葉酸是水溶性維生素B9。葉酸參與許多重要的生理反應，例如:核苷酸的生合成、胺基酸的代謝、神經傳遞物質及S-腺苷甲硫氨酸的合成。因此生物體葉酸的含量會影響細胞內的甲基化潛力(methylation potential)及高半胱氨酸(Homocysteine)的代謝。葉酸對於神經組織的發育與功能極其重要。在臨床上，葉酸不足可能造成新生兒神經管發育不全(Neural tube defect)。葉酸缺乏亦被認為是造成阿茲海默症的危險因子之一。阿茲海默症是臨床上最常見的失智症，目前並無有效的治療方法，其致病機轉亦尚未完全了解。我們的研究目標是利用斑馬魚動物模式探討細胞內葉酸含量對於發生阿茲海默症的影響。先前在實驗室中我們製作了一株轉殖螢光斑馬魚，牠們在適當的熱誘導下會產生細胞內葉酸缺乏的現象。在實驗進行前，一歲半到兩歲的年邁斑馬魚在一周內被置於35℃溫水浴三次，每次一小時，藉此引發基因轉殖魚葉酸缺乏的情形。我們使用改良過的T型水域行為學試驗來評估斑馬魚的認知及記憶能力，實驗結果顯示葉酸缺乏會造成年邁斑馬魚的認知學習能力出現明顯的衰退現象。另外在腦部切片方面，藉由免疫組織化學染色法，我們觀察到乙型－澱粉樣蛋白(β-amyloid)及磷酸化Tau蛋白在葉酸缺乏的斑馬魚腦部出現堆積，而在控制組並沒有觀察到類似的現象。利用逆轉錄聚合酶鏈式反應(RT-PCR)分析斑馬魚胚胎及成魚腦部組織的基因表現後，我們發現Cathepsin B基因的表現會因葉酸的缺乏而明顯下降。Cathepsin B是溶體(lysosome)內一種重要的半胱氨酸蛋白酶。除此之外，我們亦觀察到葉酸缺乏的斑馬魚胚胎及成魚腦部組織出現明顯上升的氧化壓力。而在缺乏葉酸的人類神經母細胞瘤-SKnSH細胞株中，亦出現Cathepsin B基因表現下降、酸性胞器的堆積，以及LC-3點狀聚集等現象，顯示葉酸缺乏導致細胞內的自噬作用(Autophagy)增加，而這樣的影響可被抗氧化劑及葉酸所補救。我們的研究結果支持利用斑馬魚模式進行阿茲海默症相關研究的可行性。同時我們發現葉酸缺乏可能透過改變Cathepsin B的基因表現，進而影響細胞的自噬作用並增加組織的氧化壓力，並使年邁斑馬魚出現類似阿茲海默症的病理徵狀。未來我們將會嘗試在斑馬魚身上，以葉酸餵食來補救上述所看到的病癥，以評估各種不同葉酸的療效及可能機制。

Folate, also known as vitamin B9, participates in various physiological functions, including nucleotides biosynthesis, amino acid metabolism, neurotransmitters synthesis and S-adenosylmethionine formation. Folate is crucial for intracellular methylation potential, cell proliferation and differentiation. Folate deficiency often causes neural tube defects in fetus. Folate deficiency is also considered a risk factor for Alzheimer’s Disease (AD) in the elderly. However, the pathomechanism underlying the folate-related AD remains unclear to date, hampering the development and application of folate-involving preventive/therapeutic regimen against AD. The aim of this study is to investigate how folate deficiency contributes to the occurrence of AD. Previously, a transgenic zebrafish line with inducible folate deficiency was developed in our lab. With this transgenic line and the modified T-maze device, we showed that the aged fish (1.5 to 2 year-old) with decreased folate content in brain displayed impeded cognitive and memory ability. Immunohistochemical staining revealed the accumulation of both amyloid plaques and Tau accumulations in the brain cryo-sections prepared from these folate deficient aged fish. The RT-PCR with the cDNA prepared from the folate deficient embryos and the brain of folate deficient aged fish revealed a decreased expression of cathepsin B, a cysteine proteases in lysosome. Increased oxidative stress was observed in both folate deficient embryos and fish brain tissues. Decreasing intracellular folate content also decreased cathepsin B transcript, increased acidic vesicles accumulations and increased LC3 punctas in human neuroblastoma SKnSH cells. All the above-described abnormalities in cells were partly reversed by adding antioxidant or folate to the culture medium. Here, we showed that the aged transgenic zebrafish with potential folate deficiency in brain displayed pathological characteristics of AD similar to those observed in mammals. Also, folate deficiency might contribute to the AD-like pathogenesis via decreasing cathepsin B expression and the interplay between the interfered intracellular autophagic-lysosomal pathway and increased oxidative stress. Future studies will be focused on evaluating the preventive efficacy of different folate adducts with/without combinatorial strategies using this folate deficient transgenic zebrafish model.

1. Alzheimer’s Disease International (2009) World Alzheimer Report (2009), http://www.alz.co.uk/reserch/files/WorldAlzheimerReport.pdf.
2. Van den Hove, D., Kompotis, K., Lardenoije, R., Kenis, G., Mill, J., Steinbusch, H., Lesch, K., Fitzsimons, C., De Strooper, B. and Rutten, B. (2014). Epigenetically regulated microRNAs in Alzheimer's disease. Neurobiology of aging, 35(4), pp.731--745.
3. Hinterberger, M. and Fischer, P. (2013). Folate and Alzheimer: when time matters. Journal of Neural Transmission, 120(1), pp.211--224.
4. Ebisch, I., Thomas, C., Peters, W., Braat, D. and Steegers-Theunissen, R. (2007). The importance of folate, zinc and antioxidants in the pathogenesis and prevention of subfertility. Human Reproduction Update, 13(2), pp.163--174.
5. Crivello, N., Spires-Jones, T., Shukitt-Hale, B., Carey, A., Roe, A., Smith, D., Blusztajn, J., Hyman, B. and Rosenberg, I. (2013). Folate deficiency accelerates Alzheimer’s disease related pathology in APP/PS1 mice. The FASEB Journal, 27, pp.1077--8.
6. Feitsma, H. and Cuppen, E. (2008). Zebrafish as a cancer model. Molecular Cancer Research, 6(5), pp.685--694.
7. Egan, R., Bergner, C., Hart, P., Cachat, J., Canavello, P., Elegante, M., Elkhayat, S., Bartels, B., Tien, A., Tien, D. and others, (2009). Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behavioural brain research, 205(1), pp.38--44.
8. Newman, M., Verdile, G., Martins, R. and Lardelli, M. (2011). Zebrafish as a tool in Alzheimer's disease research. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1812(3), pp.346--352.
9. Guyon, J., Steffen, L., Howell, M., Pusack, T., Lawrence, C. and Kunkel, L. (2007). Modeling human muscle disease in zebrafish. Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease, 1772(2), pp.205--215.
10. Schneider, E. and Ryan, T. (2006). Gamma-glutamyl hydrolase and drug resistance. Clinica chimica acta, 374(1), pp.25--32.
11. Characterization of the human gamma-glutamyl hydrolase promoter and its gene expression in human tissues and cancer cell lines. (2003). Gene, 312(17), pp.281–288.
12. Seglen, P. and Bohley, P. (1992). Autophagy and other vacuolar protein degradation mechanisms. Experientia, 48(2), pp.158--172.
13. Pivtoraiko, V., Stone, S., Roth, K. and Shacka, J. (2009). Oxidative stress and autophagy in the regulation of lysosome-dependent neuron death. Antioxidants ＼& redox signaling, 11(3), pp.481--496.
14. Cezari, M., Puzer, L., Juliano, M., Carmona, A. and Juliano, L. (2002). Cathepsin B carboxydipeptidase specificity analysis using internally quenched fluorescent peptides. Biochem. J, 368, pp.365--369.
15. Glabe, C. (2001). Intracellular mechanisms of amyloid accumulation and pathogenesis in Alzheimer’s disease. Journal of Molecular Neuroscience, 17(2), pp.137--145.
16. Westerfield, M. (1995). The zebrafish book. 1st ed. [Eugene, OR]: M. Westerfield.
17. Horne, D. and Patterson, D. (1988). Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates. Clinical chemistry, 34(11), pp.2357--2359.
18. Sigur＼dhard＼ottir, F. (2012). Effects of sleep deprivation on learning and memory in Zebrafish (Danio rerio).
19. Avdesh, A., Martin-Iverson, M., Mondal, A., Chen, M., Askraba, S., Morgan, N., Lardelli, M., Groth, D., Verdile, G. and Martins, R. (2012). Evaluation of color preference in zebrafish for learning and memory. Journal of Alzheimer's Disease, 28(2), pp.459--469.
20. MORIYAMA, Y., TAKANO, T. and OHKUMA, S. (1982). Acridine orange as a fluorescent probe for lysosomal proton pump. Journal of biochemistry, 92(4), pp.1333--1336.
21. Eichner, E., Pierce, H. and Hillman, R. (1971). Folate balance in dietary-induced megaloblastic anemia. New England Journal of Medicine, 284(17), pp.933--938.
22. Ebenezer, P., Weidner, A., LeVine, H., Markesbery, W., Murphy, M., Zhang, L., Dasuri, K., Fernandez-Kim, S., Bruce-Keller, A., Gavil＼'an, E. and others, (2010). Neuron specific toxicity of oligomeric beta amyloid : role for JUN-kinase and oxidative stress. Journal of Alzheimer's Disease, 22(3), pp.839--848.
23. Ward, S., Himmelstein, D., Lancia, J. and Binder, L. (2012). Tau oligomers and tau toxicity in neurodegenerative disease. Biochemical Society Transactions, 40(4), p.667.
24. Mueller-Steiner, S., Zhou, Y., Arai, H., Roberson, E., Sun, B., Chen, J., Wang, X., Yu, G., Esposito, L., Mucke, L. and others, (2006). Antiamyloidogenic and neuroprotective functions of cathepsin B: implications for Alzheimer's disease. Neuron, 51(6), pp.703--714.
25. MORIYAMA, Y., TAKANO, T. and OHKUMA, S. (1982). Acridine orange as a fluorescent probe for lysosomal proton pump. Journal of biochemistry, 92(4), pp.1333--1336.
26. Ebisch, I., Thomas, C., Peters, W., Braat, D. and Steegers-Theunissen, R. (2007). The importance of folate, zinc and antioxidants in the pathogenesis and prevention of subfertility. Human Reproduction Update, 13(2), pp.163--174.
27. 8-hydroxy-2′ -deoxyguanosine (8-OHdG): A Critical Biomarker of Oxidative Stress and Carcinogenesis. (2009). Journal of Environmental Science and Health, Part C: Environmental Carcinogenesis and Ecotoxicology Reviews, 27(2), pp.120-139.
28. Dasuri, K., Zhang, L. and Keller, J. (2013). Oxidative stress, neurodegeneration, and the balance of protein degradation and protein synthesis. Free Radical Biology and Medicine, 62, pp.170--185.
29. Pappolla, M., Sos, M., Omar, R. and Sambamurti, K. (1996). The heat shock/oxidative stress connection. Molecular and chemical neuropathology, 28(1-3), pp.21--34.
30. Milleron, R. and Bratton, S. (2006). Heat shock induces apoptosis independently of any known initiator caspase-activating complex. Journal of Biological Chemistry, 281(25), pp.16991--17000.
31. Alzheimer's Association, (2014). Alzheimer's & Dementia Causes, Risk Factors | Research Center| Alzheimer's Association. [online] Available at: http://www.alz.org/research/science/alzheimers_disease_causes.asp
32. Cathepsin B. (2014). The International Journal of Biochemistry & Cell Biology, 29(5), pp.715–720.
33. Glabe, C. (2001). Intracellular mechanisms of amyloid accumulation and pathogenesis in Alzheimer’s disease. Journal of Molecular Neuroscience, 17(2), pp.137--145.
34. Ibrahim, W., Tousson, E., El-Masry, T., Arafa, N. and Akela, M. (2011). The effect of folic acid as an antioxidant on the hypothalamic monoamines in experimentally induced hypothyroid rat. Toxicology and industrial health, p.0748233711410913.
35. Perry, G., Cash, A. and Smith, M. (2002). Alzheimer disease and oxidative stress. BioMed Research International, 2(3), pp.120--123.
36. Markesbery, W. (1997). Oxidative stress hypothesis in Alzheimer's disease. Free Radical Biology and Medicine, 23(1), pp.134--147.
37. Zhao, Y. and Zhao, B. (2013). Oxidative stress and the pathogenesis of Alzheimer's disease. Oxidative medicine and cellular longevity, 2013.
38. Blom, H., Shaw, G., den Heijer, M. and Finnell, R. (2006). Neural tube defects and folate: case far from closed. Nature Reviews Neuroscience, 7(9), pp.724--731.
39. Ulrich, C. (2005). Nutrigenetics in cancer research—folate metabolism and colorectal cancer. The Journal of nutrition, 135(11), pp.2698--2702.
40. D'Costa, A. and Shepherd, I. (2009). Zebrafish development and genetics: introducing undergraduates to developmental biology and genetics in a large introductory laboratory class. Zebrafish, 6(2), pp.169--177.
41. Friedrich, R., Jacobson, G. and Zhu, P. (2010). Circuit neuroscience in zebrafish. Current Biology, 20(8), pp.371--381.