[Frontiers in Bioscience, Elite, 12, 1-34, Jan 1, 2020]

Fucoidan serves a neuroprotective effect in an Alzheimer’s disease model

Mamangam Subaraja1,2, Dhanabalan Anantha Krishnan3, Varghese Edwin Hillary1, Tharsius Raja William Raja1, Pratheesh Mathew1,4, Sundaram RaviKumar5, Michael Gabriel Paulraj1, Savarimuthu Ignacimuthu1, 6

1Division of vector control, Entomology Research Institute, Loyola College, Chennai-600034, Tamil Nadu, India 2Department of Biochemistry, Vivekanandha College of Arts and Sciences for Women (Autonomous), Tiruchengode - 637 205, Tamil Nadu, India, 3Center of Advanced Study in Crystallography and Biophysics, University of Madras, Chennai-600 025, Tamil Nadu, India, 4Department of Zoology, St. Thomas College, Kottayam-686 574, Kerala, India, 5Department of Biomedical Science, Alagappa University, Karaikudi-630 003, Tamil Nadu, India, 6Xavier Research Foundation, St. Xavier’s College, Palayakottai-627001, Tamil Nadu, India


1. Abstract
2. Introduction
3. Materials and methods
    3.1. In silico studies
      3.1.1. Protein preparation
      3.1.2. Ligand preparation
      3.1.3. Induced fit docking
    3.2. In vitro studies
      3.2.1. Maintenance of cell culture
      3.2.2. Kinetic study of cholinesterase inhibition in PC12 cells
      3.2.3. Kinetic of monoamine oxidase (MAO) A and B in PC12 cells
      3.2.4. Cell viability assay
      3.2.5. Differentiation of PC12 using different concentrations of fucoidan for 7 days
      3.2.6. Blood brain barrier (BBB) permeation assay
      3.2.7. Neurite length assay
    3.3. In vivo studies
      3.3.1. Ethical statement
      3.3.2. Preparation of fly food
      3.3.3. Maintenance of D. melanogater
      3.3.4. Experimental design
      3.3.5. Food intake tracer method
      3.3.6. Dissection of D. melanogaster brains
      3.3.7. The extracts of proteins in brain
      3.3.8. Estimation of AChE, PKI3, TrkA, Tau, BAECl, APP
      3.3.9. Extraction and quantification of glutamate and gamma –aminobutyric acid (γ-GABA) contents
      3.3.10. Extraction of brain biogenic amine
    3.4. Estimation of oxidative stress indicators
      3.4.1. Determination of lipid peroxidation (LPO)
      3.4.2. Determination of catalase (CAT) activity
      3.4.3. Determination of superoxide dismutase (SOD) activity
      3.4.4. Determination of reduced glutathione (GSH) levels
      3.4.5. Estimation of immunomodulatory molecules
      3.4.6. Estimation of apoptotic regulator molecules
      3.4.7. Isolation of RNA and cDNA synthesis
      3.4.8. Quantitative real time -PCR (qRT-PCR)
      3.4.9. Semi-Qualitative RT PCR
    3.5. Neuronal behaviour analysis
      3.5.1. Climbing analysis
      3.5.2. T Maze analysis
      3.5.3. T Maze analysis
      3.5.4. Aggression analysis
    3.6. Statistical analysis
4. Results
    4.1. In silico studies
      4.1.1. Binding interactions of test compounds and target proteins
    4.2. In vitro studies
      4.2.1. Cytotoxicity in Pheochromocytoma (PC12) cell
      4.2.2. Blood–brain barrier (BBB) permeation
      4.2.3. Kinetic study of ChE and MAO in PC12 cells
      4.2.4. Neurite length analysis
    4.3. In vitro studies
      4.3.1. Mortality rate
      4.3.2. Food intake
      4.3.3. Level of ROS in brain
      4.3.4. Cholinergic esterase activities
      4.3.5. Levels of proteins in brain
      4.3.6. The levels of neurotransmitters in brain
      4.3.7. Oxidative stress induced molecular analysis
      4.3.8. Neuroinflammation molecular analysis
      4.3.9. Apopototic regulation molecular analysis
      4.3.10. mRNA levels in brain
      4.3.11. T and Y Maze
      4.3.12. Crawling behaviour
      4.3.13. Olfactory and courtship index
5. Discussion
    5.1. In silico studies
    5.2. In vitro studies
    5.3. In vivo studies
6. Conclusion
7. Acknowledgment
8. Reference


Alzheimer's disease (AD) is a progressive neurodegenerative disorder that causes memory and cognitive deficits. The present study was carried out to evaluate the protective effects of fucoidan in monocrotophos induced AD in Drosophila melanogaster. In silico studies showed that fucoidan exhibited binding energy of -9.3 kcal with proteins. Consistent with this, fucoidan, in a dose and time-dependent fashion, had inhibitory activity against cholinergic and monoamine-metabolized enzymes in vitro. Fucoidan inhibited the increase in total mRNA and protein in monocrotophos fed flies and prevented changes in biochemicals, neurochemicals and latency time of locomotor, learning and memory induced by monocrotophos. Together, the findings show that fucoidan serves a neuroprotective effect in Alzheimer’s disease model in D. melanogaster.


1. LG Apostolova. Alzheimer Disease. Continuum (Minneap Minn). 22(2 Dementia):419-34 (2016)
DOI: 10.1212/CON.0000000000000307

2. C Hendrie, BO Osuntokun, KS Hall, AO Ogunniyi (1995) Prevalence of Alzheimer's disease and dementia in two communities: Nigerian Africans and African Americans. Am. J. Psychiatry 152, 1485 (1995)
DOI: 10.1176/ajp.152.10.1485

3. CM Tanner, SM Goldman, GW Ross, SJ Grate. The disease intersection of susceptibility and exposure: chemical exposures and neurodegenerative disease risk. Alzheimers Dement. 10(3 Suppl):S213-25 (2014).
DOI: 10.1016/j.jalz.2014.04.014

4. LG Gunnarsson, L Bodin. Occupational Exposures and Neurodegenerative Diseases-A Systematic Literature Review and Meta-Analyses. Int J Environ Res Public Health. 16 (3)- pii: E337 (2019)
DOI: 10.3390/ijerph16030337

5. D Heras-Sandoval, JM Pérez-Rojas, J Hernández-Damián , J Pedraza-Chaverri J . The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration. Cell Signal 26, 2694-701 (2014)
DOI: 10.1016/j.cellsig.2014.08.019

6. D Heras-Sandoval, JM Pérez-Rojas, J Hernández-Damián , J Pedraza-Chaverri J . The role of PI3K/AKT/mTOR pathway in the modulation of autophagy and the clearance of protein aggregates in neurodegeneration. Cell Signal 26, 2694-701 (2014)
DOI: 10.1016/j.cellsig.2014.08.019

7. E Ferreiro , I Baldeiras , IL Ferreira, RO Costa, AC Rego, CF Pereira , CR Oliveira . Mitochondrial- and endoplasmic reticulum-associated oxidative stress in Alzheimer's disease: from pathogenesis to biomarkers. Int J Cell Biol 735206 (2012)
DOI: 10.1155/2012/735206

8. W Blenau, A Baumann. Molecular and pharmacological properties of insect biogenic amine receptors: Lessons from Drosophila melanogaster and Apis mellifera Arch. Insect Biochem. Physiol 48, 13– 38 (2001)
DOI: 10.1002/arch.1055

9. TK Sang, GR Jackson. Drosophila models of neurodegenerative disease. NeuroRx. 2, 438-46 (2005)
DOI: 10.1146/annurev.pathol.3.121806.151529

10. A Moloney, DB Sattelle, DA Lomas, DC Crowther Alzheimer's disease: insights from Drosophila melanogaster models. Trends Biochem Sci 35, 228-35 (2010)
DOI: 10.1016/j.tibs.2009.11.004

11. JM McCammon, H Sive. Addressing the genetics of human mental health disorders in model organisms. Annu Rev Genomics Hum Genet 16, 173-97 (2015)
DOI: 10.1146/annurev-genom-090314-050048

12. YO Ali, W Escala, K Ruan, RG Zhai. Assaying locomotor, learning, and memory deficits in Drosophila models of neurodegeneration. J Vis Exp 49, 2504 (2001)
DOI: 10.3791/2504

13. M Hamann, D Alonso, E Martin-Aparicio, A Fuertes, M Perez-Puerto, A Castro. Glycogen synthase kinase-3 (GSK-3) inhibitory activity and structure activity relationship (SAR). Studies of the manzamine alkaloids. Potential for Alzheimer’s disease. J. Nat. Prod 70, 1397–1405 (2007)
DOI: 10.1021/np060092r

14. I Santa-Maria, F Hernández , J Del Rio , FJ Moreno, J Avila . Tramiprosate, a drug of potential interest for the treatment of Alzheimer’s disease, promotes an abnormal aggregation of tau. Mol Neurodegener 2, 17 (2007)
DOI: 10.1186/1750-1326-2-17

15. O Trott, AJ Olson. AutoDock Vina: improving the speed and accuracy of docking with a new scoring function, efficient optimization, and multithreading. J. Comput. Chem 31, 455-61(2010)
DOI: 10.1002/jcc.21334

16. T Mosmann. Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays. J Immunol methods 65, 55-63 (1983)
DOI: 10.1016/0022-1759(83)90303-4

17. WM Pardridge. Alzheimer’s disease drug development and the problem of the blood–brain barrier. Alzheimers Dement 5, 427–32 (2009)
DOI: 10.1016/j.jalz.2009.06.003

18. GL Ellman, KD Courtney, V Andres, RM Fearstherstone. A new rapid colorimetric determination of acetyl cholionesterase activity. Biochem Pharmacol 7 , 88-95 (1961)
DOI: 10.1016/0006-2952(61)90145-9

19. WW Ja, GB Carvalho, EM Mak , NN de la Rosa , AY Fang , JC Liong , T Brummel, S Benzer . Prandiology of Drosophila and the CAFE assay. Proc. Natl. Acad. Sci. U.S.A 104, 8253–8256 (2007)
DOI: 10.1073/pnas.0702726104

20. AJ Beige, SD Aust. Microsomal lipid-peroxidation. Methods Enzymol 52, 302–310 (1978)
DOI: 10.1016/S0076-6879(78)52032-6

21. I Yumoto, D Ichihashi, H Iwata, A Istokovics, N Ichise, H Matsuyama, H Okuyama , K Kawasaki . Purification and characterization of a catalase from the facultatively psychrophilic bacterium Vibrio rumoiensis S-1(T) exhibiting high catalase activity. J. Bacteriol 182, 1903–1909 (2000)
DOI: 10.1128/JB.182.7.1903-1909.2000

22. S Marklund, G Marklund . Involvement of the superoxide anion radical in the autoxidation of pyrogallol and a convenient assay for superoxide dismutase. Eur. J. Biochem 47, 469–474 (1974)
DOI: 10.1111/j.1432-1033.1974.tb03714.x

23. MS Moron, JW Depierre, B Mannervik Levels of glutathione, glutathione reductase and glutathione S-transferase activities in rat lungs and liver. Biochim. Biophys. Acta 582, 67–78 (1979)
DOI: 10.1016/0304-4165(79)90289-7

24. SC Papastamatis, JF Wilkinson Paper Chromatography of Proteins. Nature 167, 724–725 (1951)
DOI: 10.1038/167724a0

25. V Erspamer, VG Boretti .Identification and characterization, by paper chromatography, of enteramine, octopamine, tyramine, histamine and allied substances in extracts of posterior salivary glands of octopoda and in other tissue extracts of vertebrates and invertebrates. Arch. Int. Pharmacodyn. Ther 88, 296 (1951)

26. RG Pendleton, A Rasheed, T Sardina, T Tully, R Hillman. Effects of tyrosine hydroxylase mutants on locomotor activity in Drosophila: a study in functional genomics. Behav. Genet.32, 89-94 (2015)
DOI: 10.1023/A:1015279221600

27. Versace, J Reisenberger. Large-scale assessment of olfactory preferences and learning in Drosophila melanogaster: behavioral and genetic components. PeerJ 3, e1214 (2015)
DOI: 10.7717/peerj.1214

28. MM Simonnet, M Berthelot-Grosjean, Y Grosjean. Testing Drosophila olfaction with a Y Maze assay. J Vis Exp 88 (2014)
DOI: 10.3791/51241

29. AA Hoffman. Territorial encounters between Drosophila males of different sizes. Anim Behav 35, 1899 – 901(1987)
DOI: 10.1016/S0003-3472(87)80085-4

30. J Cheung, MJ Rudolph. F Burshteyn, MS Cassidy, EN Gary, J Love, MC Franklin, JJ Height. Structures of human acetylcholinesterase in complex with pharmacologically important ligands. J. Med. Chem. 55, 10282-6 (2012)
DOI: 10.1021/jm300871x

31. W Huang, H Yu, R Sheng, J Li, Y Hu Identifi cation of pharmacophore model, synthesis and biological evaluation of N-phenyl-1-arylamide and N-phenylbenzenesulfonamide derivatives as BACE 1 inhibitors. Bioorg. Med. Chem 16, 10190–10197 (2008)
DOI: 10.1016/j.bmc.2008.10.059

32. M Leirós, E Alonso, ME Rateb, WE Houssen, R Ebel, M Jaspars. Gracilins:Spongionella-derived promising compounds for Alzheimer disease. Neuropharmacology 93, 285-93 (2015)
DOI: 10.1016/j.neuropharm.2015.02.015

33. ER Wood, L Kuyper, KG Petrov, RN Hunter, PA Harris, K Lackey Discovery and in vitro evaluation of potent TrkA kinase inhibitors: Oxindole and aza-oxindoles. Bioorganic Med. Chem. Lett 14, 953–957(2004)
DOI: 10.1016/j.bmcl.2003.12.002

34. A Nordberg , AL Svensson Cholinesterase inhibitors in the treatment of Alzheimer's disease: A comparison of tolerability and pharmacology. Drug Saf 19, 465–480 (1998)
DOI: 10.2165/00002018-199819060-00004

35. LJ Legoabe, A Petzer, JP Petzer. Selected c7-substituted chromone derivatives as monoamine oxidase inhibitors. Bioorg Chem 45, 1–11 (2012)
DOI: 10.1016/j.bioorg.2012.08.003

36. R Chakraborty, V Vepuri, SD Mhatre, BE Paddock, S Miller, SJ Michelson, R Delvadia, A Desai, M Vinokur, DJ Melicharek, S Utreja, P Khandelwal, P Ansaloni, LE Goldstein, RD Moir, JC Lee, LP Tabb, AJ Saunders, DR Marenda. Characterization of a Drosophila Alzheimer's disease model: pharmacological rescue of cognitive defects. PloS one 6, e20799 (2011)
DOI: 10.1371/journal.pone.0020799

37. K Iijima , HP Liu, AS Chiang, SA Hearn, M Konsolaki, Y Zhong. Dissecting the pathological effects of human Abeta40 and Abeta42 in Drosophila: a potential model for Alzheimer’s disease. Proc Natl Acad Sci USA 101, 6623–6628 (2004)
DOI: 10.1073/pnas.0400895101

38. G Li, C Kim, J Kim, H Yoon, H Zhou, J Kim . Common pesticide, dichlorodiphenyltrichloroethane (DDT), increases amyloid-β levels by impairing the function of ABCA1 and IDE: implication for Alzheimer’s disease. JAD 46, 109-22 (2015)
DOI: 10.3233/JAD-150024

39. RM Locksley, N Killeen, MJ Leonardo. The TNF and TNF receptor superfamilies: integrating mammalian biology. Cell 104, 487-501 (2001)
DOI: 10.1016/S0092-8674(01)00237-9

40. G Nilufer Yonguc, Y Dodurga, A Kurtulus, B Boz , K Acar. Caspase-1, caspase-3, TNF-alpha, p53, and Hif1-alpha gene expression status of the brain tissues and hippocampal neuron loss in short-term dichlorvos exposed rats. Mol. Biol. Rep 39, 10355-10360 (2012)
DOI: 10.1007/s11033-012-1913-4

41. S Miwa , J St-Pierre , L Partridge, MD Brand MD. Superoxide and hydrogen peroxide production by Drosophila mitochondria. Free Radic Biol Med.35, 938-48 (2003)
DOI: 10.1016/S0891-5849(03)00464-7

42. GH Kim, JE Kim, SJ Rhie, S Yoon. The Role of Oxidative Stress in Neurodegenerative Diseases. Exp Neurobiol. 24(4):325-40 (2015)
DOI: 10.5607/en.2015.24.4.325

43. OA Petroff .GABA and glutamate in the human brain. Neuroscientist. 8(6):562-73 (2002)
DOI: 10.1177/1756285612461679

44. Kolodziejczyk A, Sun X, Meinertzhagen IA, Nässel DR. Glutamate, GABA and acetylcholine signaling components in the lamina of the Drosophila visual system. PLoS One 3(5):e2110 (2008)
DOI: 10.1371/journal.pone.0002110

45. E Folkers , H-Ch Spatz. Visual learning performance of Drosophila melanogaster is altered by neuropharmaca affecting phosphodiesterase activity and acetylcholine transmission. J. Insect Physiol. 30, 957-965 (1984)
DOI: 10.1016/0022-1910(84)90074-X

46. KG. Yiannopoulou Papageorgiou SG. Current and future treatments for Alzheimer's disease. Ther Adv Neurol Disord 6, 19-33 (2013).
DOI: 10.1177/1756285612461679

47. S Saraswati , LE Fox, DR Soll, CF Wu. Tyramine and octopamine have opposite effects on the locomotion of Drosophila larvae. J. Neurosci. 58, 425-41 (2004)
DOI: 10.1002/neu.10298

48. M Schwaerzel, M Monastirioti, H Scholz , F Friggi-Grelin, S Birman, M Heisenberg. Dopamine and octopamine differentiate between aversive and appetitive olfactory memories in Drosophila. J. Neurosci. 23, 10495-502 (2003)
DOI: 10.1523/JNEUROSCI.23-33-10495.2003

Abbreviations: AD: Alzheimer disease, NDD: Neurodegenerative diseases, CNS: Central nervous system, BBB: Blood-brain barrier, TNF-α: Tumor necrosis factor alpha, Casp3: caspase 3, Bcl2: B-cell lymphoma 2, PC12: phaeochromocytoma 12, MAO-A: Monoamine oxidase A, MAO-B: Monoamine oxidase B, AchE: Acetylcholinesterase, BuchE: Butyrylcholinesterase, PI3K: Phosphoinositide 3-kinases, TrkA: Tropomyosin receptor kinase A, T Proteins: Tau proteins, BACE-1: Beta-secretase 1, APP: Amyloid precursor protein, LPO: Lipid peroxidation, CAT: Catalase, SOD: Superoxide dismutase, GSH: Glutathione, NTs: Neurotransmitters, GABA: Gamma-aminobutyric acid, Glu: Glutamate, BAs: Biogenic amines, DA: Dopamine, OA: Octopamine, TA: Tryptamine, 5-HT: 5-hydroxytryptamine, Q-RTPCR: Quantitative reverse transcription polymerase chain reaction, Semi-quantitative RTPCR: Semi-quantitative reverse-transcriptase polymerase chain reaction, ELISA: Enzyme-linked immunosorbent assay, MTT: (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide)

Key Words: Molecular docking, fucoidan, monocrotophos, PC12 human neuroblastomal cells, Drosophila melanogaster and Alzheimer’s disease

Send correspondence to: Savarimuthu Ignacimuthu, Division of vector control, Entomology Research Institute, Loyola College, Chennai, Tamil Nadu, 600034, India, Tel: 044-28178348, Fax: 004-28174644, E-mail: rajivsuba@gmail.com