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研究生：	蕭欣杰 Hsin-Chieh Shiau
論文名稱：	抑制調控果蠅5-磷酸核醣異構酶能減輕Tau引起的毒性 Down-regulation of ribose-5-phosphate isomerase attenuates Tau mediated toxicity in Drosophila
指導教授：	蘇銘燦 Su, Ming-Tsan
學位類別：	博士 Doctor
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：	62
中文關鍵詞：	5-磷酸核醣異構酶
英文關鍵詞：	ribose-5-phosphate isomerase
DOI URL：	https://doi.org/10.6345/NTNU202205436
論文種類：	學術論文
相關次數：	點閱：102 下載：1
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Tauopathy是一種多因子疾病，透過遺傳及分子方法已確認出許多致病途徑。為探究Tau引起的毒性及找出新的治療標的，我們利用眼睛及背毛模式去篩選與其他神經退化疾病有關的致病因子。在篩選研究中，靜默5-磷酸核醣異構酶(rpi)基因呈現出最佳的性狀改善結果。本研究指出在神經中抑制調控rpi基因表現可以延長tauopathy果蠅的壽命及改善運動功能。而在西方墨點實驗結果，顯示出tauopathy果蠅性狀改善不是經由改變Tau蛋白質的磷酸化。此外，進一步發現tauopathy果蠅細胞中煙草醯胺腺嘌呤二核苷酸磷酸鹽(NADPH)及還原態穀胱甘肽(GSH)濃度會上升。我們也得到過量表現TTLL1可減緩Tau的毒性及延長tauopathy果蠅的壽命。因此，在果蠅模式中，減少rpi的表現及大量表現TTLL1可以抑制Tau的毒性。

Tauopathy is a multifactorial disease in which many pathogenic pathways have been identified through genetics and molecular approaches. To better evaluate Tau induced toxicity and to find novel therapeutic targets, we screen components of pathogenic pathways that might implicated in various neurodegenerative diseases using both eye and notal bristle as model systems. We have chosen to study ribose-5-phosphate isomerase (rpi), because silencing of rpi exhibited the greatest phenotypic improvement among all other modifiers in our screening. Our data indicated that reduced neuronal expression of rpi extends the lifespan and improves the motor function of tauopathy flies. Results of immunoblotting experiments reveal that the phosphorylated Tau species were not significantly decreased when rpi were downregulated, suggesting that the beneficial effect of reduced rpi on tauopathy flies is not mediated through altering the phosphorylation of Tau. Additionally, reduced rpi increases cellular nicotinamide adenine dinucleotide phosphate (NADPH) and reduced form of glutathione (GSH) in tauopathy flies. We also found that overexpression of Tubulin tyrosine ligase-like 1 (TTLL1) attenuates Tau toxicity and extend the lifespan of tauopathy flies. Taken together our findings demonstrate that reduced rpi expression and TTLL1 overexpression suppress Tau toxicity in Drosophila.

Abstract (Chinese).....01
Abstract (English).....02
Introduction.....03
Materials and methods.....08
Fly strains and culture conditions.....08
Bristle quantification and eye morphology assessment..09
Lifespan assays.....10
Climbing ability assay.....10
Immunoblotting.....11
NADPH quantitation Assay.....11
Reduced glutathione Assay.....12
Results.....13
A targeted screen for modifiers of tauopathy in Drosophila.....13
Down regulation of rpi extends the lifespan and improves the motor function of tauopathy flies.....16
rpi downregulation does not decrease phosphor-Tau species.....17
Reduced rpi increases cellular NADPH and reduced form of glutathione in tauopathy flies.....18
Overexpression of hTTLL1 attenuates Tau toxicity and extends the lifespan of tauopathy flies.....19
Discussion.....22
Appendix.....26
Abstract (Chinese).....28
Abstract (English.....29
Introduction.....30
Materials and Methods.....35
Dual luciferase reporter constructs.....35
Cell culture, transfections, and luciferase assay.....36
Results.....37
IRES activity of ATXN8OS RNA.....37
Discussion.....39
References.....40
Figures and Tables.....46
Figure 1. Genetic modifier screening of Tau induced toxicity in Drosophila eye and notal bristle of 1-day-old adult flies.....46
Figure 2. SEM of eye micrography and notum used in the genetic modifier screens.....49
Figure 3. Downregulation of rpi extends the lifespan and improves the motor function of tauopathy flies.....50
Figure 4. The knockdown of rpi does not alter Tau phosphorylation.....52
Figure 5. Neuronal downregulation of rpi increases NADPH and the reduced form of glutathione levels in the tauopathy flies.....54
Figure 6. Reduced tubulin tyrosine ligase-like (TTLL) genes expressions enhance Tau induced toxicity.....55
Figure 7. Overexpression of hTTLL1 attenuates Tau toxicity and extends the lifespan of tauopathy flies.....57
Figure 8. Schematic diagrams of the ATXN8OS cDNA and pRF dual luciferase reporter constructs.....59
Figure 9. IRES activity of the ATXN8OS transcript.....60
Table 1. Genetic modifiers of Tau toxicity.....62

                                

1. Hong M, Zhukareva V, Vogelsberg-Ragaglia V, Wszolek Z, Reed L, et al. (1998) Mutation-specific functional impairments in distinct tau isoforms of hereditary FTDP-17. Science 282: 1914-1917.
2. Hutton M, Lendon CL, Rizzu P, Baker M, Froelich S, et al. (1998) Association of missense and 5'-splice-site mutations in tau with the inherited dementia FTDP-17. Nature 393: 702-705.
3. Spillantini MG, Murrell JR, Goedert M, Farlow MR, Klug A, et al. (1998) Mutation in the tau gene in familial multiple system tauopathy with presenile dementia. Proc Natl Acad Sci U S A 95: 7737-7741.
4. Goedert M, Jakes R (2005) Mutations causing neurodegenerative tauopathies. Biochim Biophys Acta 1739: 240-250.
5. Braak H, Braak E (1991) Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82: 239-259.
6. Arriagada PV, Growdon JH, Hedley-Whyte ET, Hyman BT (1992) Neurofibrillary tangles but not senile plaques parallel duration and severity of Alzheimer's disease. Neurology 42: 631-639.
7. Ingram EM, Spillantini MG (2002) Tau gene mutations: dissecting the pathogenesis of FTDP-17. Trends Mol Med 8: 555-562.
8. Steinhilb ML, Dias-Santagata D, Mulkearns EE, Shulman JM, Biernat J, et al. (2007) S/P and T/P phosphorylation is critical for tau neurotoxicity in Drosophila. J Neurosci Res 85: 1271-1278.
9. Alonso A, Zaidi T, Novak M, Grundke-Iqbal I, Iqbal K (2001) Hyperphosphorylation induces self-assembly of tau into tangles of paired helical filaments/straight filaments. Proc Natl Acad Sci U S A 98: 6923-6928.
10. Taniguchi T, Kawamata T, Mukai H, Hasegawa H, Isagawa T, et al. (2001) Phosphorylation of tau is regulated by PKN. J Biol Chem 276: 10025-10031.
11. Boucher D, Larcher JC, Gros F, Denoulet P (1994) Polyglutamylation of tubulin as a progressive regulator of in vitro interactions between the microtubule-associated protein Tau and tubulin. Biochemistry 33: 12471-12477.
12. Ersfeld K, Wehland J, Plessmann U, Dodemont H, Gerke V, et al. (1993) Characterization of the tubulin-tyrosine ligase. J Cell Biol 120: 725-732.
13. Rogowski K, Juge F, van Dijk J, Wloga D, Strub JM, et al. (2009) Evolutionary divergence of enzymatic mechanisms for posttranslational polyglycylation. Cell 137: 1076-1087.
14. van Dijk J, Rogowski K, Miro J, Lacroix B, Edde B, et al. (2007) A targeted multienzyme mechanism for selective microtubule polyglutamylation. Mol Cell 26: 437-448.
15. Trichet V, Ruault M, Roizes G, De Sario A (2000) Characterization of the human tubulin tyrosine ligase-like 1 gene (TTLL1) mapping to 22q13.1. Gene 257: 109-117.
16. Regnard C, Fesquet D, Janke C, Boucher D, Desbruyeres E, et al. (2003) Characterisation of PGs1, a subunit of a protein complex co-purifying with tubulin polyglutamylase. J Cell Sci 116: 4181-4190.
17. Janke C, Rogowski K, Wloga D, Regnard C, Kajava AV, et al. (2005) Tubulin polyglutamylase enzymes are members of the TTL domain protein family. Science 308: 1758-1762.
18. Wei H, Kim SJ, Zhang Z, Tsai PC, Wisniewski KE, et al. (2008) ER and oxidative stresses are common mediators of apoptosis in both neurodegenerative and non-neurodegenerative lysosomal storage disorders and are alleviated by chemical chaperones. Hum Mol Genet 17: 469-477.
19. Dias-Santagata D, Fulga TA, Duttaroy A, Feany MB (2007) Oxidative stress mediates tau-induced neurodegeneration in Drosophila. J Clin Invest 117: 236-245.
20. Wang CT, Chen YC, Wang YY, Huang MH, Yen TL, et al. (2012) Reduced neuronal expression of ribose-5-phosphate isomerase enhances tolerance to oxidative stress, extends lifespan, and attenuates polyglutamine toxicity in Drosophila. Aging Cell 11: 93-103.
21. Wentzell J, Kretzschmar D (2010) Alzheimer's Disease and tauopathy studies in flies and worms. Neurobiol Dis 40: 21-28.
22. Iijima-Ando K, Iijima K (2010) Transgenic Drosophila models of Alzheimer's disease and tauopathies. Brain Struct Funct 214: 245-262.
23. Feuillette S, Miguel L, Frebourg T, Campion D, Lecourtois M (2010) Drosophila models of human tauopathies indicate that Tau protein toxicity in vivo is mediated by soluble cytosolic phosphorylated forms of the protein. J Neurochem 113: 895-903.
24. Chen X, Li Y, Huang J, Cao D, Yang G, et al. (2007) Study of tauopathies by comparing Drosophila and human tau in Drosophila. Cell Tissue Res 329: 169-178.
25. Jackson GR, Wiedau-Pazos M, Sang TK, Wagle N, Brown CA, et al. (2002) Human wild-type tau interacts with wingless pathway components and produces neurofibrillary pathology in Drosophila. Neuron 34: 509-519.
26. Wittmann CW, Wszolek MF, Shulman JM, Salvaterra PM, Lewis J, et al. (2001) Tauopathy in Drosophila: neurodegeneration without neurofibrillary tangles. Science 293: 711-714.
27. Shulman JM, Imboywa S, Giagtzoglou N, Powers MP, Hu Y, et al. (2014) Functional screening in Drosophila identifies Alzheimer's disease susceptibility genes and implicates Tau-mediated mechanisms. Hum Mol Genet 23: 870-877.
28. Ambegaokar SS, Jackson GR (2011) Functional genomic screen and network analysis reveal novel modifiers of tauopathy dissociated from tau phosphorylation. Hum Mol Genet 20: 4947-4977.
29. Wheeler JM, Guthrie CR, Kraemer BC (2010) The role of MSUT-2 in tau neurotoxicity: a target for neuroprotection in tauopathy? Biochem Soc Trans 38: 973-976.
30. Blard O, Feuillette S, Bou J, Chaumette B, Frebourg T, et al. (2007) Cytoskeleton proteins are modulators of mutant tau-induced neurodegeneration in Drosophila. Hum Mol Genet 16: 555-566.
31. Karsten SL, Sang TK, Gehman LT, Chatterjee S, Liu J, et al. (2006) A genomic screen for modifiers of tauopathy identifies puromycin-sensitive aminopeptidase as an inhibitor of tau-induced neurodegeneration. Neuron 51: 549-560.
32. Shulman JM, Feany MB (2003) Genetic modifiers of tauopathy in Drosophila. Genetics 165: 1233-1242.
33. Yeh PA, Chien JY, Chou CC, Huang YF, Tang CY, et al. (2010) Drosophila notal bristle as a novel assessment tool for pathogenic study of Tau toxicity and screening of therapeutic compounds. Biochem Biophys Res Commun 391: 510-516.
34. Tang CY, Sun YH (2002) Use of mini-white as a reporter gene to screen for GAL4 insertions with spatially restricted expression pattern in the developing eye in drosophila. Genesis 34: 39-45.
35. Todd AM, Staveley BE (2008) Pink1 suppresses alpha-synuclein-induced phenotypes in a Drosophila model of Parkinson's disease. Genome 51: 1040-1046.
36. Zhu XC, Yu JT, Jiang T, Tan L (2013) Autophagy modulation for Alzheimer's disease therapy. Mol Neurobiol 48: 702-714.
37. Lee MJ, Lee JH, Rubinsztein DC (2013) Tau degradation: the ubiquitin-proteasome system versus the autophagy-lysosome system. Prog Neurobiol 105: 49-59.
38. Petrucelli L, Dickson D, Kehoe K, Taylor J, Snyder H, et al. (2004) CHIP and Hsp70 regulate tau ubiquitination, degradation and aggregation. Hum Mol Genet 13: 703-714.
39. Sahara N, Murayama M, Mizoroki T, Urushitani M, Imai Y, et al. (2005) In vivo evidence of CHIP up-regulation attenuating tau aggregation. J Neurochem 94: 1254-1263.
40. Wang Y, Martinez-Vicente M, Kruger U, Kaushik S, Wong E, et al. (2009) Tau fragmentation, aggregation and clearance: the dual role of lysosomal processing. Hum Mol Genet 18: 4153-4170.
41. Keck S, Nitsch R, Grune T, Ullrich O (2003) Proteasome inhibition by paired helical filament-tau in brains of patients with Alzheimer's disease. J Neurochem 85: 115-122.
42. Lee VM, Goedert M, Trojanowski JQ (2001) Neurodegenerative tauopathies. Annu Rev Neurosci 24: 1121-1159.
43. Yen SH, Liu WK, Hall FL, Yan SD, Stern D, et al. (1995) Alzheimer neurofibrillary lesions: molecular nature and potential roles of different components. Neurobiol Aging 16: 381-387.
44. Spires-Jones TL, Stoothoff WH, de Calignon A, Jones PB, Hyman BT (2009) Tau pathophysiology in neurodegeneration: a tangled issue. Trends Neurosci 32: 150-159.
45. Voss K, Combs B, Patterson KR, Binder LI, Gamblin TC (2012) Hsp70 alters tau function and aggregation in an isoform specific manner. Biochemistry 51: 888-898.
46. Gistelinck M, Lambert JC, Callaerts P, Dermaut B, Dourlen P (2012) Drosophila models of tauopathies: what have we learned? Int J Alzheimers Dis 2012: 970980.
47. Mutsuddi M, Marshall CM, Benzow KA, Koob MD, Rebay I (2004) The spinocerebellar ataxia 8 noncoding RNA causes neurodegeneration and associates with staufen in Drosophila. Curr Biol 14: 302-308.
48. Hadad M, Bresler-Musikant T, Neuman-Silberberg FS (2011) Drosophila spoonbill encodes a dual-specificity A-kinase anchor protein essential for oogenesis. Mech Dev 128: 471-482.
49. Lu Y, Lu YS, Shuai Y, Feng C, Tully T, et al. (2007) The AKAP Yu is required for olfactory long-term memory formation in Drosophila. Proc Natl Acad Sci U S A 104: 13792-13797.
50. Carnegie GK, Means CK, Scott JD (2009) A-kinase anchoring proteins: from protein complexes to physiology and disease. IUBMB Life 61: 394-406.
51. Harding AE (1982) The clinical features and classification of the late onset autosomal dominant cerebellar ataxias. A study of 11 families, including descendants of the 'the Drew family of Walworth'. Brain 105: 1-28.
52. Koob MD, Moseley ML, Schut LJ, Benzow KA, Bird TD, et al. (1999) An untranslated CTG expansion causes a novel form of spinocerebellar ataxia (SCA8). Nat Genet 21: 379-384.
53. Day JW, Schut LJ, Moseley ML, Durand AC, Ranum LP (2000) Spinocerebellar ataxia type 8: clinical features in a large family. Neurology 55: 649-657.
54. He Y, Zu T, Benzow KA, Orr HT, Clark HB, et al. (2006) Targeted deletion of a single Sca8 ataxia locus allele in mice causes abnormal gait, progressive loss of motor coordination, and Purkinje cell dendritic deficits. J Neurosci 26: 9975-9982.
55. Chen IC, Lin HY, Lee GC, Kao SH, Chen CM, et al. (2009) Spinocerebellar ataxia type 8 larger triplet expansion alters histone modification and induces RNA foci. BMC Mol Biol 10: 9.
56. Moseley ML, Zu T, Ikeda Y, Gao W, Mosemiller AK, et al. (2006) Bidirectional expression of CUG and CAG expansion transcripts and intranuclear polyglutamine inclusions in spinocerebellar ataxia type 8. Nat Genet 38: 758-769.
57. Nemes JP, Benzow KA, Moseley ML, Ranum LP, Koob MD (2000) The SCA8 transcript is an antisense RNA to a brain-specific transcript encoding a novel actin-binding protein (KLHL1). Hum Mol Genet 9: 1543-1551.
58. Benzow KA, Koob MD (2002) The KLHL1-antisense transcript ( KLHL1AS) is evolutionarily conserved. Mamm Genome 13: 134-141.
59. Chen WL, Lin JW, Huang HJ, Wang SM, Su MT, et al. (2008) SCA8 mRNA expression suggests an antisense regulation of KLHL1 and correlates to SCA8 pathology. Brain Res 1233: 176-184.
60. Robinson DN, Cooley L (1997) Drosophila kelch is an oligomeric ring canal actin organizer. J Cell Biol 138: 799-810.
61. Jiang S, Seng S, Avraham HK, Fu Y, Avraham S (2007) Process elongation of oligodendrocytes is promoted by the Kelch-related protein MRP2/KLHL1. J Biol Chem 282: 12319-12329.
62. Soltysik-Espanola M, Rogers RA, Jiang S, Kim TA, Gaedigk R, et al. (1999) Characterization of Mayven, a novel actin-binding protein predominantly expressed in brain. Mol Biol Cell 10: 2361-2375.
63. Hernandez MC, Andres-Barquin PJ, Martinez S, Bulfone A, Rubenstein JL, et al. (1997) ENC-1: a novel mammalian kelch-related gene specifically expressed in the nervous system encodes an actin-binding protein. J Neurosci 17: 3038-3051.
64. Ranum LP, Day JW (2004) Pathogenic RNA repeats: an expanding role in genetic disease. Trends Genet 20: 506-512.
65. Martinez-Salas E, Pineiro D, Fernandez N (2012) Alternative Mechanisms to Initiate Translation in Eukaryotic mRNAs. Comp Funct Genomics 2012: 391546.
66. Stoneley M, Willis AE (2004) Cellular internal ribosome entry segments: structures, trans-acting factors and regulation of gene expression. Oncogene 23: 3200-3207.
67. Spriggs KA, Bushell M, Willis AE (2010) Translational regulation of gene expression during conditions of cell stress. Mol Cell 40: 228-237.
68. Pacheco A, Martinez-Salas E (2010) Insights into the biology of IRES elements through riboproteomic approaches. J Biomed Biotechnol 2010: 458927.
69. Mokrejs M, Vopalensky V, Kolenaty O, Masek T, Feketova Z, et al. (2006) IRESite: the database of experimentally verified IRES structures (www.iresite.org). Nucleic Acids Res 34: D125-130.
70. Chen IC, Lin HY, Hsiao YC, Chen CM, Wu YR, et al. (2013) Internal ribosome entry segment activity of ATXN8 opposite strand RNA. PLoS One 8: e73885.

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