophagy identified by an image-based high-throughput screen. Proceedings of the National Academy of Sciences of the United States of America 104, 19023-19028
30. Kourtis, N., and Tavernarakis, N. (2009) Autophagy and cell death in model organisms. Cell death and differentiation 16, 21-30
31. Ravikumar, B., Sarkar, S., Davies, J. E., Futter, M., Garcia-Arencibia, M., Green-Thompson, Z. W., Jimenez-Sanchez, M., Korolchuk, V. I., Lichtenberg, M., Luo, S., Massey, D. C., Menzies, F. M., Moreau, K., Narayanan, U., Renna, M., Siddiqi, F. H., Underwood, B. R., Winslow, A. R., and Rubinsztein, D. C. (2010) Regulation of mammalian autophagy in physiology and pathophysiology. Physiological reviews 90, 1383-1435
32. Harris, H., and Rubinsztein, D. C. (2012) Control of autophagy as a therapy for neurodegenerative disease. Nature reviews. Neurology 8, 108-117
33. Taylor, J. P., Hardy, J., and Fischbeck, K. H. (2002) Toxic proteins in neurodegenerative disease. Science (New York, N.Y.) 296, 1991-1995
34. Riley, B. E., and Orr, H. T. (2006) Polyglutamine neurodegenerative diseases and regulation of transcription: assembling the puzzle. Genes & development 20, 2183-2192
35. Lemasters, J. J. (2005) Selective mitochondrial autophagy, or mitophagy, as a targeted defense against oxidative stress, mitochondrial dysfunction, and aging.
Rejuvenation research 8, 3-5
36. Ross, C. A., and Poirier, M. A. (2004) Protein aggregation and neurodegenerative disease. Nature medicine 10 Suppl, S10-17
37. Haass, C., and Selkoe, D. J. (2007) Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer's amyloid beta-peptide. Nature reviews. Molecular cell biology 8, 101-112
38. Metcalf, D. J., Garcia-Arencibia, M., Hochfeld, W. E., and Rubinsztein, D. C.
(2012) Autophagy and misfolded proteins in neurodegeneration. Experimental neurology 238, 22-28
39. Berger, Z., Ravikumar, B., Menzies, F. M., Oroz, L. G., Underwood, B. R., Pangalos, M. N., Schmitt, I., Wullner, U., Evert, B. O., O'Kane, C. J., and Rubinsztein, D. C. (2006) Rapamycin alleviates toxicity of different aggregate-prone proteins. Human molecular genetics 15, 433-442
31
40. Ravikumar, B., Berger, Z., Vacher, C., O'Kane, C. J., and Rubinsztein, D. C.
(2006) Rapamycin pre-treatment protects against apoptosis. Human molecular genetics 15, 1209-1216
41. Sarkar, S., Ravikumar, B., Floto, R. A., and Rubinsztein, D. C. (2009) Rapamycin and mTOR-independent autophagy inducers ameliorate toxicity of polyglutamine-expanded huntingtin and related proteinopathies. Cell death and differentiation 16, 46-56
42. Chikte, S., Panchal, N., and Warnes, G. (2014) Use of LysoTracker dyes: a flow cytometric study of autophagy. Cytometry. Part A : the journal of the International Society for Analytical Cytology 85, 169-178
43. Chan, W. M., Tsoi, H., Wu, C. C., Wong, C. H., Cheng, T. C., Li, H. Y., Lau, K.
F., Shaw, P. C., Perrimon, N., and Chan, H. Y. (2011) Expanded polyglutamine domain possesses nuclear export activity which modulates subcellular localization and toxicity of polyQ disease protein via exportin-1. Human molecular genetics 20, 1738-1750
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捌、圖示
Fig 1.篩選出藥物 Compound C 會誘導 autolysosome 的形成.
使用 Q36、61 與 79 的穩定細胞株，doxycycline 誘導四天，藥物處理 5、10、20 μM，流氏細胞儀(cell Quest Pro)觀察 lysotracker 染色。A.透過 lysotracker 篩選藥 物 B. compound C 誘導 autolysosome 使螢光光譜向右增強。C.以平均值進行統 計隨著 compound C 濃度上升而 autolysosome 也 dose-dependent 上升(Y 軸以 DMSO 為參考的螢光強度相對值)(student’s t-test; N=3; *p< 0.05,**p< 0.01, ***p<
0.001 ).
F luor es cence inte n si ty
N=3 N=3
A
B
C
33
Fig 2. Compound C 能增加自噬相關蛋白的增加
使用 Q79 的穩定細胞株，doxycycline 誘導四天，藥物處理 5、10、20 μM，再抽 蛋白進行 Western blot。A.在 Q79 的穩定細胞株，compound C 誘導自噬相關蛋 白的增加，Beclin1 上升與 LC3I 轉換 LC3II 的轉換率上升。B.以 western 定量進 行統計 (利用 DMSO(D)做為控制組) 。(student’s t-test; N=3; *p< 0.05,**p< 0.01,
***p< 0.001 ).
A
B
LC3 -II /LC3 -I Ra tio
N=3
34
.
Fig 3.
Compound C 能影響誘導自噬相關蛋白在 SK 及 Q79 的穩定細胞株，doxycycline 誘導四天，藥物 5、10、20 μM 處理 48 小時，進一步以螢光顯微鏡觀察。A.以螢光顯微鏡觀察，加入 compound C 誘導 autolysosome 上升，lysotracker 所標定的 lysosome(紅色)、LC3-EGFP(綠色)與雙 重標定的 autolysosome 都有隨著藥物濃度上升 dose-dependent 上升。(scale bar, 10 μm.) B.經 compound C 處理後，細胞標定 anti- LC3(紅色)的 autolysosome 有隨著 藥物處理而上升。(scale bar,25μm.)
SK-N-SH Q79
A B
35
Fig 4.
利用 Western blot 中的結果證實 Compound C 可以清除 polyQ 蛋白質的聚集利用 Q79 的穩定細胞株，先以 doxycycline 誘導四天後，加入藥物 5、
10、20 μM 處理 48 小時，再萃取蛋白進行 Western blot。A. Q79 細胞 株，隨著 compound C 藥物濃度增加會逐漸清除 polyQ 的聚集效果也 愈見明顯，另外藥物並不影響一般蛋白的 soluble form 表現或者會增 加 soluble form 表現量。(實驗以 GAPDH 作為 loading control )
A
36
Fig.5
經螢光顯微鏡確認 Compound C 可以清除堆積的多麩醯胺酸使用 Q79 的穩定細胞株，doxycycline 誘導四天，藥物 5、10、20 μM 處理 48 小 時。A.隨著 compound C 濃度上升，PolyQ 聚集減少。(scale bar, 10 μm.) B.以計算 500 個有 PolyQ-EGFP 表現的細胞中有幾個細胞表現聚集小體(綠色小點)進行統 計。compound C 濃度上升會使聚集小點下降，並且達到顯著差異。(student’s t-test;
N=3; *p< 0.05,**p< 0.01, ***p< 0.001 ).
Cel ls w ith Poly -Q a g g reg a tion
A
B
Compound C
N=3
37
Fig.6 Autophagy 抑制劑能阻斷 compound C 清除 polyQ 的能力
使用 Q79 的穩定細胞株，doxycycline 誘導四天，分別加入細胞自噬抑制劑—3-MA
使用 Q79 的穩定細胞株，doxycycline 誘導四天，分別加入細胞自噬抑制劑—3-MA