臺灣博碩士論文加值系統

背景與目標：阿茲海默症分為散發型和家族型兩種，主要病理特徵為β類澱粉蛋白質、神經纖維糾結及發炎細胞激素，β類澱粉蛋白質在血管和腦環境之間保持動態平衡，因此我們期望能藉由長期追蹤下，使用血漿中相關病理蛋白量來建立病徵初期的參考指標，提供臨床早期評估的資訊，此外我們利用誘導性多功能幹細胞分化而成的神經細胞模擬家族型阿茲海默症，藉由此兩種模式探討阿茲海默症相關病理蛋白質在散發型及家族型檢體中之變化。資料與方法：散發型阿茲海默症中的血漿來自於榮總神經內科招募的受試者，分為腦功能正常長者、輕度認知障礙及阿茲海默症患者三組，所有受試者進行為期三年的長期追蹤，每年會採取血液做後續分析，家族型阿茲海默症中細胞培養基取自誘導性多功能幹細胞分化而成的神經細胞，分為控制組，單一等位基因突變及雙等位基因突變。結果：我們發現認知功能程度越低的受試者，血漿中β類澱粉蛋白質、神經纖維糾結及發炎細胞激素含量皆較高。分析三年追蹤的結果顯示，β類澱粉蛋白質隨著追蹤時間有顯著性變化，神經纖維糾結隨著組別診斷達顯著性改變。在誘導性多功能幹細胞分化而成的神經細胞中，β類澱粉蛋白質、神經纖維糾結的蛋白質含量在APP D678H雙等位基因突變組別中都有顯著性升高。結論：我們證實阿茲海默症導致血漿中相關病理蛋白質隨著病程而有趨勢性變化。在幹細胞分化的神經細胞中，我們初步證實APP D678H突變所造成的β類澱粉蛋白質、神經纖維糾結及發炎細胞激素變化。此兩部分的研究，同步指向β類澱粉蛋白質及神經纖維糾結是致病的重要因子。

Background: Alzheimer’s disease (AD) is a progressive brain disorder that can be divided into familial and sporadic forms. AD is characterized by the presence of amyloid β (Aβ) deposition, neurofibrillary tau tangles, and neuroinflammatory cytokines. Whether these pathological hallmarks are the critical factors in the disease development has been under debate. This study aimed to compare the dynamic changes of Aβ and tau levels in both sporadic and familial AD samples. The movements of these proteins between brain and plasma are dynamic. We wonder whether plasma could be used as an early detection tool of AD in order to make a better treatment for patients. Due to the inaccessibility of the brains and the familial forms only account for a few percentages of the cases, induced pluripotent stem cells (iPSCs) were generated for modeling familial AD. Materials and Methods: To study the changes of pathological proteins in sporadic AD, we collected the plasma from healthy elderly, mild cognitive impairment and AD patients in a 3-year follow-up cohort. To study the familial AD cases, we obtained the cell lysate and medium of iPSC-derived neural cells derived from a familial AD patient with an APP D678H mutation as well as isogenic control and isogenic mutant. Results: An increase in plasma Aβ40, Aβ42, Aβ42/Aβ40 ratio, total tau, p-tau was observed in subjects with declined cognitive function. Longitudinal analysis showed Aβ-related biomarkers changed over time, and tau-related biomarkers differed across diagnostic groups. In iPSC-derived neuron, Aβ42, Aβ40, and p-tau were all significantly increased in APPD678H/D678H group. Conclusion: Our findings indicated that there is a dynamic trajectory of changes in plasma biomarkers along AD progression. Besides, we preliminarily confirm the pathologies in the AD patient iPSC-induce neuron. Both parts of the study indicated the importance of Aβ and tau in the development of AD.

Chinese Abstract_i
English Abstract_ii
Contents_iii
Figures Index_vi
Tables Index_vii
Introduction
Alzheimer’s disease (AD)_1
Pathogenesis of AD_1
Biomarker in AD_3
Association between APOE ε4, AD and cognitive decline_4
Genetics of AD_4
Modeling early-onset AD with human induced-pluripotent stem cells (iPSC)_5
Amyloid hypothesis_6
Goal of this study_6
Materials and methods
For late-onset Alzheimer’s disease study_8
Study design_8
Subjects_8
Diagnostic criteria_8
Plasma collection_9
Enzyme-linked immunosorbent assay (ELISA)_9
Apolipoprotein E genotyping_10
Statistical analysis_10
For early-onset Alzheimer’s disease study_11
Induced Pluripotent Stem Cells (iPSC)-derived neuron_11
Enzyme-linked immunosorbent assay (ELISA)_11
Western blot_12
Quantitative real-time PCR (Q-PCR)_12
Statistical analysis_12
Results
For late-onset Alzheimer’s disease study_14
Demographic and clinical data_14
Longitudinal changes in 3-year follow-up_14
Longitudinal analysis by Generalized estimating equation (GEE)_15
Risk factors in associated with MCI and AD_15
Effect of APOE ε4 allele on plasma biomarker expression_16
Individual changes of biomarkers over 3 years_16
Hypothetical model of plasma biomarkers in AD progression_16
For early-onset Alzheimer’s disease study_17
Generation of isogenic control and isogenic mutation_17
Aβ level in patient-derived cultured neuron_17
Tau level in patient-derived neuron and cultured medium_18
Inflammatory cytokine level in BV2 microglia cells_18
Discussion
Overview_19
Plasma Aβ, tau, and TNFα in normal elderly, MCI, and sporadic AD_19
Limitations in sporadic AD study_21
Pathologies in induced-neuron from a familial AD patient_22
Conclusion_24
References_25
Appendices_43

Figures Index
Figure 1.....................2
Figure 2....................36
Figure 3....................37
Figure 4....................38
Figure 5....................39
Figure 6....................40
Figure 7....................42

Tables Index
Table 1....................29
Table 2....................30
Table 3....................31
Table 4....................32
Table 5....................33
Table 6....................34
Table 7....................35

1. Asai, H., Ikezu, S., Tsunoda, S., Medalla, M., Luebke, J., Haydar, T., Wolozin, B., Butovsky, O., Kügler, S., Ikezu, T. Depletion of microglia and inhibition of exosome synthesis halt tau propagation. Nat Neurosci 18, 1584–1593 (2015).
2. Blennow, K. Cerebrospinal Fluid Protein Biomarkers for Alzheimer’s Disease. NeuroRx 1, 213–225 (2004).
3. Brosseron, F., Krauthausen, M., Kummer, M., Heneka, M. T. Body Fluid Cytokine Levels in Mild Cognitive Impairment and Alzheimer’s Disease: a Comparative Overview. Mol Neurobiol 50, 534–544 (2014).
4. Castello, M. A., Soriano, S. On the origin of Alzheimer’s disease. Trials and tribulations of the amyloid hypothesis. Ageing Res. Rev. 13, 10–12 (2014).
5. Chen, W.-T., Hong, C.-J., Lin, Y.-T., Chang, W.-H., Huang, H.-T., Liao, J.-Y., Chang, Y.-J., Hsieh, Y.-F., Cheng, C.-Y., Liu, H.-C., Chen, Y.-R., Cheng, I.-H. Amyloid-Beta (Aβ) D7H Mutation Increases Oligomeric Aβ42 and Alters Properties of Aβ-Zinc/Copper Assemblies. PLoS One 7, e35807 (2012).
6. Chourakia, V., Beiser, A., Younkin, L., Rosner, S., Weinstein, G., Hansson, O., Skoog, I., Jean-Charles, L., Au, R., Launer, L., Wolf, P. A., Younkin, S., Seshadri, S. Plasma amyloid β and risk of Alzheimer’s disease in the Framingham Heart Study. Alzheimers Dement 11, 249–257 (2016).
7. Corder, E. H., Saunders, A. M., Strittmater, W. J., Schmechel, D. E., Gaskell, P. C., Small, G. W., Roses, A. D., Hainess, J. L., Pericak-Vance, M. A. Gene Dose of Apolipoprotein E Type 4 Allele and the Risk of Alzheimer’s Disease in Late Onset Families. Science 261, 921–923 (1993).
8. Ferri C.P., Prince, M.P., Brayne. C., Brodaty, H., Fratiglioni, L., Ganguli, M., Hall, K., Hasegawa, K., Hendrie, H., Huang, Y., Jorm, a., Mathers, C., Menezes, P.R., Rimmer, E., Scazufca, M. Global prevalence of dementia: a Delphi consensus study. Lancet 366, 2112–2117 (2005).
9. Figurski, M. J., Waligórska, T., Toledo, J., Vanderstichele, H., Korecka, M., Lee, M. Y., Trojanowski, J. Q., Shaw, L. M., The Alzheimer’s Disease Neuroimaging Instiative. Improved protocol for measurement of plasma β-amyloid in longitudinal evaluation of Alzheimer’s Disease Neuroimaging Initiative study patients. Alzheimers Dement 8, 250–260 (2012).
10. Fukumoto, H., Rosene, D. L., Moss, M. B., Raju, S., Hyman, B. T., Irizarry, M. C. β- Secretase Activity Increases with Aging in Human, Monkey, and Mouse Brain. Am J Pathol 164, 719-725 (2004)
11. Fukutani, Y., Cairns, N., Rossor, M., Isaki, K. &Lantos, P. Cerebellar pathology in sporadic and familial Alzheimer’s disease including APP 717 (Val→Ile) mutation cases: A morphometric investigation. J. Neurol. Sci. 149, 177–184 (1997).
12. Hampel, H., Teipel, S. J. Total and Phosphorylated Tau Proteins: Evaluation as Core Biomarker Candidates in Frontotemporal Dementia. Dement Geriatr Cogn Disord 17, 350–354 (2004).
13. Hansson, O., Stomrun, E., Vanmechelen, E., Östling, S., Gustafson, D. R., Zetterberg, H., Blennow, K., Skoog, I. Evaluation of Plasma Aβ as Predictor of Alzheimer’s Disease in Older Individuals Without Dementia: A Population-Based Study. J. Alzheimer’s Dis. 28, 231–238 (2012).
14. Hardy, J. A., Higgins, G. A. Alzheimer’s Disease : The Amyloid Cascade Hypothesis. Science 256, 184–185 (1992).
15. Huynh, R. A., Mohan, C. Alzheimer’s Disease: Biomarkers in the Genome, Blood, and Cerebrospinal Fluid. Front. Neurol 8, 102 (2017).
16. Jung, Y.-W., Hysolli, E., Kim, K.-Y., Tanaka, Y., Park., I.-H. Human induced pluripotent stem cells and neurodegenerative disease: prospects for novel therapies. Curr Opin Neurol 25, 125–130 (2012).
17. Kim, S.-M., Song, J., Kim, S., Han, C., Park, M.-H., Koh, Y., Jo, S.-A., Kim, Y.-Y. Identification of peripheral inflammatory markers between normal control and Alzheimer’s disease. BMC Neurol 11, 51 (2011).
18. Lan, M. Y., Liu, J. S., Wu, Y. S., Peng, C. H., Chang, Y. Y. A novel APP mutation (D678H) in a Taiwanese patient exhibiting dementia and cerebral microvasculopathy. J. Clin. Neurosci. 21, 513–515 (2014).
19. Lim, S., Haque, M. M., Kim, D., Kim, D. J., Kim, Y. K. Cell-based models to investigate Tau aggregation. Comput Struct Biotechnol J 12, 7–13 (2014).
20. Lin, Y.-C., Wang, J.-Y., Wang, K.-C., Liao, J.-Y., Cheng, I.-H. Differential regulation of amyloid precursor protein sorting with pathological mutations results in a distinct effect on amyloid-β production. Journal of Neurochemistry 131, 407-412 (2014)
21. Lippa, C. F., Saunders, A. M., Smith, T. W., Swearer, J. M., Drachman, D. A., Ghetti, B., Pulaski-Salo, D., Dickson, D., Robitaille, Y., Berqeron, C., Crain, B., Benson, M. D., Farlow, M., Hyman, B. T., George-Hyslop, S. P., Roses, A. D., Pollen, D. A. Familial and sporadic Alzheimer’s disease: Neuropathology cannot exclude a final common pathway. Neurology 46, 406–412 (1996).
22. Liu, S.-L., Zhang, T., Zhang, Y.-J., Yue, W., Shi, Z.-H., Guan, Y.-L., Liu, S., Wang, X.-D., Ji, Y. Meta-analysis on correlation between genetic polymorphism of ApoE and late onset Alzheimer's disease in Chinese population. Chinese Journal of Contemporary Neurology & Neurosurgery 16, 30-37 (2016)
23. Metcalfe, M. J., Figueiredo-Pereire, M. E. Relationship Between Tau Pathology and Neuroinflammation in Alzheimer’s Disease. Mt Sinai J Med 77, 50–58 (2010).Liu, S.-L., Zhang, T., Zhang, Y.-J., Yue, W., Shi, Z.-H., Guan, Y.-L., Liu, S., Wang, X.-D., Ji, Y. Meta-analysis on correlation between genetic polymorphism of ApoE and late onset Alzheimer's disease in Chinese population. Chinese Journal of Contemporary Neurology & Neurosurgery 16, 30-37 (2016)
24. Marksteiner, J., Kemmler, G., Weiss, E. M., Knaus, G., Ullrich, C., Mechtcheriakov, S., Oberbauer, H., Auffinger, S., Hinterhölzl, J., Hinterhuber, H., Humpel, C. Five out of 16 plasma signaling proteins are enhanced in plasma of patients with mild cognitive impairment and Alzheimer’s disease. Neurobiol Aging 32, 539–540 (2011).
25. Mayeux, R., Honig, L. S., Tang, M.-X., Manly, J., Stern, Y., Schupf, N., Mehta, P. D. Plasma Aβ40 and Aβ42 and Alzheimer’s disease: Relation to age, mortality, and risk. Neurology 61, 1185–1190 (2003).
26. Meraz-Ríos, M. A., Toral-Rios, D., Franco-Bocanegra, D., Villeda-Hernández, J., Campos-Peña, V. Inflammatory process in Alzheimer's Disease. Front Integr Neurosci 7,59 (2013).
27. McKhann, G., Drachman, D., Folstein, M., Katzman, R., Price, D., Stadlan, E. M. Clinical diagnosis of Alzheimer's disease. Neurology 34, 939 (1984)
28. Murphy, M. P., LeVineIll, H. Alzheimer’s Disease and the β-Amyloid Peptide. J Alzheimers Dis 19, 1–17 (2010).
29. Nicolas, M., Hassan, B. A. Amyloid precursor protein and neural development. Development 141, 2543–2548 (2014).
30. Nochlin, D., vanBelle, G., Bird, T. D., Sumi, S. M. Comparison of the severity of neuropathologic changes in familial and sporadic Alzheimer’s disease. Alzheimer Dis. Assoc. Disord. 7, 212–222 (1993).
31. Okereke, O. I., Xia, W., Selkoe, D. J., Grodstein, F. Ten-year change in plasma amyloid β levels and late-life cognitive decline. Arch Neurol 66, 1247–1253 (2009).
32. Rissman, R. A., Trojanowski, J. Q., Shaw, L. M., Aisen, P. S. Longitudinal plasma amyloid beta as a biomarker of Alzheimer’s disease. J Neural Transm 119, 843–850 (2012).
33. Rosenberg, G. Blood-Brain Barrier Permeability in Aging and Alzheimer’s Disease. J Prev Alzheimers Dis 1, 138–139 (2014).
34. Sato, Y., Bernier, F., Yamanaka, Y., Aoshima, K., Oda, Y., Ingelsson M., Lannfelt, L., Miyashita, A., Kuwano, R., Ikeuch, T. Reduced plasma desmosterol-to-cholesterol ratio and longitudinal cognitive decline in Alzheimer’s disease. Alzheimers Dement (Amst) 1, 67–74 (2015).
35. Schraen-Maschke, S., Sergeant, N., Dhaenens, C.-M., Bombois, S., Deramecourt, V., Caillet-Boudin, M.-L., Pasquier, F., Maurage, C. A., Sablonniere, B., Vanmechelen, E., Buee, L. Tau as a biomarker of neurodegenerative diseases. Biomark Med 2, 363–384 (2008).
36. Schupf, N., Tang, M. X., Fukuyama, H., Manly, J., Andrews, H., Mehta, P., Ravetch, J., Mayeux, R. Peripheral Aβ subspecies as risk biomarkers of Alzheimer’s disease. Proc Natl Acad Sci 105, 14052–14057 (2008).
37. Selkoe, D. J., Hardy, J. The amyloid hypothesis of Alzheimer’s disease at 25 years. EMBO Mol Med 8, 1–14 (2016).
38. Sharma, N., Singh, A. N. Exploring Biomarkers for Alzheimer’s Disease. J Clin Diagn Res 10, KE01-KE06 (2016).
39. Sundelöf, J., Giedraitis, V., Irzarry, M. C., Sundström, J., Ingelsson, E., Rönnemaa, E., Ärnlöv, J., Gunnarsson, M. D., Hyman, B. T., Basun, H., Ingelsson, M., Lannfelt, L., Kilander, L. Plasma β Amyloid and the Risk of Alzheimer Disease and Dementia in Elderly Men. Arch Neurol 65, 256–263 (2008).
40. Takeda, S., Sato, N., Uchio-Yamada, K., Sawada, K., Kunieda, T., Takeuchi, D., Kurinami, H., Shinohara, M., Rakugi, H., Morishita, R. Elevation of plasma β-amyloid level by glucose loading in Alzheimer mouse models. Biochem Biophys Res Commun 385, 193–197 (2009).
41. Toledo, T. B., Zetterberg, H., Harten, C., Glodzik, L., Martinez-Lage, P., Bocchio-Chiavetto, L., Rami, L., Hansson, O., Sperling, R., Engelborghs, S., Osorio, R. S, Vanderstichele, H., Vandikck, M., Hampel, H., Teipl, S., Moghekar, A., Ablert, M., Hu, W. T., Argilés, J. A. M., Gorostidi, A., Teunissen, C. E., Deyn, P. P., Hyman, B. T., Molinuevo, J. L, Frisoni, G. B., Linazasoro, G., Leon, M. J., Filer, W. M., Scheltens, P., Blennow, K., Shaw, L. M., Trojanowski, J. Q. Alzheimer’s disease cerebrospinal fluid biomarker in cognitively normal subjects. Brain 138, 2701–2715 (2015).
42. Turana, Y., Suzy Handajani, Y., Widjaja, N. Association between APOE ε4 Genotype and Memory Impairment in Elderly with Normal Global Cognitive Assessment. Diagnostics 5, 615–623 (2015).
43. VanCauwenberghe, C., VanBroeckhoven, C., Sleegers, K. The genetic landscape of Alzheimer disease: clinical implications and perspectives. Genet. Med 18, 421–430 (2016).
44. VanOijen, M., Hofman, A., Soares, H. D., Koudstaal, P. J., Breteler, M. M. Plasma Aβ1-40 and Aβ1-42 and the risk of dementia: a prospective case-cohort study. Lancet Neurololy 5, 655–660 (2006).
45. Verghese, P. B., Castellano, J. M., Holtzman, D. M. Roles of Apolipoprotein E in Alzheimer’s disease and Other Neurological Disorders. Lancet Neurololy 10, 241–252 (2011).
46. Wang, T., Xiao, S., Liu, Y., Lin, Z., Su, N., Li, X, Li, G., Zhang, M, Fang, Y. The efficacy of plasma biomarkers in early diagnosis of Alzheimer’s disease. Geriatr. Psychiatry 29, 713–719 (2014).
47. Wang, W.-Y., Tan, M., Yu, J., Tan, L. Role of pro-inflammatory cytokines released from microglia in Alzheimer’s disease. Ann Transl Med 3, 1–15 (2015).
48. Wisdom, N. M., Callahan, J. L., Hawkins, K. A. The effects of apolipoprotein E on non-impaired cognitive functioning: A meta-analysis. Neurobiol Aging 32, 63–74 (2011).
49. Yasutake, C., Kuroda, K., Yanagawa, T., Okamura, T., Yoneda, H. Serum BDNF, TNF-α and IL-1β levels in dementia patients: Comparison between Alzheimer’s disease and vascular dementia. Eur Arch Psychiatry Clin Neurosci 256, 402–406 (2006).
50. Young, A. B. Four decades of neurodegenerative disease: How far we have come! J Neurosci 29, 12722–12728 (2009).
51. Zetterberg, H., Wilson, D., Andreasson, U., Minthon, L., Blennow, K., Randll, J. Hansson, O. Plasma tau levels in Alzheimer’s disease. Alzheimer’s Res. Ther 5, 4–6 (2013).
52. Zhang, Y, Pak, C.-H., Han, Y., Ahlenius, H., Zhang, Z., Chanda, S., Marro, S., Patzke, C., Acuna, C., Covy, J., Xu, W., Yang, N., Danko, T., Chen, L., Wernig, M. Südhof, T. C. Rapid Single-Step Induction of Functional Neurons from Human Pluripotent Stem Cells. Neuron 78, 785–798 (2013).