漸凍人症的病理特徵，TDP-43沉積，在阿茲海默症的病人中相當常見，這代表著TDP-43可能在阿茲海默症中是除了兩個為人所知的病理特徵乙型類澱粉蛋白 (Aβ)以及tau蛋白之外，第三個重要的病理特徵。根據陳韻如老師實驗室過去的研究結果，TDP-43可以影響Aβ40的堆疊、使之保持在寡聚體的階段、並證實兩者間有交互作用，這些研究結果促使我想去探討TDP-43與Aβ40之間的分子交互作用。
首先，在Thioflavin-T檢測中，藉由讓特定幾個抗TDP-43寡聚體的單株抗體或市售的抗TDP-43的單株抗體與TDP-43結合，TDP-43抑制Aβ40聚集的效能大幅降低，因此我發現TDP-43藉由其N端或是透過第二個與核糖核酸結合的位置(RRM2)和Aβ40產生交互作用。此外，雖然小鼠和人類的TDP-43主要差異在RRM2的序列，兩種蛋白都會影響Aβ40的堆疊。第二，在蛋白質核磁共振實驗中，我發現準備Aβ40所使用的緩衝液會造成TDP-43蛋白質構型改變，並可能進而使TDP-43無法和Aβ40有交互作用。但在改變Aβ40的準備方式後，我們仍發現氮15同位素標定的TDP-43 N端核磁共振圖譜並沒有發生改變，顯示在此條件下兩者無穩定作用。第三，在酵素免疫分析法中，藉由讓特定幾個抗TDP-43寡聚體的自製單株抗體或市售的抗TDP-43的單株抗體先與TDP-43結合，biotin-Aβ40就無法或降低與TDP-43的結合，因此我發現TDP-43藉由其C端以及RRM1+2與biotin-Aβ40有交互作用。由此我們推測，Aβ40與TDP-43有著多個交互作用的位置，確切位置仍需後續證明。

ALS disease hallmarks, TDP-43 pathology, were rather common in Alzheimer’s disease (AD), which suggested that TDP-43 might be the third important disease hallmarks in AD besides the well-known AD hallmarks, amyloid-β (Aβ) and tau protein. From the previous results of Dr. Yun-Ru Chen’s lab, we found that TDP-43 could affect Aβ40 aggregation, retain it in its oligomeric state, and interact with it, which motivated me to investigate how did they interact in molecular level.
Firstly, in Thioflavin-T assay we demonstrated TDP-43 might interact with Aβ40 by its N-terminal or RNA recognition motif 2 (RRM2) region with the aid of selected self-prepared TDP-O antibodies and commercial antibodies. In addition, both mouse and human TDP-43 could affect Aβ40 aggregation although mouse TDP-43 mainly differed with human TDP-43 in RRM2 motif. Secondly, in 1H-15N HSQC protein NMR experiments we found that 15N-Aβ40 preparation buffer altered TDP-43 conformation and might interrupt the interaction between 15N-Aβ40 and TDP-43. But we still demonstrated no significant changes in N-terminal of 15N-TDP-43 NMR graph after Aβ40 preparation was changed. The results showed that in the experimental condition, the two proteins did not stably interact. Thirdly, in ELISA assay, we demonstrated TDP-43 interacted with biotin-Aβ40 by its C-terminal or RRM1+2 domain with the aid of our TDP-O antibodies and the commercial antibodies. Our results suggested that Aβ40 could interact with multiple domains of TDP-43 and the specific interaction sites need further examination.

誌謝 i
中文摘要 ii
Abstract iii
Contents iv
List of Figures vi
Chapter 1 Introduction: 1
1.1 Dementia: 1
1.2 Alzheimer’s disease 1
1.2.1 The discovery of Alzheimer’s disease 1
1.2.2 Main hypothesis in AD and drug development 2
1.2.3 Aβ production and the structural characteristics 3
1.3 TAR DNA-binding protein 43 (TDP-43) 4
1.3.1 The discovery of TDP-43 4
1.3.2 The physiological function of TDP-43 5
1.3.3 The discovery and importance of TDP-43 in Alzheimer’s disease 6
1.3.4 The generation of monoclonal TDP-O antibodies 6
1.3.5 Possible extracellular and intracellular interaction between TDP-43 and Aβ40 7
1.4 Aim of my research 8
Chapter 2 Material and methods 10
2.1 Protein expression and purification 10
2.1.1 TDP-43 (transactive response DNA binding protein 43 kDa) 10
2.1.2 Aβ40 expression and purification 13
2.2 dot blot 16
2.3 Size exclusion chromatography 17
2.4 Western blot 17
2.5 Thioflavin T assay 18
2.6 1H-15N Heteronuclear Single-Quantum Coherence (HSQC) nuclear magnetic resonance of protein (protein NMR) 19
2.6.1 15N labeled Aβ40 19
2.6.2 15N labeled TDP-43 (1-100) 20
2.7 ELISA 21
Chapter 3 Results 23
3.1 Protein purification 23
3.1.1 Full-length and truncated human TDP-43 23
3.1.2 Aβ40 purification 26
3.1.3 Characterization of monoclonal anti-human TDP-43 oligomer antibodies 26
3.2 Examination of interactions between TDP-43 and Aβ40 28
3.2.1 ThT assay 29
3.2.2 1H-15N Heteronuclear Single-Quantum Coherence nuclear magnetic resonance of protein (1H-15N HSQC protein NMR) 31
3.2.3 Enzyme-linked immunosorbent assay (ELISA) 34
Chapter 4. Discussion 37
Figures 41
References: 62

1.Thompson, C.A., et al., Systematic review of information and support interventions for caregivers of people with dementia. BMC Geriatrics, 2007. 7(1): p. 18.
2.Wimo, A., et al., The worldwide costs of dementia 2015 and comparisons with 2010. Alzheimer''s & Dementia, 2017. 13(1): p. 1-7.
3.Bondi, M.W., E.C. Edmonds, and D.P. Salmon, Alzheimer’s Disease: Past, Present, and Future. Journal of the International Neuropsychological Society, 2017. 23(9-10): p. 818-831.
4.Du, X., X. Wang, and M. Geng, Alzheimer’s disease hypothesis and related therapies. Translational Neurodegeneration, 2018. 7(1): p. 2.
5.Haass, C. and D.J. Selkoe, Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer''s amyloid β-peptide. Nature Reviews Molecular Cell Biology, 2007. 8: p. 101.
6.Shankar, G.M., et al., Amyloid-β protein dimers isolated directly from Alzheimer''s brains impair synaptic plasticity and memory. Nature Medicine, 2008. 14: p. 837.
7.Zhao, L.N., et al., The Toxicity of Amyloid ß Oligomers. International Journal of Molecular Sciences, 2012. 13(6).
8.van der Kant, R. and Lawrence S.B. Goldstein, Cellular Functions of the Amyloid Precursor Protein from Development to Dementia. Developmental Cell, 2015. 32(4): p. 502-515.
9.Gu, L. and Z. Guo, Alzheimer''s Aβ42 and Aβ40 peptides form interlaced amyloid fibrils. Journal of Neurochemistry, 2013. 126(3): p. 305-311.
10.Pauwels, K., et al., Structural Basis for Increased Toxicity of Pathological Aβ42:Aβ40 Ratios in Alzheimer Disease. Journal of Biological Chemistry, 2012. 287(8): p. 5650-5660.
11.Chen, G.-f., et al., Amyloid beta: structure, biology and structure-based therapeutic development. Acta Pharmacologica Sinica, 2017. 38: p. 1205.
12.Balbach, J.J., et al., Supramolecular Structure in Full-Length Alzheimer''s β-Amyloid Fibrils: Evidence for a Parallel β-Sheet Organization from Solid-State Nuclear Magnetic Resonance. Biophysical Journal, 2002. 83(2): p. 1205-1216.
13.Ou, S.H., et al., Cloning and characterization of a novel cellular protein, TDP-43, that binds to human immunodeficiency virus type 1 TAR DNA sequence motifs. Journal of Virology, 1995. 69(6): p. 3584.
14.Buratti, E. and F.E. Baralle, Characterization and Functional Implications of the RNA Binding Properties of Nuclear Factor TDP-43, a Novel Splicing Regulator ofCFTR Exon 9. Journal of Biological Chemistry, 2001. 276(39): p. 36337-36343.
15.Arai, T., et al., TDP-43 is a component of ubiquitin-positive tau-negative inclusions in frontotemporal lobar degeneration and amyotrophic lateral sclerosis. Biochemical and Biophysical Research Communications, 2006. 351(3): p. 602-611.
16.Kabashi, E., et al., TARDBP mutations in individuals with sporadic and familial amyotrophic lateral sclerosis. Nature Genetics, 2008. 40: p. 572.
17.Josephs, K.A., et al., Staging TDP-43 pathology in Alzheimer’s disease. Acta Neuropathologica, 2014. 127(3): p. 441-450.
18.Chanson, J.B., et al., TDP43-Positive Intraneuronal Inclusions in a Patient with Motor Neuron Disease and Parkinson’s Disease. Neurodegenerative Diseases, 2010. 7(4): p. 260-264.
19.Davidson, Y., et al., TDP-43 in ubiquitinated inclusions in the inferior olives in frontotemporal lobar degeneration and in other neurodegenerative diseases: a degenerative process distinct from normal ageing. Acta Neuropathologica, 2009. 118(3): p. 359-369.
20.Mompeán, M., et al., Point mutations in the N-terminal domain of transactive response DNA-binding protein 43 kDa (TDP-43) compromise its stability, dimerization, and functions. Journal of Biological Chemistry, 2017. 292(28): p. 11992-12006.
21.Lukavsky, P.J., et al., Molecular basis of UG-rich RNA recognition by the human splicing factor TDP-43. Nature Structural &Amp; Molecular Biology, 2013. 20: p. 1443.
22.Cohen, T.J., V.M.Y. Lee, and J.Q. Trojanowski, TDP-43 functions and pathogenic mechanisms implicated in TDP-43 proteinopathies. Trends in Molecular Medicine, 2011. 17(11): p. 659-667.
23.Davis, S.A., et al., TDP-43 expression influences amyloidβ plaque deposition and tau aggregation. Neurobiology of Disease, 2017. 103: p. 154-162.
24.James, B.D., et al., TDP-43 stage, mixed pathologies, and clinical Alzheimer’s-type dementia. Brain, 2016. 139(11): p. 2983-2993.
25.Gu, J., et al., TDP-43 suppresses tau expression via promoting its mRNA instability. Nucleic Acids Research, 2017. 45(10): p. 6177-6193.
26.Fang, Y.-S., et al., Full-length TDP-43 forms toxic amyloid oligomers that are present in frontotemporal lobar dementia-TDP patients. Nature Communications, 2014. 5: p. 4824.
27.LaFerla, F.M., K.N. Green, and S. Oddo, Intracellular amyloid-β in Alzheimer''s disease. Nature Reviews Neuroscience, 2007. 8: p. 499.
28.Wang, P., et al., TDP-43 induces mitochondrial damage and activates the mitochondrial unfolded protein response. PLOS Genetics, 2019. 15(5): p. e1007947.
29.Querfurth, H.W. and F.M. LaFerla, Alzheimer''s Disease. New England Journal of Medicine, 2010. 362(4): p. 329-344.
30.Iguchi, Y., et al., Exosome secretion is a key pathway for clearance of pathological TDP-43. Brain, 2016. 139(12): p. 3187-3201.
31.Gao, J., et al., Pathomechanisms of TDP-43 in neurodegeneration. Journal of Neurochemistry, 2018. 146(1): p. 7-20.
32.Hawe, A., M. Sutter, and W. Jiskoot, Extrinsic Fluorescent Dyes as Tools for Protein Characterization. Pharmaceutical Research, 2008. 25(7): p. 1487-1499.
33.Ni, C.-L., et al., Folding stability of amyloid-β 40 monomer is an important determinant of the nucleation kinetics in fibrillization. The FASEB Journal, 2011. 25(4): p. 1390-1401.
34.Tjernberg, L.O., et al., Amyloid β-peptide polymerization studied using fluorescence correlation spectroscopy. Chemistry & Biology, 1999. 6(1): p. 53-62.
35.Xue, C., et al., Thioflavin T as an amyloid dye: fibril quantification, optimal concentration and effect on aggregation. Royal Society Open Science. 4(1): p. 160696.
36.Kumar, S., et al., Foldamer-Mediated Structural Rearrangement Attenuates Aβ Oligomerization and Cytotoxicity. Journal of the American Chemical Society, 2017. 139(47): p. 17098-17108.
37.Janowska, M.K., K.-P. Wu, and J. Baum, Unveiling transient protein-protein interactions that modulate inhibition of alpha-synuclein aggregation by beta-synuclein, a pre-synaptic protein that co-localizes with alpha-synuclein. Scientific Reports, 2015. 5: p. 15164.
38.Fezoui, Y., et al., An improved method of preparing the amyloid β-protein for fibrillogenesis and neurotoxicity experiments. Amyloid, 2000. 7(3): p. 166-178.
39.Hou, L., et al., Solution NMR Studies of the Aβ(1−40) and Aβ(1−42) Peptides Establish that the Met35 Oxidation State Affects the Mechanism of Amyloid Formation. Journal of the American Chemical Society, 2004. 126(7): p. 1992-2005.
40.Hou, L. and M.G. Zagorski, NMR Reveals Anomalous Copper(II) Binding to the Amyloid Aβ Peptide of Alzheimer''s Disease. Journal of the American Chemical Society, 2006. 128(29): p. 9260-9261.
41.Afroz, T., et al., Functional and dynamic polymerization of the ALS-linked protein TDP-43 antagonizes its pathologic aggregation. Nature Communications, 2017. 8(1): p. 45.
42.Reyes Barcelo, A.A., F.J. Gonzalez-Velasquez, and M.A. Moss, Soluble aggregates of the amyloid-β peptide are trapped by serum albumin to enhance amyloid-β activation of endothelial cells. Journal of Biological Engineering, 2009. 3(1): p. 5.