葉酸是水溶性的維生素B9。葉酸影響很多人體的生理功能，例如對去氧核醣核酸、核醣核酸的甲基化，核苷酸、蛋白質的生合成，去氧核醣核酸的修復…等。研究指出葉酸缺乏可能造成新生兒神經管缺損，也是阿茲海默症的危險因子。但葉酸缺乏在阿茲海默症中所扮演的角色目前並不清楚。在先前的研究中，在葉酸缺乏斑馬魚的腦部切片中曾觀察到阿茲海默症也會有的乙型–澱粉樣蛋白及磷酸化Tau蛋白的堆積，也觀察到認知學習能力受損。在本篇研究中，我們探討在年老斑馬魚體內葉酸含量對神經病變上的影響，同時利用管餵給予年老斑馬魚氧化態以及還原態的葉酸，並評估其認知行為能力。我們亦量測腦及肝臟中葉酸含量，並進行腦組織切片免疫染色與觀察。結果顯示，在給予一小時熱休克刺激下，組織中葉酸含量的變化並不明顯，但葉酸缺乏斑馬魚的認知能力似有受損的現象。當給予5–甲酰四氫葉酸(5-CHO-THF)及較低劑量(45 µg)的葉酸(FA)時，可以部分恢復受損的認知能力。然而，當給予較高劑量(400 µg)的FA、維生素C及三磷酸腺苷(ATP)並不能得到同樣的結果。這些結果顯示，當給予還原態葉酸及適當劑量的氧化態葉酸可以對於年老斑馬魚的認知能力具有保護作用。為了加強葉酸缺乏的嚴重程度，我們將熱休克刺激增長為12小時。組織中的葉酸含量則呈現較明顯的下降。同時亦具有認知能力受損的現象。我們發現在認知能力受損的斑馬魚的腦部葉酸含量較低。然而目前利用免疫組織化學染色觀察乙型–澱粉樣蛋白沉積的結果卻顯示乙型–澱粉樣蛋白的沉積與認知能力似無顯著相關。因此，目前的結果顯示葉酸缺乏對年老斑馬魚認知能力的影響可能不是藉由促成乙型–澱粉樣蛋白沉積所造成的。未來我們將利用8-oxoguanine抗體去進行染色，探討葉酸缺乏對斑馬魚腦部的氧化壓力是否造成影響。並測試其他抗氧化劑與單碳提供者對減緩認知能力受損的效果。

Folate, as known as vitamin B9, is a water-soluble nutrient which is essential for many physiological functions such as methylation, nucleotide synthesis and DNA repair. Folate deficiency (FD) had been reported as risk factor for neurological disease such as neuro tube defects and Alzheimer’s disease (AD). The role of FD in AD patients remains unclear. In the previous study, the deposition of β-amyloid (Aβ) and phosphorylated-tau, the pathological hallmarks of AD, were observed in the brain of aged FD zebrafish. The impaired cognitive ability was also observed in aged FD zebrafish. In this study, we investigated the impacts of folate status on the neuropathy of aged zebrafish. Simultaneously, aged zebrafish were tube feeding with either oxidized or reduced forms of folate and evaluated for their cognitive and memory ability. Then we measured the folate content in brain and liver, also applied immunohistochemistry staining in brain sections and then observed. The result showed that the tissue folate content was not significantly changed under the one-hour heat shock treatment, but the cognitive and memory ability of FD zebrafish was affected. The impairments were partially restored by supplementing with 5-formyltetrahydrofolate (5-CHO-THF) and lower dose (45 µg) of folic acid (FA). However, higher dose (400 µg) of FA, vitamin C, and ATP did not exert significant improvement. These findings suggested that supplementing with reduced folate and lower dose of FA had protective effect to the cognitive ability of aged zebrafish. To enhance the intensity of folate deficiency, we extended the heat shock treatment to 14 hours. The tissue folate content was significantly decreased in FD and the cognitive ability was also impaired. We found that the brain folate content was lower in cognitive ability impaired zebrafish. However, the result of Aβ deposition by the immunohistochemistry staining showed that there was no significant correlation between Aβ deposition and cognitive ability. Hence, we thought that the impact of FD on cognitive ability of aged zebrafish might not be due to Aβ deposition. In the future, the 8-OHdG stain was applied to investigate whether the oxidative stress in brain of zebrafish was affected by FD. Moreover, to examine the effect of other antioxidants and one-carbon provider on relieving the impairment of cognitive ability.

[1] Mankodi B. S. and Rege D. V. (1966). Biosynthesis of folic acid. ii. Utilization of exogenous intermediates for the biosynthesis of folic acid by bacillus subtitis enzymes. Arch Mikrobiol, 53(3), 208–217.
[2] Carmel R (2005). Folic Acid. Modern Nutrition in Health and Disease. M. Shils, M. Shike, A. Ross, B. Caballero and R. Cousins. Baltimore, MD, Lippincott Williams & Wilkins: 470–481.
[3] Selhub J (2002). Folate, vitamin B12 and vitamin B6 and one carbon metabolism. J Nutr Health Aging, 6(1), 39–42.
[4] "Folate deficiency: MedlinePlus Medical Encyclopedia". www.nlm.nih.gov. Retrieved 2015-11-16.
[5] Wien, T. N., Pike, E., Wisløff, T., Staff, A., Smeland, S., & Klemp, M. (2012). Cancer risk with folic acid supplements: a systematic review and meta-analysis. BMJ Open, 2(1), e000653. http://doi.org/10.1136/bmjopen-2011-000653
[6] Wang, R., Zheng, Y., Huang, J.-Y., Zhang, A.-Q., Zhou, Y.-H., & Wang, J.-N. (2014). Folate intake, serum folate levels, and prostate cancer risk: a meta-analysis of prospective studies. BMC Public Health, 14, 1326. http://doi.org/10.1186/1471-2458-14-1326
[7] Dana Foundation, Nicky Penttila (August 31, 2015). From the Archives: Reducing the Risks of Alzheimer’s. https://danablog.org/2015/08/31/from-the-archives-reducing-risks-of-alzheimers/
[8] Reitz, C., and Mayeux, R. (2014). Alzheimer disease: Epidemiology, Diagnostic Criteria, Risk Factors and Biomarkers. Biochemical Pharmacology, 88(4), 640–651. http://doi.org/10.1016/j.bcp.2013.12.024
[9] Burns A, Iliffe S (5 February 2009). "Alzheimer's disease". The BMJ 338: b158.
[10] C. Ballard, S. Gauthier, A. Corbett, C. Brayne, D. Aarsland, E. Jones (2011). Alzheimer’s disease. Lancet, 377, 1019–1031
[11] Alberto Serrano-Pozo, Matthew P. Frosch, Eliezer Masliah and Bradley T. Hyman (2011). Neuropathological Alterations in Alzheimer Disease. Cold Spring Harb. Perspect. Med. 1(1), a006189. http://doi.org/10.1101/cshperspect.a006189
[12] Lacor PN, Buniel MC, Furlow PW, Clemente AS, Velasco PT, Wood M, Viola KL, Klein WL. (2007) Aß Oligomer-Induced Aberrations in Synapse Composition, Shape, and Density Provide a Molecular Basis for Loss of Connectivity in Alzheimer's Disease. The Journal of Neuroscience, 27(4), 796–807.
[13] Laurén J, Gimbel DA, Nygaard HB, Gilbert JW, Strittmatter SM. (2009). Cellular Prion Protein Mediates Impairment of Synaptic Plasticity by Amyloid-β Oligomers. Nature, 457(7233), 1128–32.
[14] Goedert M, Spillantini MG, Crowther RA. (1991). Tau Proteins and Neurofibrillary Degeneration. Brain Pathology, 1(4), 279–86.
[15] Chun W, Johnson GV. (2007). The Role of Tau Phosphorylation and Cleavage in Neuronal Cell Death. Frontiers in Bioscience, 12, 733–56.
[16] Chong MS, Sahadevan S. (2005). Preclinical Alzheimer's disease: diagnosis and prediction of progression. Lancet Neurology, 4(9), 576–9.
[17] Marksteiner J, Hinterhuber H, Humpel C. (2007). Cerebrospinal Fluid Biomarkers for Diagnosis of Alzheimer's Disease: Beta-amyloid(1–42), Tau, Phospho-tau-181 and Total Protein. Drugs of Today, 43(6), 423–31.
[18] Eskes TK. (1998). Open or closed? A world of difference: a history of homocysteine research. Nutr Rev. 56(8), 236–244.
[19] Wen-Ni Chang, Jen-Ning Tsai, Bing-Hung Chen, Huei-Sheng Huang, and Tzu-Fun Fu. (2007). Serine hydroxymethyltransferase isoforms are differentially inhibited by leucovorin: characterization and comparison of recombinant zebrafish serine hydroxymethyltransferases. Drug Metab Dispos., 35(11), 2127–37.
[20] Czeizel, A. E., Dudás, I., Vereczkey, A., & Bánhidy, F. (2013). Folate Deficiency and Folic Acid Supplementation: The Prevention of Neural-Tube Defects and Congenital Heart Defects. Nutrients, 5(11), 4760–4775. http://doi.org/10.3390/nu5114760
[21] Pei I. Ho, David Ashline, Sirikarnt Dhitavat, Daniela Ortiz, Scott C. Collins, Thomas B. Shea, Eugene Rogers (2003). Folate deprivation induces neurodegeneration: roles of oxidative stress and increased homocysteine. Neurobiol Dis. 14(1), 32–42.
[22] Hughes TF, Andel R, Small BJ, et al. (2010). Midlife fruit and vegetable consumption and risk of dementia in later life in Swedish twins. Am J Geriatr Psychiatry. 18(5), 413–420.
[23] Reynolds, E. H. (2002). Folic acid, ageing, depression, and dementia. BMJ : British Medical Journal, 324(7352), 1512–1515.
[24] Faux NG, Ellis KA, Porter L, et al. (2011). Homocysteine, vitamin B12, and folic acid levels in Alzheimer's disease, mild cognitive impairment, and healthy elderly: baseline characteristics in subjects of the Australian Imaging Biomarker Lifestyle study. J Alzheimers Dis. 27(4), 909-922.
[25] "Zebra Danio". Aquatics To Your Door. Retrieved April 10, 2013.
[26] Howe, K., Clark, M. D., Torroja, C. F., Torrance, J., Berthelot, C., Muffato, M., … Stemple, D. L. (2013). The zebrafish reference genome sequence and its relationship to the human genome. Nature, 496(7446), 498–503. http://doi.org/10.1038/nature12111
[27] "Fish for Science". University of Sheffield. 2011. Retrieved March 19, 2011.
[28] Egan, R. J., Bergner, C. L., Hart, P. C., Cachat, J. M., Canavello, P. R., Elegante, M. F., … Kalueff, A. V. (2009). Understanding behavioral and physiological phenotypes of stress and anxiety in zebrafish. Behavioural Brain Research, 205(1), 38–44. http://doi.org/10.1016/j.bbr.2009.06.022
[29] Newman M, Verdile G, Martins RN, Lardelli M. (2011). Zebrafish as a tool in Alzheimer's disease research. Biochim Biophys Acta, 1812(3), 346–52.
[30] Moussavi Nik SH, Wilson L, Newman M, Croft K, Mori TA, Musgrave I, Lardelli M (2012). The BACE1-PSEN-AβPP regulatory axis has an ancient role in response to low oxygen/oxidative stress. J Alzheimers Dis, 28(3), 515–30.
[31] Avdesh A, Martin-Iverson MT, Mondal A, Chen M, Askraba S, Morgan N, Lardelli M, Groth DM, Verdile G, Martins RN. (2012). Evaluation of color preference in zebrafish for learning and memory. J Alzheimers Dis, 28(2), 459–69.
[32] E.L. Serra, C.C. Medalha, Rosana Mattioli (1999). Natural preference of zebrafish (Danio rerio) for a dark environment. Braz J Med Biol Res, 32(12), 1551–3.
[33] Schneider, E. and Ryan, T. (2006). Gamma-glutamyl hydrolase and drug resistance. Clinica chimica acta, 374(1), 25–32.
[34] Tseng-Ting Kao, Chia-Yi Chu, Gang-Hui Lee, Tsun-Hsien Hsiao, Nai-Wei Cheng, Nan-Shan Chang, Bing-Hung Chen, Tzu-Fun Fu (2014). Folate deficiency-induced oxidative stress contributes to neuropathy in young and aged zebrafish--Implication in neural tube defects and Alzheimer's diseases. Neurobiol Dis. 71, 234–44.
[35] Westerfield, M. (1995) The Zebrafish Book. A Guide for the Laboratory Use of Zebrafish (Danio rerio), 3rd Edition. Eugene, OR, University of Oregon Press, 385.
[36] Sigurðardóttir, F.D. (2012). Effects of sleep deprivation on learning and memory in zebrafish (Danio rerio). University of Iceland, Reykjavík.
[37] Fischer AH, Jacobson KA, Rose J, Zeller R. (2008). Cryosectioning tissues. Cold Spring Harb Protoc 2008:pdb.prot4991.
[38] Horne, D. and Patterson, D. (1988). Lactobacillus casei microbiological assay of folic acid derivatives in 96-well microtiter plates. Clinical Chemistry, 34(11), 2357–2359.
[39] Dieter Lütjohann; Sabrina Meichsner; Hanna Pettersson (2012). Lipids in Alzheimer's disease and their potential for therapy. Clin Lipidology, 7(1), 65–78.
[40] Michael W King, PhD (2016) Vitamins: Water and Fat Soluble http://themedicalbiochemistrypage.org/vitamins.php
[41] Patric J. Stover (2009) One-carbon metabolism-genome interactions in folate-associated pathologies. Journal of Nutrition, 139, 2402–2405.
[42] Allison D'Costa and Iain Shepherd (2009). Zebrafish Development and Genetics: Introducing Undergraduates to Developmental Biology and Genetics in a Large Introductory Laboratory Class. Zebrafish, 6(2), 169–177.