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完整後設資料紀錄
DC 欄位 | 值 | 語言 |
---|---|---|
dc.contributor.advisor | 戴桓青(Hwan-Ching Tai) | |
dc.contributor.author | Yu-Ling Huang | en |
dc.contributor.author | 黃于玲 | zh_TW |
dc.date.accessioned | 2021-06-08T03:46:36Z | - |
dc.date.copyright | 2019-08-07 | |
dc.date.issued | 2018 | |
dc.date.submitted | 2019-02-08 | |
dc.identifier.citation | 1. Jessell, T.M. & Kandel, E.R. Synaptic transmission: a bidirectional and self-modifiable form of cell-cell communication. Cell 72 Suppl, 1-30 (1993).
2. Ferrante, M., Migliore, M. & Ascoli, G.A. Functional impact of dendritic branch point morphology. J. Neurosci. 33, 2156-2165 (2013). 3. Ishibashi, T. et al. Astrocytes Promote Myelination in Response to Electrical Impulses. Neuron 49, 823-832 (2006). 4. Fields, R.D. & Stevens-Graham, B. New Insights into Neuron-Glia Communication. Science 298, 556-562 (2002). 5. Hanisch, U.K. & Kettenmann, H. Microglia: active sensor and versatile effector cells in the normal and pathologic brain. Nat. Neurosci. 10, 1387-1394 (2007). 6. Sofroniew, M.V. & Vinters, H.V. Astrocytes: biology and pathology. Acta Neuropathol 119, 7-35 (2010). 7. Araque, A. & Navarrete, M. Glial cells in neuronal network function. Philos Trans R Soc Lond B Biol Sci 365, 2375-2381 (2010). 8. Cabezas, R. et al. Astrocytic modulation of blood brain barrier: perspectives on Parkinson’s disease. Front Cell Neurosci 8 (2014). 9. Drachman, D.A. Do we have brain to spare? Neurology 64, 2004-2005 (2005). 10. Sherrington, C.S. The integrative action of the nervous system. (Yale University Press, New Haven, CT, US; 1906). 11. Finger, S. Origins of neuroscience: A history of explorations into brain function. (Oxford University Press, New York, NY, US; 1994). 12. Valenstein, E.S. The Discovery of Chemical Neurotransmitters. Brain Cogn. 49, 73-95 (2002). 13. Hormuzdi, S.G., Filippov, M.A., Mitropoulou, G., Monyer, H. & Bruzzone, R. Electrical synapses: a dynamic signaling system that shapes the activity of neuronal networks. Biochim. Biophys. Acta 1662, 113-137 (2004). 14. Pereda, A.E. et al. Gap junction-mediated electrical transmission: regulatory mechanisms and plasticity. Biochim. Biophys. Acta 1828, 134-146 (2013). 15. Kandel, E.R., Schwartz, J.H., Jessell, T.M., Siegelbaum, S.A. & Hudspeth, A.J. Principles of neural science, Vol. 4. (McGraw-hill New York, 2000). 16. Si, B. & Song, E. Recent Advances in the Detection of Neurotransmitters. Chemosensors 6, 1 (2018). 17. Halterman, M.W. Neuroscience, 3rd Edition. Neurology 64, 769-769-a (2005). 18. Berchtold, N.C. & Cotman, C.W. Evolution in the conceptualization of dementia and Alzheimer's disease: Greco-Roman period to the 1960s. Neurobiol. Aging 19, 173-189 (1998). 19. 2018 Alzheimer's disease facts and figures. Alzheimers Dement 14, 367-429 (2018). 20. Brookmeyer, R., Johnson, E., Ziegler-Graham, K. & Arrighi, H.M. Forecasting the global burden of Alzheimer's disease. Alzheimers Dement 3, 186-191 (2007). 21. Bae, J.R. & Kim, S.H. Synapses in neurodegenerative diseases. BMB Rep 50, 237-246 (2017). 22. Scheff, S.W., Price, D.A., Schmitt, F.A., DeKosky, S.T. & Mufson, E.J. Synaptic alterations in CA1 in mild Alzheimer disease and mild cognitive impairment. Neurology 68, 1501-1508 (2007). 23. Holmes, C. et al. Long-term effects of Abeta42 immunisation in Alzheimer's disease: follow-up of a randomised, placebo-controlled phase I trial. Lancet 372, 216-223 (2008). 24. Nicolas, M. & Hassan, B.A. Amyloid precursor protein and neural development. Development 141, 2543-2548 (2014). 25. Braak, H. & Braak, E. Neuropathological stageing of Alzheimer-related changes. Acta Neuropathol 82, 239-259 (1991). 26. Iqbal, K. et al. Tau pathology in Alzheimer disease and other tauopathies. Biochim. Biophys. Acta 1739, 198-210 (2005). 27. Brundin, P., Melki, R. & Kopito, R. Prion-like transmission of protein aggregates in neurodegenerative diseases. Nat. Rev. Mol. Cell Biol. 11, 301-307 (2010). 28. Saman, S. et al. Exosome-associated tau is secreted in tauopathy models and is selectively phosphorylated in cerebrospinal fluid in early Alzheimer disease. J. Biol. Chem. 287, 3842-3849 (2012). 29. Clavaguera, F. et al. Transmission and spreading of tauopathy in transgenic mouse brain. Nat. Cell Biol. 11, 909-913 (2009). 30. Mahley, R.W. Apolipoprotein E: cholesterol transport protein with expanding role in cell biology. Science 240, 622-630 (1988). 31. Liu, C.C., Liu, C.C., Kanekiyo, T., Xu, H. & Bu, G. Apolipoprotein E and Alzheimer disease: risk, mechanisms and therapy. Nat Rev Neurol 9, 106-118 (2013). 32. Bales, K.R., Du, Y., Holtzman, D., Cordell, B. & Paul, S.M. Neuroinflammation and Alzheimer's disease: critical roles for cytokine/Abeta-induced glial activation, NF-kappaB, and apolipoprotein E. Neurobiol. Aging 21, 427-432; discussion 451-423 (2000). 33. Koffie, R.M. et al. Apolipoprotein E4 effects in Alzheimer's disease are mediated by synaptotoxic oligomeric amyloid-beta. Brain 135, 2155-2168 (2012). 34. Dumanis, S.B., DiBattista, A.M., Miessau, M., Moussa, C.E. & Rebeck, G.W. APOE genotype affects the pre-synaptic compartment of glutamatergic nerve terminals. J. Neurochem. 124, 4-14 (2013). 35. Costa, C.J. & Willis, D.E. To the end of the line: Axonal mRNA transport and local translation in health and neurodegenerative disease. Dev Neurobiol 78, 209-220 (2018). 36. Glock, C., Heumuller, M. & Schuman, E.M. mRNA transport & local translation in neurons. Curr. Opin. Neurobiol. 45, 169-177 (2017). 37. Mohr, E., Fehr, S. & Richter, D. Axonal transport of neuropeptide encoding mRNAs within the hypothalamo-hypophyseal tract of rats. EMBO J. 10, 2419-2424 (1991). 38. Conner, J.M., Lauterborn, J.C., Yan, Q., Gall, C.M. & Varon, S. Distribution of brain-derived neurotrophic factor (BDNF) protein and mRNA in the normal adult rat CNS: evidence for anterograde axonal transport. J. Neurosci. 17, 2295-2313 (1997). 39. Gomes, C., Merianda, T.T., Lee, S.J., Yoo, S. & Twiss, J.L. Molecular determinants of the axonal mRNA transcriptome. Dev Neurobiol 74, 218-232 (2014). 40. Knowles, R.B. et al. Translocation of RNA granules in living neurons. J. Neurosci. 16, 7812-7820 (1996). 41. Köhrmann, M. et al. Microtubule-dependent Recruitment of Staufen-Green Fluorescent Protein into Large RNA-containing Granules and Subsequent Dendritic Transport in Living Hippocampal Neurons. Mol. Biol. Cell 10, 2945-2953 (1999). 42. Dynes, J.L. & Steward, O. Dynamics of bidirectional transport of Arc mRNA in neuronal dendrites. J. Comp. Neurol. 500, 433-447 (2007). 43. Dictenberg, J.B., Swanger, S.A., Antar, L.N., Singer, R.H. & Bassell, G.J. A direct role for FMRP in activity-dependent dendritic mRNA transport links filopodial-spine morphogenesis to fragile X syndrome. Dev. Cell 14, 926-939 (2008). 44. Doyle, M. & Kiebler, M.A. Mechanisms of dendritic mRNA transport and its role in synaptic tagging. EMBO J. 30, 3540-3552 (2011). 45. Tesseur, I. et al. Prominent axonopathy and disruption of axonal transport in transgenic mice expressing human apolipoprotein E4 in neurons of brain and spinal cord. Am J Pathol 157, 1495-1510 (2000). 46. Christensen, D.Z., Huettenrauch, M., Mitkovski, M., Pradier, L. & Wirths, O. Axonal degeneration in an Alzheimer mouse model is PS1 gene dose dependent and linked to intraneuronal Aβ accumulation. Front Aging Neurosci. 6, 139 (2014). 47. Khan, U.A. et al. Molecular drivers and cortical spread of lateral entorhinal cortex dysfunction in preclinical Alzheimer's disease. Nat. Neurosci. 17, 304-311 (2014). 48. Khalil, B., Morderer, D., Price, P.L., Liu, F. & Rossoll, W. mRNP assembly, axonal transport, and local translation in neurodegenerative diseases. Brain Res. 1693, 75-91 (2018). 49. Cotman, C., Brown, D.H., Harrell, B.W. & Anderson, N.G. Analytical differential centrifugation: An analysis of the sedimentation properties of synaptosomes, mitochondria and lysosomes from rat brain homogenates. Arch. Biochem. Biophys. 136, 436-447 (1970). 50. Gray, E.G. & Whittaker, V.P. The isolation of nerve endings from brain: an electron-microscopic study of cell fragments derived by homogenization and centrifugation. J. Anat. 96, 79-88 (1962). 51. Hollingsworth, E.B. et al. Biochemical characterization of a filtered synaptoneurosome preparation from guinea pig cerebral cortex: cyclic adenosine 3':5'-monophosphate-generating systems, receptors, and enzymes. J. Neurosci. 5, 2240-2253 (1985). 52. Johnson, M.W., Chotiner, J.K. & Watson, J.B. Isolation and characterization of synaptoneurosomes from single rat hippocampal slices. J. Neurosci. Methods 77, 151-156 (1997). 53. Booth, R.F. & Clark, J.B. A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain. Biochem. J. 176, 365-370 (1978). 54. Jhou, J.F. & Tai, H.C. The Study of Postmortem Human Synaptosomes for Understanding Alzheimer's Disease and Other Neurological Disorders: A Review. Neurol Ther 6, 57-68 (2017). 55. Brown, M. & Wittwer, C. Flow Cytometry: Principles and Clinical Applications in Hematology. Clin. Chem. 46, 1221-1229 (2000). 56. Murphy, R.F. Analysis and isolation of endocytic vesicles by flow cytometry and sorting: demonstration of three kinetically distinct compartments involved in fluid-phase endocytosis. Proc. Natl. Acad. Sci. U.S.A. 82, 8523-8526 (1985). 57. Wolf, M.E. & Kapatos, G. Flow cytometric analysis of rat striatal nerve terminals. J. Neurosci. 9, 94-105 (1989). 58. Gylys, K.H., Fein, J.A. & Cole, G.M. Quantitative characterization of crude synaptosomal fraction (P-2) components by flow cytometry. J. Neurosci. Res. 61, 186-192 (2000). 59. Gylys, K.H. et al. Synaptic changes in Alzheimer's disease: increased amyloid-beta and gliosis in surviving terminals is accompanied by decreased PSD-95 fluorescence. Am J Pathol 165, 1809-1817 (2004). 60. Bilousova, T. et al. Synaptic Amyloid-beta Oligomers Precede p-Tau and Differentiate High Pathology Control Cases. Am J Pathol 186, 185-198 (2016). 61. Levy, M. & Miller, S.L. The stability of the RNA bases: Implications for the origin of life. Proc. Natl. Acad. Sci. U.S.A. 95, 7933-7938 (1998). 62. Vomelova, I., Vanickova, Z. & Sedo, A. Methods of RNA purification. All ways (should) lead to Rome. Folia Biol (Praha) 55, 243-251 (2009). 63. Arya, M. et al. Basic principles of real-time quantitative PCR. Expert Rev Mol Diagn 5, 209-219 (2005). 64. Navarro, E., Serrano-Heras, G., Castano, M.J. & Solera, J. Real-time PCR detection chemistry. Clin Chim Acta 439, 231-250 (2015). 65. Kim, J., Lim, J. & Lee, C. Quantitative real-time PCR approaches for microbial community studies in wastewater treatment systems: Applications and considerations. Biotechnol. Adv. 31, 1358-1373 (2013). 66. Sanger, F. et al. Nucleotide sequence of bacteriophage φX174 DNA. Nature 265, 687 (1977). 67. Sanger, F., Nicklen, S. & Coulson, A.R. DNA sequencing with chain-terminating inhibitors. Proc. Natl. Acad. Sci. U.S.A. 74, 5463-5467 (1977). 68. Staden, R. Automation of the computer handling of gel reading data produced by the shotgun method of DNA sequencing. Nucleic Acids Res. 10, 4731-4751 (1982). 69. Md, F. et al. Pyrosequencing-principles and applications. Int. j. life sci. pharma res. 2, 65-76 (2012). 70. Bahassi el, M. & Stambrook, P.J. Next-generation sequencing technologies: breaking the sound barrier of human genetics. Mutagenesis 29, 303-310 (2014). 71. Mutz, K.-O., Heilkenbrinker, A., Lönne, M., Walter, J.-G. & Stahl, F. Transcriptome analysis using next-generation sequencing. Curr. Opin. Biotechnol. 24, 22-30 (2013). 72. Narlikar, L. & Jothi, R. ChIP-Seq Data Analysis: Identification of Protein–DNA Binding Sites with SISSRs Peak-Finder. Methods Mol Biol. 802, 305-322 (2012). 73. Sarda, S. & Hannenhalli, S. Next-Generation Sequencing and Epigenomics Research: A Hammer in Search of Nails. Genomics Inform. 12, 2-11 (2014). 74. Han, Y., Gao, S., Muegge, K., Zhang, W. & Zhou, B. Advanced Applications of RNA Sequencing and Challenges. Bioinform Biol Insights 9, 29-46 (2015). 75. Gagan, J. & Van Allen, E.M. Next-generation sequencing to guide cancer therapy. Genome Med 7, 80 (2015). 76. Twine, N.A., Janitz, K., Wilkins, M.R. & Janitz, M. Whole Transcriptome Sequencing Reveals Gene Expression and Splicing Differences in Brain Regions Affected by Alzheimer's Disease. PLoS ONE 6, e16266 (2011). 77. Gylys, K.H., Fein, J.A., Yang, F. & Cole, G.M. Enrichment of presynaptic and postsynaptic markers by size-based gating analysis of synaptosome preparations from rat and human cortex. Cytometry A 60, 90-96 (2004). 78. Zou, Y.F. Analysis of synaptic Apolipoprotein E localization by immunofluorescence microscopy and flow cytometry. Master thesis of Depatment of Chemistry, College of Science, National Taiwan University, Taipei, Taiwan (2016). 79. Steward, O., Farris, S., Pirbhoy, P.S., Darnell, J. & Driesche, S.J.V. Localization and local translation of Arc/Arg3.1 mRNA at synapses: some observations and paradoxes. Frontiers in Molecular Neuroscience 7 (2015). 80. Moreton, J., Izquierdo, A. & Emes, R.D. Assembly, Assessment, and Availability of De novo Generated Eukaryotic Transcriptomes. Front Genet 6, 361 (2015). 81. Li, Z. et al. Comparison of the two major classes of assembly algorithms: overlap-layout-consensus and de-bruijn-graph. Brief Funct Genomics 11, 25-37 (2012). 82. Lischer, H.E.L. & Shimizu, K.K. Reference-guided de novo assembly approach improves genome reconstruction for related species. BMC Bioinformatics 18, 474 (2017). 83. Robinson, M.D., McCarthy, D.J. & Smyth, G.K. edgeR: a Bioconductor package for differential expression analysis of digital gene expression data. Bioinformatics 26, 139-140 (2010). 84. Budnik, V., Ruiz-Cañada, C. & Wendler, F. Extracellular vesicles round off communication in the nervous system. Nat. Rev. Neurosci. 17, 160-172 (2016). 85. Ben-Shachar, D. & Laifenfeld, D. Mitochondria, synaptic plasticity, and schizophrenia. Int. Rev. Neurobiol. 59, 273-296 (2004). 86. Poon, M.M., Choi, S.H., Jamieson, C.A., Geschwind, D.H. & Martin, K.C. Identification of process-localized mRNAs from cultured rodent hippocampal neurons. J. Neurosci. 26, 13390-13399 (2006). 87. Bi, J., Tsai, N.P., Lin, Y.P., Loh, H.H. & Wei, L.N. Axonal mRNA transport and localized translational regulation of kappa-opioid receptor in primary neurons of dorsal root ganglia. Proc Natl Acad Sci U S A 103, 19919-19924 (2006). 88. Willis, D.E. et al. Extracellular stimuli specifically regulate localized levels of individual neuronal mRNAs. J. Cell Biol. 178, 965-980 (2007). 89. Raj, A., van den Bogaard, P., Rifkin, S.A., van Oudenaarden, A. & Tyagi, S. Imaging individual mRNA molecules using multiple singly labeled probes. Nat. Methods 5, 877 (2008). 90. Taylor, A.M. et al. Axonal mRNA in uninjured and regenerating cortical mammalian axons. J. Neurosci. 29, 4697-4707 (2009). 91. Zivraj, K.H. et al. Subcellular Profiling Reveals Distinct and Developmentally Regulated Repertoire of Growth Cone mRNAs. J. Neurosci. 30, 15464-15478 (2010). 92. Gumy, L.F. et al. Transcriptome analysis of embryonic and adult sensory axons reveals changes in mRNA repertoire localization. RNA 17, 85-98 (2011). 93. Cajigas, I.J. et al. The local transcriptome in the synaptic neuropil revealed by deep sequencing and high-resolution imaging. Neuron 74, 453-466 (2012). 94. Merianda, T.T., Vuppalanchi, D., Yoo, S., Blesch, A. & Twiss, J.L. Axonal transport of neural membrane protein 35 mRNA increases axon growth. J. Cell Sci. 126, 90-102 (2013). 95. Minis, A. et al. Subcellular transcriptomics-dissection of the mRNA composition in the axonal compartment of sensory neurons. Dev Neurobiol 74, 365-381 (2014). 96. Ainsley, J.A., Drane, L., Jacobs, J., Kittelberger, K.A. & Reijmers, L.G. Functionally diverse dendritic mRNAs rapidly associate with ribosomes following a novel experience. Nat Commun 5, 4510 (2014). 97. Taliaferro, J.M. et al. Distal Alternative Last Exons Localize mRNAs to Neural Projections. Mol. Cell 61, 821-833 (2016). 98. Shigeoka, T. et al. Dynamic Axonal Translation in Developing and Mature Visual Circuits. Cell 166, 181-192 (2016). 99. Briese, M. et al. Whole transcriptome profiling reveals the RNA content of motor axons. Nucleic Acids Res. 44, e33 (2016). 100. Bigler, R.L., Kamande, J.W., Dumitru, R., Niedringhaus, M. & Taylor, A.M. Messenger RNAs localized to distal projections of human stem cell derived neurons. Sci. Rep. 7, 611 (2017). 101. Aschrafi, A., Gioio, A.E., Dong, L. & Kaplan, B.B. Disruption of the Axonal Trafficking of Tyrosine Hydroxylase mRNA Impairs Catecholamine Biosynthesis in the Axons of Sympathetic Neurons. eneuro 4 (2017). 102. Vidaki, M. et al. A Requirement for Mena, an Actin Regulator, in Local mRNA Translation in Developing Neurons. Neuron 95, 608-622.e605 (2017). 103. Terenzio, M. et al. Locally translated mTOR controls axonal local translation in nerve injury. Science 359, 1416-1421 (2018). | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/21783 | - |
dc.description.abstract | 許多神經系統疾病例如癡呆症,自閉症,精神分裂症,精神發育遲滯等疾病的根本原因皆與突觸功能障礙有關。其中,最常見的老年性癡呆症─阿茲海默症,其慢性的神經退化過程中會伴隨著神經突觸的喪失,進而導致神經凋亡。與老年斑、神經元纖維纏結、β-澱粉樣蛋白斑、過度磷酸化的Tau蛋白等相比,突觸喪失與阿茲海默症中的認知能力下降更有密切的關聯性。阿茲海默症的致病機制主要與三種重要的蛋白質相關,分別為β-澱粉樣蛋白、Tau蛋白和ApoE載脂蛋白,而這三種蛋白皆為神經突觸蛋白。
當人們將阿茲海默症的病徵研究著重在位於突觸末梢表現異常的蛋白質時,更值得注意的是在神經及突觸的末端亦存在著大量的RNA分子,包含了mRNA、microRNA、以及長鏈非編碼 RNA。這些突觸通常含有多核醣體,蛋白質轉譯也在突觸內發生,這些新合成的蛋白質在突觸的可塑性上扮演了相當重要的角色。然而,哺乳動物大腦中典型的興奮性突觸非常小,直徑只有約500奈米,且它們僅有非常低的mRNA拷貝數。因此,通過傳統的螢光原位雜交技術(FISH)鑑定定位於突觸的mRNAs是非常具有挑戰性的,再加上若要以FISH鑑定數千種基因在突觸中的功能定位,實際上是難以實行的。 為此我們開發了一種方法,利用次世代測序(NGS)的靈敏度和高通量以確定突觸末端的mRNA組成。我們通過簡單的沉澱法(粗製備方法)和蔗糖梯度離心法(蔗糖製備方法)來製備具有較高濃度突觸的樣品,從非常少量的小鼠皮質突觸末端作為初始材料,我們利用設計用於單細胞的高靈敏度轉錄組擴增試劑組將這些突觸的mRNA轉換為cDNA文庫。綜上所述,我們通過對小鼠基因組進行生物資訊比對,約可獲得500個轉錄體,再使用幾種生物信息學方法分析這些基因的關係,包括基因本體論和同源群簇等方法,發現這些基因主要與主要與突觸,細胞外囊泡,核糖核蛋白和線粒體有關。 我們從次世代測序中一共獲得約16億次讀數,約有9.5%的比對率。根據結果顯示,相較於粗製備法,蔗糖梯度製備法可獲得更多的突觸,雖然兩種方法所製備的樣品仍會含有其他胞器導致樣品有其他污染。此外,低比對率顯示出小鼠蛋白質數據庫仍不足以完整分析小鼠轉錄組,因為轉錄組中可能含有其他表現異常的的基因異構物和選擇性剪接上之變異。因此,在未來我們將利用螢光染色來進行分選以獲取高純度突觸末端,並通過建立客製化基因數據庫進行更深入的轉錄組分析。 | zh_TW |
dc.description.abstract | Synaptic dysfunction underlies many neurological disorders such as dementia, autism, schizophrenia, mental retardation, etc. In Alzheimer’s disease (AD), the most common form of senile dementia, synaptic loss precedes neuronal death in the prolonged process of neurodegeneration. The loss of synapse shows a very strong correlation with cognitive decline in AD, stronger than the levels of senile plaques, neurofibrillary tangles, beta-amyloid (Aβ) oligomers, or hyperphosphorylated tau. There are three important proteins involved in the pathogenesis of AD—Aβ, tau, and apolipoprotein E, all of which are synaptic proteins.
While much attention has been paid to protein abnormalities at synaptic terminals, it should also be noted that nerve endings and synaptic terminals also contain numerous RNA molecules. These include mRNAs, microRNAs, and long non-coding RNAs. The synapse where proteins are translated often contains polyribosomes, and the newly synthesized proteins play critical roles in synaptic plasticity. However, the typical excitatory synapses in mammalian brains are very small, only about 500 nm in diameter, and they contain a very low copy number of mRNAs. It is therefore extremely challenging to identify mRNAs localized to synapses by conventional fluorescence in situ hybridization (FISH) techniques. To optimize FISH assays for thousands of genes to probe their synaptic localization is largely impractical. We devised a strategy to identify the mRNA composition of synaptic terminals that takes advantage of the sensitivity and high throughput of next generation sequencing (NGS). We prepared synaptically enriched biochemical fractions by simple sedimentation (crude preparation) and by sucrose gradient centrifugation (sucrose preparation). Starting with very small amounts of mouse cortical synaptic terminals, we utilized highly sensitive transcriptome amplification kits designed for single cells to convert their mRNAs into cDNA libraries. Altogether, we identified about 500 transcripts in these synaptic preparations by blasting against mouse genome. Several bioinformatics methods were used to analyze the relationship of these genes, including Gene Ontology and Clusters of Orthologous Groups. They are mostly associated with synapses, extracellular vesicles, ribonucleoproteins, and mitochondria. In total, we obtained about 1.6 billion reads from sequencing runs with 9.5% alignment rate after BLAST. The sucrose preparation shows better synaptic enrichment than the crude preparation but both still contain various contaminating organelles. The low alignment rate suggests that mouse protein databases are insufficient for the full analysis for the mouse transcriptome, which may express unusual gene isoforms and alternative splicing variants. In the future, we will acquire high-purity synaptic terminals by fluorescence activated cell sorting and carry out deeper transcriptome analysis by building custom gene databases. | en |
dc.description.provenance | Made available in DSpace on 2021-06-08T03:46:36Z (GMT). No. of bitstreams: 1 ntu-107-R05223209-1.pdf: 8194446 bytes, checksum: ada1548e63ba423b6ab619ea32582604 (MD5) Previous issue date: 2018 | en |
dc.description.tableofcontents | 誌謝 i
中文摘要 ii ABSTRACT iv CONTENTS vi LIST OF FIGURES ix LIST OF TABLES x Abbreviations xi Chapter 1 Introduction 1 1.1 An Overview of the Brain 1 1.2 The synapse 3 1.3 Synaptic dysfunction in Alzheimer's disease 6 1.4 Localization of mRNA in synapse 11 1.5 Isolation of synaptosome 15 1.6 Flow cytometry of synaptosome 16 1.7 Procedures for RNA extraction and quality control 17 1.8 Next generation sequencing 19 1.9 Aim of study 21 Chapter 2 Materials and Methods 22 2.1 Materials 22 2.1.1 Mouse brains 22 2.1.2 Chemicals, consumables, materials and kits 22 2.1.3 Buffers 23 2.2 Instruments 24 2.2.1 BD FACS CantoII 24 2.2.2 CFX96 Touch Real-Time PCR Detection System 25 2.3 Subcellular fractionation of mouse brain 26 2.3.1 Crude synaptosome preparation 26 2.3.2 Synaptoneurosomes preparation with sucrose gradient 26 2.4 Flow cytometry for counting synapses 27 2.5 RNA extraction and double-strand cDNA synthesis 27 2.6 Quality control by RT-qPCR and Bioanalyzer 28 2.7 RNA sequencing and data analysis 28 Chapter 3 Results and Discussion 29 3.1 Qualification of extracted mRNA and cDNA libraries 29 3.2 The assembly status of RNA sequencing 33 3.3 The efficiency of two isolation methods 37 3.4 Brief function classification of identified transcriptomes 39 3.5 A deeper look at synaptic mRNA localization 42 3.5.1 Visualization of molecular interaction networks 42 3.5.2 The differential expression analysis 45 3.6 Comparison with prior studies 46 Chapter 4 Conclusions 49 4.1 Comparisons of two isolation methods 49 4.2 Database construction with identified synaptic mRNA 49 4.3 Future research on isolation method and customized sequencing 49 REFERENCE 50 APPENDIX 61 1.1 Preparing mouse crude synaptosome 61 1.2 Mouse synaptoneurosomes preparation 63 1.3 Sucrose gradient isolation 65 1.4 REPLI-g WTA single cell kit protocol 67 1.5 RT-qPCR protocol 69 | |
dc.language.iso | en | |
dc.title | 利用高通量定序技術分析神經突觸內mRNA之分布 | zh_TW |
dc.title | Transcriptome analysis of mRNAs localized to synapses by high-throughput sequencing | en |
dc.type | Thesis | |
dc.date.schoolyear | 107-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 黃憲松(Hsien-Sung Haung),陳平(Richard Ping Cheng) | |
dc.subject.keyword | 阿茲海默症,突觸體,mRNA定位,次世代定序,流式細胞儀,蔗糖梯度, | zh_TW |
dc.subject.keyword | Alzheimer’s disease,synaptosome,mRNA localization,NGS,flow cytometry,sucrose gradient, | en |
dc.relation.page | 69 | |
dc.identifier.doi | 10.6342/NTU201802050 | |
dc.rights.note | 未授權 | |
dc.date.accepted | 2019-02-12 | |
dc.contributor.author-college | 理學院 | zh_TW |
dc.contributor.author-dept | 化學研究所 | zh_TW |
顯示於系所單位: | 化學系 |
文件中的檔案:
檔案 | 大小 | 格式 | |
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ntu-107-1.pdf 目前未授權公開取用 | 8 MB | Adobe PDF |
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