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研究生：	鄭瑀萱 Yu-Hsuan Cheng
論文名稱：	鑑定新藥物NH040-1透過抑制神經細胞內質網壓力以治療第十七型脊髓小腦共濟失調症 Identification of a novel compound NH040-1 to attenuate endoplasmic reticulum stress in spinocerebellar ataxia type 17
指導教授：	吳忠信 Wu, Chung-Hsin 林榮耀 Lin, Jung-Yaw
學位類別：	碩士 Master
系所名稱：	生命科學系 Department of Life Science
論文出版年：	2015
畢業學年度：	103
語文別：	英文
論文頁數：	68
中文關鍵詞：	第十七型小腦脊髓共濟失調症、內質網壓力、活性氧、細胞凋亡、金銀花
英文關鍵詞：	spinocerebellar ataxia type 17, endoplasmic reticulum stress, reactive oxygen species, apoptosis, Lonicera japonica
DOI URL：	https://doi.org/10.6345/NTNU202205254
論文種類：	學術論文
相關次數：	點閱：185 下載：1
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第十七型小腦脊髓共濟失調症 (Spinocerebellar ataxia type 17; SCA17) 是一種由多麩醯胺(polyglutamine; polyQ)蛋白異常擴增所引起的神經退化性疾病，致病原因是在TATA box binding protein (TBP) 基因上CAG三核苷序列重複出現不正常擴增，因而轉譯出異常的polyQ蛋白表現；polyQ蛋白異常擴增造成蛋白質錯誤折疊及聚集(Aggregation)，另外未折疊及錯誤折疊蛋白若在內質網內堆積會引發內質網壓力 (Endoplasmic reticulum stress; ER stress)，而細胞會啟動未折疊蛋白反應 (Unfolded protein response; UPR) 保護機制以修復內質網功能，一旦壓力持續未解決，反而會誘發由內質網介導的凋亡路徑進而造成細胞死亡。此外內質網功能失調也會增加活性氧 (Reactive oxygen species; ROS) 的生成，誘發氧化壓力而加劇傷害。近年來，越來越多研究認為內質網壓力及氧化壓力可能參與第十七型小腦脊髓共濟失調症致病過程，且許多文獻指出，中草藥的使用具有治療退化性疾病之潛力。因此，本研究設計 (I)人類神經瘤母細胞 (Neuroblastoma SH-SY5Ycells) 以毒蘿蔔素 (Thapsigargin; TG) 及衣黴素 (Tunicamycin; TM) 誘導的內質網壓力、(II) SCA17 nTBP/Q79-EGFP細胞以多西環素 (Doxycycline)誘導出polyQ蛋白表現 (III) SCA17小鼠模式三階段實驗，鑑定金銀花 (Lonicera japonica)之有效成分NH040-1是否具有保護神經細胞的作用。實驗結果顯示NH040-1可增加由TG或TM所造成細胞死亡的生存率、可抑制神經細胞凋亡、可降低內質網壓力路徑相關蛋白的表現並可減少由內質網壓力產生的活性氧生成量，顯示NH040-1對內質網壓力誘導的細胞死亡具有改善效果；並進一步以多西環素誘導出的SCA17 nTBP/Q79-EGFP細胞，我們發現NH040-1可對Q79細胞型態退化有顯著改善效果、減少Q79細胞聚集的現象、且可降低內質網壓力誘導的細胞凋亡表現。實驗進一步轉移至SCA17基因轉殖小鼠上，藉由腹腔注射NH040-1藥物，透過滾輪、步行等實驗觀察其行為，結果顯示NH040-1能夠有效地促進其滾輪上之行為分析以及改善步行印跡，在其運動失調的症狀上發揮療效，並且可以減少基因轉殖小鼠小腦中TBP/polyQ 蛋白的不正常聚集及內質網壓力介導的細胞凋亡現象，以及改善Purkinje cells失序分佈。綜合以上實驗結果，NH040-1具有透過抑制內質網壓力及活性氧生成的表現，進而治療第十七型小腦脊髓共濟失調症的潛在能力。

Spinocerebellar ataxia type 17 (SCA17), one of the polyglutamine (polyQ) diseases, is a neurodegenerative disorders caused by the expansion of the polyQ tract (＞43 CAG repeat) in human TATA box binding protein (TBP) gene, leading to an abnormal expansion of a polyQ stretch in the corresponding protein. Previous study indicated that the endoplasmic reticulum (ER) stress or unfolded protein response (UPR) may contribute to the pathogenesis of SCA17, by the accumulation of misfolded proteins, alterations in the calcium homeostasis and reactive oxygen species (ROS) production.
This study identified NH040-1 from Lonicera japonica that protecting neurons from Thapsigargin (TG) or Tunicamycin (TM)-induced ER stress and ROS generation in the in vitro SH-SY5Y and SCA17 cell models and the in vivo transgenic mice model.
We showed NH040-1 was effective against TG or TM-induced ER stress by increasing the cell viability, supressed ER-mediated apoptotic protein expression and ROS production in SH-SY5Y cell model. Additionally, the results indicated that NH040-1 enhanced the neurite overgrowth and decrease misfolded protein aggregation in SCA 17 cell model. Furthermore, the application of NH040-1 in SCA17 mice model showed that NH040-1: (I) ameliorated motor coordinationin deficits in rotarod analysis and footprint patterns experiments, (II) reduced the polyQ aggregation as well as ER stress-induced apoptotic protein expression, and (III) ameliorated Purkinje neuron degeneration in the cerebella of transgenic mice. Therefore, NH040-1 could be a potentially therapeutic drug to attenuate ER stress and neurodegeneration in SCA17.

誌謝    V
中文摘要    VII
Abstract    IX
Figure list    1
Introduction    3
1 Polyglutamine (polyQ) diseases    3
2 Spinocerebellar ataxia type 17 (SCA17)    4
3 ER stress    5
4 Reactive oxygen species (ROS)    7
5 Chinese herbal medicines (CHMs)    8
6 Lonicera japonica flower    9
Research aims    10
Materials and Methods    11
1. Materials    11
2. Cell culture    12
3. MTT assay    12
4. Western blotting analysis    13
4.1. Preparation of cell lysates    13
4.2. Quantification of protein concentrations    13
4.3. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)    14
4.3.1. Preparation of protein sample    14
4.3.2. Preparation of the SDS-polyacrylamide gels    14
4.4. Procedure of wet and Semi-Dry transfer system    16
4.5. Immunoblotting    17
5. Reactive oxygen species (ROS) analysis    18
6. 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radical scavenging assay    19
7. Crystal violet staining of neurite outgrowth    19
8. Dot-Blot Filter retardation assay of TBP/polyQ aggregation    20
9. Animal model    22
10. Genotyping of transgenic mice    23
10.1. Polymerase Chain Reaction (PCR)    24
10.1.1. Preparation of the PCR master mix    24
10.1.2. Running the PCR    25
10.2. Agarose Gel Electrophoresis for PCR product analysis    25
11. Motor behavioral assessments    26
12. Footprint patterns analysis    27
13. Western blot analysis of the cerebellum of tested mice    27
14. Immunohistochemistry (IHC) of paraffin-embedded mice cerebella    28
15. Statistical analysis    29
Results    30
1. NH040-1 protects SH-SY5Y cells against TG or TM-induced cell death.    30
2. NH040-1 suppresses the unfolded protein response (UPR) in SH-SY5Y cells.    31
3. NH040-1 suppresses the ER stress-mediated apoptosis in SH-SY5Y cells.    31
4. NH040-1 attenuates ER stress-induced ROS production in SH-SY5Y cells.    32
5. NH040-1 enhances the neurite outgrowth of SCA17 (nTBP/ Q79-EGFP) cells.    33
6. NH040-1 reduces TBP-polyQ aggregation in SCA17 cells.    34
7. NH040-1 inhibits ER stress in SCA17 cells.    34
8. NH040-1 ameliorates the neurological behavior of SCA 17 transgenic mice.    35
9. NH040-1 attenuates the TBP aggregation and ER stress-mediated apoptosis in the cerebella of SCA17 transgenic mice.    36
10. NH040-1 ameliorates Purkinje neuron degeneration in the cerebella of SCA17 transgenic mice.    37
Discussion    38
References    44
Figures    51
Figure 1. Cell cytotoxicity of NH040-1 and its inhibition of cell death induced by TG or TM in SH-SY5Y cells.    51
Figure 2. NH040-1 decreases the activation of ER stress associated proteins in SH-SY5Y cells.    52
Figure 3. NH040-1 suppresses ER stress-mediated apoptotic proteins induced by TG or TM in SH-SY5Y cells.    53
Figure 4. Effects of NH040-1 on the expression of Bcl-2 and Bax induced by TG or TM in SH-SY5Y cells.    54
Figure 5. NH040-1 has a high ROS scavenging activity.    55
Figure 6. NH040-1 attenuates ROS production induced by TG or TM in SH-SY5Y cells.    56
Figure 7. NH040-1 enhances the neurite outgrowth of SCA17 cells.    58
Figure 8. NH040-1 decreases TBP-polyQ aggregation in SCA17 cells.    59
Figure 9. NH040-1 inhibits ER stress in SCA17 cells.    60
Figure 10. Effects of NH040-1 on body weight and motor performance in SCA17 transgenic (Tg) mice    61
Figure 11. Effects of NH040-1 on footprint patterns of SCA17 transgenic mice    63
Figure 12. Effects of NH040-1 on TBP aggregation and ER stress-induced apoptosis in the cerebella of SCA17 transgenic mice    65
Figure 13. Effects of NH040-1 on Purkinje cells in cerebella of SCA17 transgenic mice    66
Figure 14. Scheme of the proposed neuroprotective of NH040-1    67
Supplementary figure    68
Supplementary figure 1. PCR screening of SCA17 transgenic mice    68

                                

1 Lindholm, D., Wootz, H. & Korhonen, L. ER stress and neurodegenerative diseases. Cell death and differentiation 13, 385-392, doi:10.1038/sj.cdd.4401778 (2006).
2 Blum, E. S., Schwendeman, A. R. & Shaham, S. PolyQ disease: misfiring of a developmental cell death program? Trends in cell biology 23, 168-174, doi:10.1016/j.tcb.2012.11.003 (2013).
3 Shao, J. & Diamond, M. I. Polyglutamine diseases: emerging concepts in pathogenesis and therapy. Human molecular genetics 16, R115-R123, doi:Doi 10.1093/Hmg/Ddm213 (2007).
4 Stevanin, G. & Brice, A. Spinocerebellar ataxia 17 (SCA17) and Huntington's disease-like 4 (HDL4). Cerebellum 7, 170-178, doi:10.1007/s12311-008-0016-1 (2008).
5 Toyoshima, Y., Onodera, O., Yamada, M., Tsuji, S. & Takahashi, H. in GeneReviews(R) (eds R. A. Pagon et al.) (1993).
6 Duennwald, M. L. & Lindquist, S. Impaired ERAD and ER stress are early and specific events in polyglutamine toxicity. Genes & development 22, 3308-3319, doi:10.1101/gad.1673408 (2008).
7 Fujita, E. et al. Caspase-12 processing and fragment translocation into nuclei of tunicamycin-treated cells. Cell death and differentiation 9, 1108-1114, doi:Doi 10.1038/Sj.Cdd.4401080 (2002).
8 Kouroku, Y. et al. Polyglutamine aggregates stimulate ER stress signals and caspase-12 activation. Human molecular genetics 11, 1505-1515 (2002).
9 Lee, L. C. et al. Role of the CCAAT-binding protein NFY in SCA17 pathogenesis. PloS one 7, e35302, doi:10.1371/journal.pone.0035302 (2012).
10 Zoghbi, H. Y. & Orr, H. T. Glutamine repeats and neurodegeneration. . Annu. Rev. Neurosci 23, 217-247 (2000).
11 Lipinski, M. M. & Yuan, J. Mechanisms of cell death in polyglutamine expansion diseases. Current opinion in pharmacology 4, 85-90, doi:10.1016/j.coph.2003.09.008 (2004).
12 Paschen, W. & Frandsen, A. Endoplasmic reticulum dysfunction--a common denominator for cell injury in acute and degenerative diseases of the brain? Journal of neurochemistry 79, 719-725 (2001).
13 Lin, J. H., Walter, P. & Yen, T. S. Endoplasmic reticulum stress in disease pathogenesis. Annual review of pathology 3, 399-425, doi:10.1146/annurev.pathmechdis.3.121806.151434 (2008).
14 Ni, M. & Lee, A. S. ER chaperones in mammalian development and human diseases. FEBS letters 581, 3641-3651, doi:10.1016/j.febslet.2007.04.045 (2007).
15 Ron, D. & Walter, P. Signal integration in the endoplasmic reticulum unfolded protein response. Nature reviews. Molecular cell biology 8, 519-529, doi:10.1038/nrm2199 (2007).
16 Yoshida, H. Identification of the cis-Acting Endoplasmic Reticulum Stress Response Element Responsible for Transcriptional Induction of Mammalian Glucose-regulated Proteins. Journal of Biological Chemistry 273, 33741-33749, doi:10.1074/jbc.273.50.33741 (1998).
17 Gorbatyuk, M. S. et al. Glucose regulated protein 78 diminishes alpha-synuclein neurotoxicity in a rat model of Parkinson disease. Molecular therapy : the journal of the American Society of Gene Therapy 20, 1327-1337, doi:10.1038/mt.2012.28 (2012).
18 Cullinan, S. B. & Diehl, J. A. Coordination of ER and oxidative stress signaling: the PERK/Nrf2 signaling pathway. The international journal of biochemistry & cell biology 38, 317-332, doi:10.1016/j.biocel.2005.09.018 (2006).
19 Szegezdi, E., Logue, S. E., Gorman, A. M. & Samali, A. Mediators of endoplasmic reticulum stress-induced apoptosis. EMBO reports 7, 880-885, doi:10.1038/sj.embor.7400779 (2006).
20 Ding, W., Yang, L., Zhang, M. & Gu, Y. Reactive oxygen species-mediated endoplasmic reticulum stress contributes to aldosterone-induced apoptosis in tubular epithelial cells. Biochemical and biophysical research communications 418, 451-456, doi:10.1016/j.bbrc.2012.01.037 (2012).
21 Kim, H. R. et al. Bax inhibitor 1 regulates ER-stress-induced ROS accumulation through the regulation of cytochrome P450 2E1. J Cell Sci 122, 1126-1133, doi:Doi 10.1242/Jcs.038430 (2009).
22 Breckenridge, D. G., Germain, M., Mathai, J. P., Nguyen, M. & Shore, G. C. Regulation of apoptosis by endoplasmic reticulum pathways. Oncogene 22, 8608-8618, doi:10.1038/sj.onc.1207108 (2003).
23 Chinta, S. J. et al. Coupling endoplasmic reticulum stress to the cell death program in dopaminergic cells: effect of paraquat. Neuromolecular medicine 10, 333-342, doi:10.1007/s12017-008-8047-9 (2008).
24 Degterev, A., Boyce, M. & Yuan, J. A decade of caspases. Oncogene 22, 8543-8567, doi:10.1038/sj.onc.1207107 (2003).
25 Li, H., Wu, S., Shi, N., Lian, S. & Lin, W. Nrf2/HO-1 pathway activation by manganese is associated with reactive oxygen species and ubiquitin-proteasome pathway, not MAPKs signaling. Journal of applied toxicology : JAT 31, 690-697, doi:10.1002/jat.1654 (2011).
26 Song, J. X. et al. Anti-Parkinsonian drug discovery from herbal medicines: what have we got from neurotoxic models? Journal of ethnopharmacology 139, 698-711, doi:10.1016/j.jep.2011.12.030 (2012).
27 Sucher, N. J. Insights from molecular investigations of traditional Chinese herbal stroke medicines: implications for neuroprotective epilepsy therapy. Epilepsy & behavior : E&B 8, 350-362, doi:10.1016/j.yebeh.2005.11.015 (2006).
28 Chik, S. C. C., Or, T. C. T., Luo, D., Yang, C. L. H. & Lau, A. S. Y. Pharmacological Effects of Active Compounds on Neurodegenerative Disease with Gastrodia and Uncaria Decoction, a Commonly Used Poststroke Decoction. Sci World J, doi:Artn 896873
Doi 10.1155/2013/896873 (2013).
29 Weon, J. B. et al. Neuroprotective activity of the methanolic extract of Lonicera japonica in glutamate-injured primary rat cortical cells. Pharmacognosy magazine 7, 284-288, doi:10.4103/0973-1296.90404 (2011).
30 Feng, R. et al. Inhibition of activator protein-1, NF-kappaB, and MAPKs and induction of phase 2 detoxifying enzyme activity by chlorogenic acid. The Journal of biological chemistry 280, 27888-27895, doi:10.1074/jbc.M503347200 (2005).
31 Huang, S. M., Chuang, H. C., Wu, C. H. & Yen, G. C. Cytoprotective effects of phenolic acids on methylglyoxal-induced apoptosis in Neuro-2A cells. Molecular nutrition & food research 52, 940-949, doi:10.1002/mnfr.200700360 (2008).
32 Kwon, S. H. et al. Neuroprotective effects of chlorogenic acid on scopolamine-induced amnesia via anti-acetylcholinesterase and anti-oxidative activities in mice. European journal of pharmacology 649, 210-217, doi:10.1016/j.ejphar.2010.09.001 (2010).
33 Ito, H., Sun, X. L., Watanabe, M., Okamoto, M. & Hatano, T. Chlorogenic acid and its metabolite m-coumaric acid evoke neurite outgrowth in hippocampal neuronal cells. Bioscience, biotechnology, and biochemistry 72, 885-888, doi:10.1271/bbb.70670 (2008).
34 Oboh, G., Agunloye, O. M., Akinyemi, A. J., Ademiluyi, A. O. & Adefegha, S. A. Comparative study on the inhibitory effect of caffeic and chlorogenic acids on key enzymes linked to Alzheimer's disease and some pro-oxidant induced oxidative stress in rats' brain-in vitro. Neurochemical research 38, 413-419, doi:10.1007/s11064-012-0935-6 (2013).
35 Santos, R. M. M., Hunter, T., Wright, N. & Lima, D. R. A. Caffeine and Chlorogenic Acids in Coffee and Effects on Selected Neurodegenerative Diseases. Journal of Pharmaceutical and Scientific Innovation 2, 9-17, doi:10.7897/2277-4572.02443 (2013).
36 Kung, P. J. et al. Indole and synthetic derivative activate chaperone expression to reduce polyQ aggregation in SCA17 neuronal cell and slice culture models. Drug design, development and therapy 8, 1929-1939, doi:10.2147/DDDT.S67376 (2014).
37 Wang, X., Fan, Z., Wang, B., Luo, J. & Ke, Z. J. Activation of double-stranded RNA-activated protein kinase by mild impairment of oxidative metabolism in neurons. Journal of neurochemistry 103, 2380-2390, doi:10.1111/j.1471-4159.2007.04978.x (2007).
38 Chien, C. T. et al. Glucagon-like peptide-1 receptor agonist activation ameliorates venous thrombosis-induced arteriovenous fistula failure in chronic kidney disease. Thrombosis and haemostasis 112, 1051-1064, doi:10.1160/TH14-03-0258 (2014).
39 Beauquis, J., Roig, P., De Nicola, A. F. & Saravia, F. Short-term environmental enrichment enhances adult neurogenesis, vascular network and dendritic complexity in the hippocampus of type 1 diabetic mice. PloS one 5, e13993, doi:10.1371/journal.pone.0013993 (2010).
40 Wanker, E. E. et al. Membrane filter assay for detection of amyloid-like polyglutamine-containing protein aggregates. Methods in enzymology 309, 375-386 (1999).
41 Chang, Y. C. et al. Neuroprotective effects of granulocyte-colony stimulating factor in a novel transgenic mouse model of SCA17. Journal of neurochemistry 118, 288-303, doi:10.1111/j.1471-4159.2011.07304.x (2011).
42 Friedman, M. J. et al. Polyglutamine domain modulates the TBP-TFIIB interaction: implications for its normal function and neurodegeneration. Nature neuroscience 10, 1519-1528, doi:10.1038/nn2011 (2007).
43 Yang, S., Huang, S., Gaertig, M. A., Li, X. J. & Li, S. Age-dependent decrease in chaperone activity impairs MANF expression, leading to Purkinje cell degeneration in inducible SCA17 mice. Neuron 81, 349-365, doi:10.1016/j.neuron.2013.12.002 (2014).
44 Kelp, A. et al. A novel transgenic rat model for spinocerebellar ataxia type 17 recapitulates neuropathological changes and supplies in vivo imaging biomarkers. The Journal of neuroscience : the official journal of the Society for Neuroscience 33, 9068-9081, doi:10.1523/JNEUROSCI.5622-12.2013 (2013).
45 Figiel, M., Szlachcic, W. J., Switonski, P. M., Gabka, A. & Krzyzosiak, W. J. Mouse models of polyglutamine diseases: review and data table. Part I. Molecular neurobiology 46, 393-429, doi:10.1007/s12035-012-8315-4 (2012).
46 Oyadomari, S. & Mori, M. Roles of CHOP/GADD153 in endoplasmic reticulum stress. Cell death and differentiation 11, 381-389, doi:10.1038/sj.cdd.4401373 (2004).
47 Sollberger, G., Strittmatter, G. E., Kistowska, M., French, L. E. & Beer, H. D. Caspase-4 Is Required for Activation of Inflammasomes. J Immunol 188, 1992-2000, doi:Doi 10.4049/Jimmunol.1101620 (2012).
48 Wang, X. Z. et al. Identification of novel stress-induced genes downstream of chop. The EMBO journal 17, 3619-3630, doi:10.1093/emboj/17.13.3619 (1998).
49 Malhotra, J. D. & Kaufman, R. J. Endoplasmic reticulum stress and oxidative stress: a vicious cycle or a double-edged sword? Antioxidants & redox signaling 9, 2277-2293, doi:10.1089/ars.2007.1782 (2007).
50 Soto, C. Unfolding the role of protein misfolding in neurodegenerative diseases. Nature reviews. Neuroscience 4, 49-60, doi:10.1038/nrn1007 (2003).
51 Roshan, R., Ghosh, T., Gadgil, M. & Pillai, B. Regulation of BACE1 by miR-29a/b in a cellular model of Spinocerebellar Ataxia 17. RNA biology 9, 891-899, doi:10.4161/rna.19876 (2012).
52 Huang, S. S., Ling, J. J., Yang, S., Li, X. J. & Li, S. H. Neuronal expression of TATA box-binding protein containing expanded polyglutamine in knock-in mice reduces chaperone protein response by impairing the function of nuclear factor-Y transcription factor. Brain 134, 1943-1958, doi:Doi 10.1093/Brain/Awr146 (2011).
53 Nishitoh, H. et al. ASK1 is essential for endoplasmic reticulum stress-induced neuronal cell death triggered by expanded polyglutamine repeats. Genes & development 16, 1345-1355, doi:10.1101/gad.992302 (2002).
54 Sakahira, H., Breuer, P., Hayer-Hartl, M. K. & Hartl, F. U. Molecular chaperones as modulators of polyglutamine protein aggregation and toxicity. P Natl Acad Sci USA 99, 16412-16418, doi:Doi 10.1073/Pnas.182426899 (2002).

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