Towards identifying novel modulators and targets for alzheimers disease therapy

TOWARDS IDENTIFYING NOVEL MODULATORS AND TARGETS FOR ALZHEIMER’S DISEASE THERAPY BAHETY PRITI BALDEODAS (B. Pharm, Nirma University) A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY DEPARTMENT OF PHARMACY NATIONAL UNIVERSITY OF SINGAPORE 2014 DECLARATION I hereby declare that the thesis is my original work and it has been written by me in its entirety. I have duly acknowledged all the sources of information which have been used in the thesis. This thesis has also not been submitted for any degree in any university previously. . _________________________________ Bahety Priti Baldeodas 03 November 2014 i ii ACKNOWLEDGEMENTS The completion of this thesis would not have been possible without the support and encouragement of a number of people around me. Thanking them in these few pages is definitely not enough, but I would like to express my deep gratitude to all those people who have helped me to move forward and fulfil this dream of mine. Foremost, I would like to express my sincerest gratitude to my supervisor Dr Ee Pui Lai Rachel for her constant guidance and supervision throughout this research journey. Her patience, unwavering support and encouragement and unreserved nature gave me countless opportunities to learn and try new things, to explore different projects outside our own lab and truly understand the science behind little things. I am also indebted to my co-supervisor A/Prof Chan Chun Yong Eric for his unreserved help, motivation and inspiring discussions, especially for the metabonomics portion of my work. I have learned a lot from both my supervisors and will be forever grateful to them for this valuable and enjoyable experience. I would like to extend my appreciation to all the past and present members of Ee lab: Pay Chin, Zhan Yuin, Li Yan, Luqi, Jasmeet, Wang Ying and Sybil for making this long journey a memorable experience. I also wish to thank my other lab family at Metabolic Profiling Research Group: James, Lian Yee and Hui Ting for being there whenever I needed them. A heartfelt gratitude to Yee Min and Yanjun for teaching me the very basics of chromatography and guiding me throughout the metabonomics work. Thank you to the other friends in the Pharmaceutical Biology Laboratory and department for their help and friendly support. Thank you to Yuanjie too for being a great friend since day-1 of this roller-coaster ride. The time spent with you all not only helped me to solve my scientific difficulties but also gave me moral support when I needed it the most. In addition, I would like to thank my FYP and UROPS students, Hai Van and Jia Ni for helping out with my experiments and iii unknowingly teaching me how to be a good mentor. Appreciations are also due to the people behind the scenes, making everything possible: Winnie, Sek Eng and Pey Pey for making the lab work much smoother and easier. The invaluable support and assistance provided by the academic and administrative staff of the Department of Pharmacy is also gratefully acknowledged. A big thank you to all my friends in India, Singapore and elsewhere for being my other family away from home; for always being patient to listen to my grumbling about my experiments and crack jokes for the same to lighten me up. Your encouragement and moral support have been instrumental in the completion of this thesis and the maintenance of my sanity. A very special appreciation is due to National University of Singapore for giving me the NUS Graduate Scholarship and the Industrial Partnership Programme Scholarship, which enabled me to undertake this study and get valuable industrial internship. This work is made possible by the generous support of the NUS Academic Research Grant. Lastly, I would like to thank my family, specially my parents and my brother, who have always been the pillars of my strength, encouraging and pushing me to follow my adventurous dreams, specially this one. They taught me to never give up and made me what I am today. I dedicate this thesis to them. Thank you for believing in me, always!! iv LIST OF PUBLICATIONS AND PRESENTATIONS Publications and submitted Manuscripts: 1. Bahety P, Zhang Luqi and Ee PLR. Dihydrofolate reductase enzyme inhibition synergizes with a glycogen synthase kinase-3β inhibitor for enhanced neuroprotective effect in SH-SY5Y neuroblastoma cells. Manuscript under preparation. 2. Bahety P, Nguyen THV, Hong Y, Chan ECY and Ee PLR. Targeted metabonomic profiling of cholesterol metabolism pathway in a DHA treated Alzheimer’s disease cell model using gas chromatography single quadrupole mass spectrometry. Manuscript under preparation. 3. Zhang Luqi, Bahety P and Ee PLR. Protective role of Wnt signaling co-receptors LRP5/6 against hydrogen peroxide-induced neurotoxicity and tau phosphorylation in SH-SY5Y neuroblastoma cells. Manuscript under preparation. 4. Bahety P, Tan YM, Hong Y, Zhang L, Chan ECY and Ee PLR. Metabotyping of docosahexaenoic acid - treated Alzheimer’s disease cell model. PLoS ONE, 2014, 9(2): e90123. doi: 10.1371/journal.pone.0090123. 5. Wang Y, Ke XY, Khara JS, Bahety P, Liu S, Seow SV, Yang YY and Ee PLR. Synthetic modifications of the immunomodulating peptide thymopentin to confer anti-mycobacterial activities. Biomaterials, 2014, 35(9); 3102-3109. 6. Leow PC, Bahety P, Boon CP, Lee CY, Tan KL, Yang T and Ee PLR. Functionalized curcumin analogues as potent modulators of the Wnt/β-catenin signaling pathway. European Journal of Medicinal Chemistry, 2014, 71; 6780. Conference Proceedings: 1. Bahety P, Tan YM, Hong Y, Chan ECY and Ee PLR. Understanding the effects of docosahexaenoic acid in mitigating amyloid precursor protein-induced mitochondrial dysfunctions using metabonomics approach. Neurodegenerative Diseases (11th International Conference AD/PD, Florence, March 2013: Abstracts), 2013, 11 (Suppl. 1). v Conference Presentations (Oral): 1. Bahety P, Zhang L, and Ee PLR. Exploring the neuroprotective effects of dual DHFR and GSK-3β enzyme inhibition in an Alzheimer’s disease cell model. 9th PharmSci@Asia Symposium, 5-6 June 2014, Shanghai, China - Best Presentation and Student Travel Grant Award. 2. Bahety P, Tan YM, Hong Y, Chan ECY and Ee PLR. Understanding the effects of docosahexaenoic acid in mitigating amyloid precursor protein-induced mitochondrial dysfunctions using metabonomics approach. 18th Biological Sciences Graduate Congress, 6-8 Jan 2014, Kuala Lumpur, Malaysia – Student Travel and Housing Grant Award. 3. Bahety P, Tan YM, Hong Y, Chan ECY and Ee PLR. Understanding the effects of docosahexaenoic acid in mitigating amyloid precursor protein-induced mitochondrial dysfunctions using metabonomics approach. 2nd ITB-NUS Scientific Symposium, 12 Nov 2013, Singapore. 4. Bahety P and Ee PLR. Dual inhibition of the dihydrofolate reductase and glycogen synthase kinase enzymes enhances Wnt/β-catenin signaling for improved neuronal survival. 7th PharmaSci@Asia Symposium, 6-7 Jun 2012, Singapore - Student Travel Grant Award. 5. Bahety P, Go ML and Ee PLR. Investigating the role of glycogen synthase kinase - 3β inhibitors as Wnt/β-catenin signaling pathway inducers on SH-SY5Y neuroblastoma cells as a therapeutic strategy for Alzheimer’s disease. 2nd PharmSci@India Conference, 3-4 Sep 2011, Hyderabad, India - Student Travel Grant Award. Conference Presentations (Poster): 1. Bahety P, Nguyen THV, Hong Y, Chan ECY and Ee PLR. Targeted metabonomic profiling of the cholesterol metabolism pathway in a docosahexaenoic acid treated Alzheimer’s disease cell model. Humboldt Kolleg International Symposium on Environment and Health, 22 Sep 2014, Singapore. 2. Bahety P, Tan YM, Hong Y, Chan ECY and Ee PLR. Metabotyping of docosahexaenoic acid treated Alzheimer’s disease cell model. The Yong Loo Lin School of Medicine Annual Graduate Scientific Congress, 11 Mar 2014, Singapore. vi 3. Bahety P, Zhang L and Ee PLR. Inhibition of dihydrofolate reductase enzyme enhances neuroprotective effects mediated by glycogen synthase kinase-3β inhibition in an Alzheimer’s disease cell model. 13th International Geneva/Springfield International Symposium on Advances in Alzheimer's Therapy, 26-29 Mar 2014, Geneva, Switzerland. 4. Bahety P, Tan YM, Hong Y, Chan ECY and Ee PLR. Understanding the effects of docosahexaenoic acid in mitigating amyloid precursor protein-induced mitochondrial dysfunctions using metabonomics approach. 11th International Conference on Alzheimer’s and Parkinson’s Diseases, 6-10 Mar 2013, Florence, Italy. 5. Bahety P, Tan YM, Hong Y, Chan ECY and Ee PLR. Gas chromatography/timeof-flight mass spectrometry metabotyping of docosahexaenoic acid-treated Alzheimer’s disease cell model. Globalization of Pharmaceutics Education Network, 28 Nov – Dec 2012, Melbourne, Australia - Biota and Teikoku Seiyaku Co. Ltd Housing Grant and Travel Grant Award. 6. Bahety P and Ee PLR. Dual inhibition of the dihydrofolate reductase and glycogen synthase kinase enzymes enhances Wnt/β-catenin signaling for improved neuronal survival. NUS Annual Pharmacy Research Symposium, April 2012, Singapore. vii viii 36. Zhang S, Hedskog L, Petersen CA, Winblad B, Ankarcrona M (2010) Dimebon (latrepirdine) enhances mitochondrial function and protects neuronal cells from death. Journal of Alzheimers Disease 21: 389-402. 37. Hooper C, Killick R, Lovestone S (2008) The GSK3 hypothesis of Alzheimer’s disease. Journal of Neurochemistry 104: 1433-1439. 38. Vanleuven F (2011) GSK3 and Alzheimer's disease: facts & fiction. Frontiers in Molecular Neuroscience 4. 39. Mondragón-Rodríguez S, Perry G, Zhu X, Moreira PI, Williams S (2012) Glycogen Synthase Kinase 3: A Point of Integration in Alzheimer's Disease and a Therapeutic Target? International Journal of Alzheimer's Disease: 4. 40. Giese KP (2009) GSK-3: A key player in neurodegeneration and memory. IUBMB Life 61: 516-521. 41. Kang DE, Soriano S, Frosch MP, Collins T, Naruse S, et al. (1999) Presenilin facilitates the constitutive turnover of beta-catenin: differential activity of Alzheimer's disease-linked PS1 mutants in the beta-catenin-signaling pathway. Journal of Neuroscience 19: 4229-4237. 42. Nishimura M, Yu G, Levesque G, Zhang DM, Ruel L, et al. (1999) Presenilin mutations associated with Alzheimer disease cause defective intracellular trafficking of beta-catenin, a component of the presenilin protein complex. Nature Medicine 5: 164-169. 43. Lucas JJ, Hernández F, Gómez‐Ramos P, Morán MA, Hen R, et al. (2001) Decreased nuclear β‐catenin, tau hyperphosphorylation and neurodegeneration in GSK‐3β conditional transgenic mice. The EMBO Journal 20: 27-39. 44. Asuni AA, Hooper C, Reynolds CH, Lovestone S, Anderton BH, et al. (2006) GSK3α exhibits β-catenin and tau directed kinase activities that are modulated by Wnt. European Journal of Neuroscience 24: 3387-3392. 45. Nakajima K, Kohsaka S (1993) Functional roles of microglia in the brain. Neuroscience Research 17: 187-203. 46. Martin M, Rehani K, Jope RS, Michalek SM (2005) Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nature Immunology 6: 777-784. 47. Hooper C, Markevich V, Plattner F, Killick R, Schofield E, et al. (2007) Glycogen synthase kinase-3 inhibition is integral to long-term potentiation. European Journal of Neuroscience 25: 81-86. 48. Peineau S, Bradley C, Taghibiglou C, Doherty A, Bortolotto ZA, et al. (2008) The role of GSK-3 in synaptic plasticity. British Journal of Pharmacology 153: 2. 49. Peineau S, Taghibiglou C, Bradley C, Wong TP, Liu L, et al. (2007) LTP inhibits LTD in the hippocampus via regulation of GSK3beta. Neuron 53: 703-717. 50. Dewachter I, Ris L, Jaworski T, Seymour CM, Kremer A, et al. (2009) GSK3beta, a centre-staged kinase in neuropsychiatric disorders, modulates long term memory by inhibitory phosphorylation at serine-9. Neurobiological Disorders 35: 193-200. 51. Pap M, Cooper GM (1998) Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-Kinase/Akt cell survival pathway. Journal of Biological Chemistry 273: 19929-19932. 52. Sun X, Sato S, Murayama O, Murayama M, Park JM, et al. (2002) Lithium inhibits amyloid secretion in COS7 cells transfected with amyloid precursor protein C100. Neuroscience Letters 321: 61-64. 103 53. Phiel CJ, Wilson CA, Lee VM, Klein PS GSK-3alpha regulates production of Alzheimer's disease amyloid-beta peptides. Nature 2003 423: 435-439. 54. Alvarez G, Munoz-Montano JR, Satrustegui J, Avila J, Bogonez E, et al. (1999) Lithium protects cultured neurons against beta-amyloid-induced neurodegeneration. FEBS Letter 453: 260-264. 55. Cross DA, Culbert AA, Chalmers KA, Facci L, Skaper SD, et al. (2001) Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. Journal of Neurochemistry 77: 94-102. 56. Huang WC, Lin YS, Wang CY, Tsai CC, Tseng HC, et al. (2009) Glycogen synthase kinase-3 negatively regulates anti-inflammatory interleukin-10 for lipopolysaccharide-induced iNOS/NO biosynthesis and RANTES production in microglial cells. Immunology 128: 1365-2567. 57. Ramirez SH, Fan S, Zhang M, Papugani A, Reichenbach N, et al. (2010) Inhibition of Glycogen Synthase Kinase 3β (GSK3β) Decreases Inflammatory Responses in Brain Endothelial Cells. The American journal of pathology 176: 881-892. 58. Van Dyk K, Sano M (2007) The Impact of Nutrition on Cognition in the Elderly. Neurochemical Research 32: 893-904. 59. Yamada K, Tanaka T, Han D, Senzaki K, Kameyama T, et al. (1999) Protective effects of idebenone and α-tocopherol on β-amyloid-(1–42)-induced learning and memory deficits in rats: implication of oxidative stress in β-amyloid-induced neurotoxicity in vivo. European Journal of Neuroscience 11: 83-90. 60. Morris MC, Beckett LA, Scherr PA, Hebert LE, Bennett DA, et al. (1998) Vitamin E and Vitamin C Supplement Use and Risk of Incident Alzheimer Disease. Alzheimer Disease & Associated Disorders 12: 121-126. 61. Engelhart MJ, Geerlings MI, Ruitenberg A, et al. (2002) DIetary intake of antioxidants and risk of alzheimer disease. JAMA 287: 3223-3229. 62. Luchsinger JA, Tang M, Shea S, Mayeux R (2003) ANtioxidant vitamin intake and risk of alzheimer disease. Archives of Neurology 60: 203-208. 63. Isaac MG, Quinn R, Tabet N (2008) Vitamin E for Alzheimer's disease and mild cognitive impairment. Cochrane Database Systems Review 16. 64. Morris MC, Evans DA, Bienias JL, Tangney CC, Bennett DA, et al. (2003) Consumption of fish and n-3 fatty acids and risk of incident Alzheimer disease. Archives of Neurology 60: 940-946. 65. Kalmijn S, Launer LJ, Ott A, Witteman JCM, Hofman A, et al. (1997) Dietary fat intake and the risk of incident dementia in the Rotterdam study. Annals of Neurology 42: 776-782. 66. Muskiet FAJ, van Goor SA, Kuipers RS, Velzing-Aarts FV, Smit EN, et al. (2006) Long-chain polyunsaturated fatty acids in maternal and infant nutrition. Prostaglandins, leukotrienes, and essential fatty acids 75: 135-144. 67. Bazan NG, Molina MF, Gordon WC (2011) Docosahexaenoic acid signalolipidomics in nutrition: Significance in aging, neuroinflammation, macular degeneration, Alzheimer’s, and other neurodegenerative diseases. Annual Review of Nutrition 31: 321-351. 68. Kyle DJ, Schaefer E, Patton G, Beiser A (1999) Low serum docosahexaenoic acid is a significant risk factor for Alzheimer’s dementia. Lipids 34: S245-S245. 69. Mao P (2013) Oxidative stress and its clinical applications in dementia. Journal of Neurodegenerative Diseases 2013: 15. 104 70. Lim GP, Calon F, Morihara T, Yang F, Teter B, et al. (2005) A diet enriched with the omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in an aged Alzheimer mouse model. The Journal of Neuroscience 25: 3032-3040. 71. Calon F, Lim GP, Yang F, Morihara T, Teter B, et al. (2004) Docosahexaenoic acid protects from dendritic pathology in an Alzheimer's disease mouse model. Neuron 43: 633-645. 72. Hashimoto M, Hossain S, Agdul H, Shido O (2005) Docosahexaenoic acidinduced amelioration on impairment of memory learning in amyloid β-infused rats relates to the decreases of amyloid β and cholesterol levels in detergent-insoluble membrane fractions. Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 1738: 91-98. 73. Hashimoto M, Tanabe Y, Fujii Y, Kikuta T, Shibata H, et al. (2005) Chronic administration of docosahexaenoic acid ameliorates the impairment of spatial cognition learning ability in amyloid β–infused rats. The Journal of Nutrition 135: 549-555. 74. Green KN, Martinez-Coria H, Khashwji H, Hall EB, Yurko-Mauro KA, et al. (2007) Dietary Docosahexaenoic Acid and Docosapentaenoic Acid Ameliorate Amyloid-β and Tau Pathology via a Mechanism Involving Presenilin Levels. The Journal of Neuroscience 27: 4385-4395. 75. Weldon SM, Mullen AC, Loscher CE, Hurley LA, Roche HM (2007) Docosahexaenoic acid induces an anti-inflammatory profile in lipopolysaccharidestimulated human THP-1 macrophages more effectively than eicosapentaenoic acid. The Journal of Nutritional Biochemistry 18: 250-258. 76. Vedin I, Cederholm T, Freund Levi Y, Basun H, Garlind A, et al. (2008) Effects of docosahexaenoic acid–rich n−3 fatty acid supplementation on cytokine release from blood mononuclear leukocytes: the OmegAD study. The American Journal of Clinical Nutrition 87: 1616-1622. 77. Mullen A, Loscher CE, Roche HM (2010) Anti-inflammatory effects of EPA and DHA are dependent upon time and dose-response elements associated with LPS stimulation in THP-1-derived macrophages. The Journal of Nutritional Biochemistry 21: 444-450. 78. Laurin D, Verreault R, Lindsay J, Dewailly E, Holub BJ (2003) Omega-3 fatty acids and risk of cognitive impairment and dementia. Journal of Alzheimers Disease 5: 315-322. 79. Freund-Levi Y, Eriksdotter-Jonhagen M, Cederholm T, Basun H, Faxen-Irving G, et al. (2006) Omega-3 fatty acid treatment in 174 patients with mild to moderate Alzheimer disease: OmegAD study: a randomized double-blind trial. Archives of Neurology 63: 1402-1408. 80. Quinn JF, Raman R, Thomas RG, et al. (2010) Docosahexaenoic acid supplementation and cognitive decline in alzheimer disease: A randomized trial. JAMA 304: 1903-1911. 81. Grimm MOW, Kuchenbecker J, Grösgen S, Burg VK, Hundsdörfer B, et al. (2011) Docosahexaenoic Acid Reduces Amyloid β Production via Multiple Pleiotropic Mechanisms. Journal of Biological Chemistry 286: 14028-14039. 82. Zhao Y, Calon F, Julien C, Winkler JW, Petasis NA, et al. (2011) Docosahexaenoic Acid-Derived Neuroprotectin D1 Induces Neuronal Survival via Secretase- and PPARγ-Mediated Mechanisms in Alzheimer's Disease Models. PLoS One 6: e15816. 105 83. Lukiw WJ, Cui J-G, Marcheselli VL, Bodker M, Botkjaer A, et al. (2005) A role for docosahexaenoic acid–derived neuroprotectin D1 in neural cell survival and Alzheimer disease. The Journal of Clinical Investigation 115: 2774-2783. 84. Cole GM, Frautschy SA (2010) DHA may prevent age-related dementia. The Journal of Nutrition 140: 869-874. 85. Trushina E, Mielke MM (2014) Recent advances in the application of metabolomics to Alzheimer's Disease. Biochimica et Biophysica Acta (BBA) Molecular Basis of Disease 1842: 1232-1239. 86. Oresic M, Hyotylainen T, Herukka SK, Sysi-Aho M, Mattila I, et al. (2011) Metabolome in progression to Alzheimer's disease. Translational Psychiatry 1: e57. 87. Motsinger-Reif A, Zhu H, Kling M, Matson W, Sharma S, et al. (2013) Comparing metabolomic and pathologic biomarkers alone and in combination for discriminating Alzheimer's disease from normal cognitive aging. Acta Neuropathologica Communications 1: 28. 88. Greenberg N, Grassano A, Thambisetty M, Lovestone S, Legido-Quigley C (2009) A proposed metabolic strategy for monitoring disease progression in Alzheimer's disease. Electrophoresis 30: 1235-1239. 89. Li N-j, Liu W-t, Li W, Li S-q, Chen X-h, et al. (2010) Plasma metabolic profiling of Alzheimer's disease by liquid chromatography/mass spectrometry. Clinical Biochemistry 43: 992-997. 90. Klinghoffer RA, Frazier J, Annis J, Berndt JD, Roberts BS, et al. (2009) A lentivirus-mediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of beta-catenin/GSK3 signaling. PLoS One 4: 0006892. 91. Lim GP, Calon F, Morihara T, Yang F, Teter B, et al. (2005) A diet enriched with the omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in an aged Alzheimer mouse model. J Neurosci 25: 3032-3040. 92. Calon F, Lim GP, Yang F, Morihara T, Teter B, et al. (2004) Docosahexaenoic acid protects from dendritic pathology in an Alzheimer's disease mouse model. Neuron 43: 633-645. 93. Hashimoto M, Hossain S, Agdul H, Shido O (2005) Docosahexaenoic acidinduced amelioration on impairment of memory learning in amyloid beta-infused rats relates to the decreases of amyloid beta and cholesterol levels in detergentinsoluble membrane fractions. Biochimica et Biophysica Acta 30: 1-3. 94. Chapkin RS, Kim W, Lupton JR, McMurray DN (2009) Dietary docosahexaenoic and eicosapentaenoic acid: Emerging mediators of inflammation. Prostaglandins, Leukotrienes and Essential Fatty Acids 81: 187-191. 95. Kang D, Soriano S, Frosch M, Collins T, Naruse S, et al. (1999) Presenilin facilitates the constitutive turnover of beta-catenin: differential activity of Alzheimer's disease-linked PS1 mutants in the beta-catenin-signaling pathway. J Neurosci 19: 4229-4237. 96. Nishimura M, Yu G, Levesque G, Zhang D, Ruel L, et al. (1999) Presenilin mutations associated with Alzheimer disease cause defective intracellular trafficking of beta-catenin, a component of the presenilin protein complex. Nat Med 5: 164-169. 97. Yu G, Chen F, Levesque G, Nishimura M, Zhang D, et al. (1998) The presenilin protein is a component of a high molecular weight intracellular complex that contains β-catenin. J Biol Chem 273: 16470-16475. 106 98. Asuni AA, Hooper C, Reynolds H, Lovestone S, Anderton BH, et al. (2006) GSK3α exhibits β-catenin and tau directed kinase activities that are modulated by Wnt. Eur J Neurosci 24: 3387-3392. 99. Caricasole A, Bakker A, Copani A, Nicoletti F, Gaviraghi G, et al. (2005) Two sides of the same coin: Wnt signaling in neurodegeneration and neuro-oncology. Biosci Rep 25: 309-327. 100. Lucas J, Hernandez F, Gomez-Ramos P, Moran M, Hen R, et al. (2001) Decreased nuclear β-catenin, tau hyperphosphorylation and neurodegeneration in GSK-3β conditional transgenic mice. Embo J 20: 27-39. 101. Martin M, Rehani K, Jope R, Michalek S (2005) Toll-like receptor-mediated cytokine production is differentially regulated by glycogen synthase kinase 3. Nat Immunol 6: 777-784. 102. Nakajima K, Kohsaka S (1993) Functional roles of microglia in the brain. Neurosci Res 17: 187-203. 103. Rossi F, Bianchini E (1996) Synergistic induction of nitric oxide by betaamyloid and cytokines in astrocytes. Biochem Biophys Res Commun 225: 474478. 104. Pap M, Cooper GM (1998) Role of glycogen synthase kinase-3 in the phosphatidylinositol 3-kinase/Akt cell survival pathway. J Biol Chem 273: 1992919932. 105. Cross DA, Culbert AA, Chalmers KA, Facci L, Skaper SD, et al. (2001) Selective small-molecule inhibitors of glycogen synthase kinase-3 activity protect primary neurones from death. J Neurochem 77: 94-102. 106. Bhat R, Xue Y, Berg S, Hellberg S, Ormö M, et al. (2003) Structural insights and biological effects of glycogen synthase kinase 3-specific inhibitor ARA014418. J Biol Chem 278: 45937-45945. 107. Huang W, Lin Y, Wang C, Tsai C, Tseng H, et al. (2009) Glycogen synthase kinase-3 negatively regulates anti-inflammatory interleukin-10 for lipopolysaccharide-induced iNOS/NO biosynthesis and RANTES production in microglial cells. Immun 128: e275-286. 108. Medina M, Garrido JJ, Wandosell FG (2011) Modulation of GSK-3 as a therapeutic strategy on Tau pathologies. Frontiers in Molecular Neuroscience 4. 109. Klinghoffer R, Frazier J, Annis J, Berndt J, Roberts B, et al. (2009) A lentivirusmediated genetic screen identifies dihydrofolate reductase (DHFR) as a modulator of β-catenin/GSK3 signaling. PLoS One 4: 0006892. 110. Meijer L, Skaltsounis A-L, Magiatis P, Polychronopoulos P, Knockaert M, et al. (2003) GSK-3-Selective Inhibitors Derived from Tyrian Purple Indirubins. Chemistry & Biology 10: 1255-1266. 111. Zhang X, Yin W-k, Shi X-d, Li Y (2011) Curcumin activates Wnt/β-catenin signaling pathway through inhibiting the activity of GSK-3β in APPswe transfected SY5Y cells. European Journal of Pharmaceutical Sciences 42: 540546. 112. Llorens-Marítin M, Jurado J, Hernández F, Ávila J (2014) GSK-3β, a pivotal kinase in Alzheimer disease. Frontiers in Molecular Neuroscience 7. 113. Muñoz-Montaño JR, Moreno FJ, Avila J, Dıá z-Nido J (1997) Lithium inhibits Alzheimer's disease-like tau protein phosphorylation in neurons. FEBS Letters 411: 183-188. 107 114. Georgievska B, Sandin J, Doherty J, Mörtberg A, Neelissen J, et al. (2013) AZD1080, a novel GSK3 inhibitor, rescues synaptic plasticity deficits in rodent brain and exhibits peripheral target engagement in humans. Journal of Neurochemistry 125: 446-456. 115. Clancy RM, Abramson SB (1995) Nitric oxide: a novel mediator of inflammation. Exp Biol Med 210: 93-101. 116. Gross S, Levi R (1992) Tetrahydrobiopterin synthesis. An absolute requirement for cytokine-induced nitric oxide generation by vascular smooth muscle. J Biol Chem 267: 25722-25729. 117. Chang Y-T, Chen C-L, Lin C-F, Lu S-L, Cheng M-H, et al. (2013) Regulatory role of GSK-3β on NF-κB, nitric oxide, and TNF-α in group A streptococcal Infection. Mediators Inflamm 2013: 10. 118. Huang X, Atwood CS, Hartshorn MA, Multhaup G, Goldstein LE, et al. (1999) The Aβ peptide of Alzheimer's disease directly produces hydrogen peroxide through metal ion reduction. Biochem 38: 7609-7616. 119. Nikoulina S, Ciaraldi T, Mudaliar S, Carter L, Johnson K, et al. (2002) Inhibition of glycogen synthase kinase improves insulin action and glucose metabolism in human skeletal muscle. Diabetes 51: 2190-2198. 120. Cline G, Johnson K, Regittnig W, Perret P, Tozzo E, et al. (2002) Effects of a novel glycogen synthase kinase-3 inhibitor on insulin-stimulated glucose metabolism in Zucker diabetic fatty (fa/fa) rats. Diabetes 51: 2903-2910. 121. Henriksen E, Dokken B (2006) Role of glycogen synthase kinase-3 in insulin resistance and type diabetes. Curr Drug Targets 7: 1435-1441. 122. Ciaraldi T, Carter L, Mudaliar S, Henry R (2010) GSK-3β and control of glucose metabolism and insulin action in human skeletal muscle. Mol Cell Endocrinol 315: 153-158. 123. Lasalvia-Prisco E, Cucchi S, Vazquez J, Lasalvia-Galante E, Golomar W, et al. (2004) Insulin-induced enhancement of antitumoral response to methotrexate in breast cancer patients. Cancer Chemother Pharmacol 53: 220-224. 124. Schilsky R, Ordway F (1985) Insulin effects on methotrexate polyglutamate synthesis and enzyme binding in cultured human breast cancer cells. Cancer Chemother Pharmacol 15: 272-277. 125. Alabaster O, Vonderhaar BK, Shafie SM (1981) Metabolic modification by insulin enhances methotrexate cytotoxicity in MCF-7 human breast cancer cells. Eur J Cancer Clin Oncol 17: 1223-1228. 126. Devi L, Prabhu BM, Galati DF, Avadhani NG, Anandatheerthavarada HK (2006) Accumulation of amyloid precursor protein in the mitochondrial import channels of human Alzheimer’s disease brain is associated with mitochondrial dysfunction The Journal of Neuroscience 26: 9057-9068. 127. Mao P, Reddy PH (2011) Aging and amyloid beta-induced oxidative DNA damage and mitochondrial dysfunction in Alzheimer's disease: Implications for early intervention and therapeutics. Biochimica et Biophysica Acta (BBA) Molecular Basis of Disease 1812: 1359-1370. 128. Reddy PH (2006) Amyloid precursor protein-mediated free radicals and oxidative damage: implications for the development and progression of Alzheimer's disease. Journal of Neurochemistry 96: 1-13. 108 129. Reddy PH, Manczak M, Mao P, Calkins MJ, Reddy AP, et al. (2010) Amyloid-β and mitochondria in aging and Alzheimer’s disease: Implications for synaptic damage and cognitive decline. Journal of Alzheimers Disease 20 S499-S512. 130. Reddy PH (2009) Amyloid beta, mitochondrial structural and functional dynamics in Alzheimer's disease. Experimental Neurology 218: 286-292. 131. Florent-Béchard S, Malaplate-Armand C, Koziel V, Kriem B, Olivier J-L, et al. (2007) Towards a nutritional approach for prevention of Alzheimer's disease: Biochemical and cellular aspects. Journal of the Neurological Sciences 262: 2736. 132. Ruxton CHS, Reed SC, Simpson MJA, Millington KJ (2004) The health benefits of omega-3 polyunsaturated fatty acids: a review of the evidence. Journal of Human Nutrition and Dietetics 17: 449-459. 133. Serhan CN (2009) Systems approach to inflammation resolution: identification of novel anti-inflammatory and pro-resolving mediators. Journal of Thrombosis and Haemostasis 7: 44-48. 134. Chan ECY, Pasikanti KK, Nicholson JK (2011) Global urinary metabolic profiling procedures using gas chromatography-mass spectrometry. Nature Protocols 6: 1483-1499. 135. Ma Q-H, Futagawa T, Yang W-L, Jiang X-D, Zeng L, et al. (2008) A TAG1APP signalling pathway through Fe65 negatively modulates neurogenesis. Nature Cell Biology 10: 283-294. 136. Kopka J, Schauer N, Krueger S, Birkemeyer C, Usadel B, et al. (2005) GMD@CSB.DB: the Golm Metabolome Database. Bioinformatics 21: 1635-1638. 137. Wishart DS, Tzur D, Knox C, Eisner R, Guo AC, et al. (2007) HMDB: the human metabolome database. Nucleic Acids Research 35: D521-D526. 138. Pedro M (2002) Emerging bioinformatics for the metabolome. Briefings in Bioinformatics 3: 134-145. 139. Zhang YW, Thompson R, Zhang H, Xu H (2011) APP processing in Alzheimer's disease. Molecular Brain 4: 1756-6606. 140. Chen K-p, Dou F (2012) Selective interaction of amyloid precursor protein with different isoforms of neural cell adhesion molecule. Journal of Molecular Neuroscience 46: 203-209. 141. Lakshmana MK, Yoon I-S, Chen E, Bianchi E, Koo EH, et al. (2009) Novel role of RanBP9 in BACE1 processing of amyloid precursor protein and amyloid β peptide generation. Journal of Biological Chemistry 284: 11863-11872. 142. Shioi J, Georgakopoulos A, Mehta P, Kouchi Z, Litterst CM, et al. (2007) FAD mutants unable to increase neurotoxic Aβ 42 suggest that mutation effects on neurodegeneration may be independent of effects on Aβ. Journal of Neurochemistry 101: 674-681. 143. Zhou S, Zhou H, Walian PJ, Jap BK (2005) CD147 is a regulatory subunit of the γ-secretase complex in Alzheimer's disease amyloid β-peptide production. Proceedings of National Academy of Sciences USA 102: 7499-7504. 144. Phiel CJ, Wilson CA, Lee VMY, Klein PS (2003) GSK-3[alpha] regulates production of Alzheimer's disease amyloid-[beta] peptides. Nature 423: 435-439. 145. J N, A S, U H, L L, AD R, et al. (1994) Relative abundance of Alzheimer A beta amyloid peptide variants in Alzheimer disease and normal aging. Proceedings of National Academy of Sciences USA 91: 8378–8382. 109 146. Oksman M, Iivonen H, Hogyes E, Amtul Z, Penke B, et al. (2006) Impact of different saturated fatty acid, polyunsaturated fatty acid and cholesterol containing diets on beta-amyloid accumulation in APP/PS1 transgenic mice. Neurobiology of Disease 23: 563-572. 147. Lim GP, Calon F, Morihara T, Yang F, Teter B, et al. (2005) A diet enriched with the omega-3 fatty acid docosahexaenoic acid reduces amyloid burden in an aged Alzheimer mouse model. Journal of Neuroscience 25: 3032-3040. 148. Grimm MO, Kuchenbecker J, Grosgen S, Burg VK, Hundsdorfer B, et al. (2011) Docosahexaenoic acid reduces amyloid beta production via multiple pleiotropic mechanisms. Journal of Biological Chemistry 286: 14028-14039. 149. Anandatheerthavarada HK, Biswas G, Robin M-A, Avadhani NG (2003) Mitochondrial targeting and a novel transmembrane arrest of Alzheimer's amyloid precursor protein impairs mitochondrial function in neuronal cells. The Journal of Cell Biology 161: 41-54. 150. Bubber P, Haroutunian V, Fisch G, Blass JP, Gibson GE (2005) Mitochondrial abnormalities in Alzheimer brain: Mechanistic implications. Annals of Neurology 57: 695-703. 151. Labrousse VF, Nadjar A, Joffre C, Costes L, Aubert A, et al. (2012) Short-term long chain omega3 diet protects from neuroinflammatory processes and memory impairment in aged mice. PLoS ONE 7: e36861. 152. Stonehouse W, Conlon CA, Podd J, Hill SR, Minihane AM, et al. (2013) DHA supplementation improved both memory and reaction time in healthy young adults: a randomized controlled trial. The American Journal of Clinical Nutrition 97: 1134-1143. 153. Fotuhi M, Mohassel P, Yaffe K (2009) Fish consumption, long-chain omega-3 fatty acids and risk of cognitive decline or Alzheimer disease: a complex association. Nature Clinical Practice Neurology 5: 140-152. 154. Shobab LA, Hsiung G-YR, Feldman HH (2005) Cholesterol in Alzheimer's disease. The Lancet Neurology 4: 841-852. 155. Barrett PJ, Song Y, Van Horn WD, Hustedt EJ, Schafer JM, et al. (2012) The amyloid precursor protein has a flexible transmembrane domain and binds cholesterol. Science 336: 1168-1171. 156. Fassbender K, Simons M, Bergmann C, Stroick M, Lütjohann D, et al. (2001) Simvastatin strongly reduces levels of Alzheimer's disease beta-amyloid peptides Abeta 42 and Abeta 40 in vitro and in vivo. Proceedings of National Academy of Sciences USA 98: 5856-5861. 157. Bodovitz S, Klein WL (1996) Cholesterol modulates alpha-secretase cleavage of amyloid precursor protein. Journal of Biological Chemistry 271: 4436-4440. 158. Frears ER, Stephens DJ, Walters CE, Davies H, Austen BM (1999) The role of cholesterol in the biosynthesis of beta-amyloid. Neuroreport 10: 1699-1705. 159. Petanceska SS, DeRosa S, Olm V, Diaz N, Sharma A, et al. (2002) Statin therapy for Alzheimer’s disease. Journal of Molecular Neuroscience 19: 155-161. 160. Kandiah N, Feldman HH (2009) Therapeutic potential of statins in Alzheimer's disease. Journal of the Neurological Sciences 283: 230-234. 161. Refolo LM, Pappolla MA, LaFrancois J, Malester B, Schmidt SD, et al. (2001) A cholesterol-lowering drug reduces β-amyloid pathology in a transgenic mouse model of Alzheimer's disease. Neurobiology of Disease 8: 890-899. 110 162. Davies P, Bailey PJ, Goldenberg MM, Ford-Hutchinson AW (1984) The role of arachidonic acid oxygenation products in pain and inflammation. Annual Review of Immunology 2: 335-357. 163. Samuelsson B (1983) Leukotrienes: mediators of immediate hypersensitivity reactions and inflammation. Science (New York, NY) 220: 568-575. 164. Kuehl FA, Egan RW (1980) Prostaglandins, arachidonic acid, and inflammation. Science (New York, NY) 210: 978-984. 165. Wilson DM, Binder LI (1997) Free fatty acids stimulate the polymerization of tau and amyloid beta peptides. In vitro evidence for a common effector of pathogenesis in Alzheimer's disease. The American Journal of Pathology 150: 2181-2195. 166. Esposito G, Giovacchini G, Liow J-S, Bhattacharjee AK, Greenstein D, et al. (2008) Imaging neuroinflammation in Alzheimer disease with radiolabeled arachidonic acid and PET. Journal of Nuclear Medicine 49: 1414-1421. 167. Quinn JF, Raman R, Thomas RG, Yurko-Mauro K, Nelson EB, et al. (2010) Docosahexaenoic acid supplementation and cognitive decline in Alzheimer disease. JAMA: the journal of the American Medical Association 304: 1903-1911. 168. Sparks DL, Scheff SW, Hunsaker JC, 3rd, Liu H, Landers T, et al. (1994) Induction of Alzheimer-like beta-amyloid immunoreactivity in the brains of rabbits with dietary cholesterol. Exp Neurol 126: 88-94. 169. Notkola IL, Sulkava R, Pekkanen J, Erkinjuntti T, Ehnholm C, et al. (1998) Serum total cholesterol, apolipoprotein E epsilon allele, and Alzheimer's disease. Neuroepidemiology 17: 14-20. 170. Kivipelto M, Helkala E-L, Laakso MP, Hänninen T, Hallikainen M, et al. (2001) Midlife vascular risk factors and Alzheimer's disease in later life: longitudinal, population based study. Bmj 322: 1447-1451. 171. Barrett PJ, Song Y, Horn WDV, Hustedt EJ, Schafer JM, et al. (2012) The Amyloid Precursor Protein has a Flexible Transmembrane Domain and Binds Cholesterol. Science 336: 1168-1171. 172. Silva T, Teixeira J, Remiao F, Borges F (2013) Alzheimer's disease, cholesterol, and statins: the junctions of important metabolic pathways. Angew Chem Int Ed Engl 52: 1110-1121. 173. Lutjohann D, Papassotiropoulos A, Bjorkhem I, Locatelli S, Bagli M, et al. (2000) Plasma 24S-hydroxycholesterol (cerebrosterol) is increased in Alzheimer and vascular demented patients. Journal of Lipid Research 41: 195-198. 174. Schonknecht P, Lutjohann D, Pantel J, Bardenheuer H, Hartmann T, et al. (2002) Cerebrospinal fluid 24S-hydroxycholesterol is increased in patients with Alzheimer's disease compared to healthy controls. Neurosci Letters 324: 83-85. 175. Avdulov NA, Chochina SV, Igbavboa U, Warden CS, Vassiliev AV, et al. (1997) Lipid Binding to Amyloid β-Peptide Aggregates: Preferential Binding of Cholesterol as Compared with Phosphatidylcholine and Fatty Acids. Journal of Neurochemistry 69: 1746-1752. 176. Carlsson CM, Gleason CE, Hess TM, Moreland KA, Blazel HM, et al. (2008) Effects of simvastatin on cerebrospinal fluid biomarkers and cognition in middleaged adults at risk for Alzheimer's disease. J Alzheimers Dis 13: 187-197. 177. Connor WE, Connor SL (2007) The importance of fish and docosahexaenoic acid in Alzheimer disease. Am J Clin Nutr 85: 929-930. 111 178. Li G, Shofer JB, Rhew IC, Kukull WA, Peskind ER, et al. (2010) Age-varying association between statin use and incident Alzheimer's disease. J Am Geriatr Soc 58: 1311-1317. 179. van Gelder BM, Tijhuis M, Kalmijn S, Kromhout D (2007) Fish consumption, n-3 fatty acids, and subsequent 5-y cognitive decline in elderly men: the Zutphen Elderly Study. Am J Clin Nutr 85: 1142-1147. 180. Feldman HH, Doody RS, Kivipelto M, Sparks DL, Waters DD, et al. (2010) Randomized controlled trial of atorvastatin in mild to moderate Alzheimer disease: LEADe. Neurology 74: 956-964. 181. Freund-Levi Y, Eriksdotter-Jonhagen M, Cederholm T, Basun H, Faxen-Irving G, et al. (2006) Omega-3 fatty acid treatment in 174 patients with mild to moderate Alzheimer disease: OmegAD study: a randomized double-blind trial. Arch Neurol 63: 1402-1408. 182. Kotani S, Sakaguchi E, Warashina S, Matsukawa N, Ishikura Y, et al. (2006) Dietary supplementation of arachidonic and docosahexaenoic acids improves cognitive dysfunction. Neurosci Res 56: 159-164. 183. Sano M, Bell KL, Galasko D, Galvin JE, Thomas RG, et al. (2011) A randomized, double-blind, placebo-controlled trial of simvastatin to treat Alzheimer disease. Neurology 77: 556-563. 184. Simons M, Schwarzler F, Lutjohann D, von Bergmann K, Beyreuther K, et al. (2002) Treatment with simvastatin in normocholesterolemic patients with Alzheimer's disease: A 26-week randomized, placebo-controlled, double-blind trial. Ann Neurol 52: 346-350. 185. Belyaev ND, Kellett KA, Beckett C, Makova NZ, Revett TJ, et al. (2010) The transcriptionally active amyloid precursor protein (APP) intracellular domain is preferentially produced from the 695 isoform of APP in a {beta}-secretasedependent pathway. J Biol Chem 285: 41443-41454. 186. Grimm MO, Kuchenbecker J, Grosgen S, Burg VK, Hundsdorfer B, et al. (2011) Docosahexaenoic acid reduces amyloid beta production via multiple pleiotropic mechanisms. J Biol Chem 286: 14028-14039. 187. Farooqui AA, Ong W-Y, Horrocks LA, Chen P, Farooqui T (2007) Comparison of biochemical effects of statins and fish oil in brain: The battle of the titans. Brain Research Reviews 56: 443-471. 188. Acimovic J, Lovgren-Sandblom A, Monostory K, Rozman D, Golicnik M, et al. (2009) Combined gas chromatographic/mass spectrometric analysis of cholesterol precursors and plant sterols in cultured cells. J Chromatogr B Analyt Technol Biomed Life Sci 877: 2081-2086. 189. Axelson M, Larsson O (1996) 27-hydroxylated low density lipoprotein (LDL) cholesterol can be converted to 7alpha,27-dihydroxy-4-cholesten-3-one (cytosterone) before suppressing cholesterol production in normal human fibroblasts. Evidence that an altered metabolism of ldl cholesterol can underlie a defective feedback control in malignant cells. J Biol Chem 271: 12724-12736. 190. Le Fur Y, Maume G, Feuillat M, Maume BF (1999) Characterization by gas chromatography/mass spectrometry of sterols in saccharomyces cerevisiae during autolysis. J Agric Food Chem 47: 2860-2864. 191. Wolf C, Chevy F, Pham J, Kolf-Clauw M, Citadelle D, et al. (1996) Changes in serum sterols of rats treated with 7-dehydrocholesterol-delta 7-reductase 112 inhibitors: comparison to levels in humans with Smith-Lemli-Opitz syndrome. J Lipid Res 37: 1325-1333. 192. FDA, US Department of Health and Human Services. Guidance for industry: bioanalytical method validation, 2001. 193. International Conference on Harmonization (ICH). Validation of analytical methods: methodology. ICH Q2 B, Geneva, 1996. 194. Rockwood K, Kirkland S, Hogan DB, et al. (2002) Use of lipid-lowering agents, indication bias, and the risk of dementia in community-dwelling elderly people. Archives of Neurology 59: 223-227. 195. Wolozin B, Kellman W, Ruosseau P, Celesia GG, Siegel G (2000) Decreased prevalence of alzheimer disease associated with 3-hydroxy-3-methyglutaryl coenzyme a reductase inhibitors. Archives of Neurology 57: 1439-1443. 196. Grimm MOW, Zimmer VC, Lehmann J, Grimm HS, Hartmann T (2013) The Impact of Cholesterol, DHA, and Sphingolipids on Alzheimer's Disease. BioMed Research International 2013: 16. 197. Hooff GP, Wood WG, Müller WE, Eckert GP (2010) Isoprenoids, small GTPases and Alzheimer's disease. Biochimica et Biophysica Acta (BBA) Molecular and Cell Biology of Lipids 1801: 896-905. 198. Zhou Y, Su Y, Li B, Liu F, Ryder JW, et al. (2003) Nonsteroidal antiinflammatory drugs can lower amyloidogenic Abeta42 by inhibiting Rho. Science 302: 1215-1217. 199. Wood WG, Schroeder F, Igbavboa U, Avdulov NA, Chochina SV (2002) Brain membrane cholesterol domains, aging and amyloid beta-peptides. Neurobiology of aging 23: 685-694. 200. Cordle A, Landreth G (2005) 3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase Inhibitors Attenuate β-Amyloid-Induced Microglial Inflammatory Responses. The Journal of Neuroscience 25: 299-307. 201. Buhaescu I, Izzedine H (2007) Mevalonate pathway: A review of clinical and therapeutical implications. Clinical Biochemistry 40: 575-584. 202. Shafaati M, Marutle A, Pettersson H, Lovgren-Sandblom A, Olin M, et al. (2011) Marked accumulation of 27-hydroxycholesterol in the brains of Alzheimer's patients with the Swedish APP 670/671 mutation. Journal of Lipid Research 52: 1004-1010. 203. Prasanthi J, Huls A, Thomasson S, Thompson A, Schommer E, et al. (2009) Differential effects of 24-hydroxycholesterol and 27-hydroxycholesterol on betaamyloid precursor protein levels and processing in human neuroblastoma SHSY5Y cells. Molecular Neurodegeneration 4: 1. 204. Kramer T, Schmidt B, Lo Monte F (2012) Small-Molecule Inhibitors of GSK-3: Structural Insights and Their Application to Alzheimer's Disease Models. International Journal of Alzheimer's Disease 2012: 32. 205. Leost M, Schultz C, Link A, Wu Y-Z, Biernat J, et al. (2000) Paullones are potent inhibitors of glycogen synthase kinase-3β and cyclin-dependent kinase 5/p25. European Journal of Biochemistry 267: 5983-5994. 206. Conde S, Pérez DI, Martínez A, Perez C, Moreno FJ (2003) Thienyl and Phenyl α-Halomethyl Ketones:  New Inhibitors of Glycogen Synthase Kinase (GSK-3β) from a Library of Compound Searching. Journal of Medicinal Chemistry 46: 4631-4633. 113 207. Kim H-J, Choo H, Cho YS, No KT, Pae AN (2008) Novel GSK-3β inhibitors from sequential virtual screening. Bioorganic & Medicinal Chemistry 16: 636643. 208. Gleissman H, Yang R, Martinod K, Lindskog M, Serhan CN, et al. (2010) Docosahexaenoic acid metabolome in neural tumors: identification of cytotoxic intermediates. The FASEB Journal 24: 906-915. 209. Trushina E, Dutta T, Persson X-MT, Mielke MM, Petersen RC (2013) Identification of Altered Metabolic Pathways in Plasma and CSF in Mild Cognitive Impairment and Alzheimer’s Disease Using Metabolomics. PLoS One 8: e63644. 114 APPENDICES Appendix A: Knockdown neuroblastoma cells efficiency of DHFR siRNA in SH-SY5Y (a) (b) Figure S1: Knockdown efficiency of DHFR siRNA in SH-SY5Y neuroblastoma cells. (a) SH-SY5Y cells were with either negative control siRNA or DHFR siRNA sequence A/B/C at 10 nM and 20 nM concentrations and cell lysates were subjected to western blot analysis. The blots shown are representative of three different experiments; (b) Cellular DHFR protein expression quantified from the western blot results, expressed as percentage of control samples. 115 Appendix B: Marker metabolites identified from medium and lysate samples of DHA-treated and vehicle-treated CHO-wt and CHO-APP695 cells. Vehicle-treated DHA-treated Identified Kovats -4 c Metabolite Sample Normalized peak area(x 10 ) Normalized peak area (x 10-4)c Fold Fold bya RIb ∆d ∆d CHO-wt CHO-APP695 CHO-wt CHO-APP695 Propanoic acid medium NIST 1032.0 97.6 ± 22.1 131.2 ± 11.2 * 1.34 83.0 ± 26.3 126.0 ± 16.6* 1.52 * Lactic acid medium NIST 1059.6 902.3 ± 10.4 1371.1 ± 49.1 1.52 844.5 ± 94.6 1157.8 ± 36.1ns 1.37 * * Alanine medium NIST 1100.2 280.3 ± 48.7 465.8 ± 70.1 1.66 345.5 ± 75.2 568.9 ± 86.5 1.65 * * Oxalic acid medium NIST 1125.8 2.6 ± 0.1 2.9 ± 0.1 1.12 2.5 ± 0.3 3.0 ± 0.3 1.23 Urea medium NIST 1221.3 65.5 ± 24.2 78.2 ± 20.7ns 1.19 88.2 ±19.7 114.3 ± 17.1* 1.30 4-hydroxybutyric acid medium NIST 1234.8 2.8 ± 0.3 5.3 ± 1.1* 1.85 3.3 ± 0.2 5.5 ± 0.7* 1.68 * * Niacin medium NIST 1269.8 0.4 ± 0.0 0.5 ± 0.0 1.21 0.5 ± 0.0 0.6 ± 0.0 1.34 Glycerol medium NIST 1292.5 26.7 ± 2.1 32.9 ± 0.9* 1.23 24.6 ± 3.3 31.6 ± 1.4* 1.28 Threonine medium NIST 1294.7 49.8 ± 12.7 53.5 ± 5.2 ns 1.07 61.3 ± 10.6 80.2 ± 12.6* 1.31 ns * Succinic acid medium NIST 1305.9 3.2 ± 0.5 3.5 ± 0.1 1.09 3.4 ± 0.3 4.1 ± 0.3 1.20 Glycine medium NIST 1315.8 80.8 ± 20.9 102.1 ± 29.4 ns 1.26 181.4 ± 58.3 284.6 ± 47.6* 1.57 Uracil medium NIST 1328.4 2.9 ± 0.2 4.1 ± 0.1* 1.41 2.9 ± 0.3 4.3 ± 0.2* 1.49 * * Itaconic acid medium NIST 1334.9 1.9 ± 0.3 6.4 ± 0.6 3.25 1.6 ± 0.2 5.6 ± 0.3 3.43 Serine medium NIST 1373.9 11.9 ± 3.1 5.2 ± 1.0* 0.44 15.0 ± 0.4 5.4 ± 0.8* 0.36 Aspartic acid medium NIST 1482.2 0.3 ± 0.1 0.5 ± 0.0 ns 1.67 0.5 ± 0.1 0.8 ± 0.2* 1.76 ns * Butylated hydroxytoluene medium NIST 1492.3 2.0 ± 0.0 1.3 ± 0.6 0.65 1.7 ± 0.3 1.3 ± 0.1 0.77 Malic acid medium NIST 1501.2 1.3 ± 0.1 1.2 ± 0.3 ns 0.92 1.4 ± 0.1 1.6 ±0.1* 1.17 Pyroglutamic acid medium NIST 1506.6 1043.4 ± 15.8 737.4 ± 43.9* 0.71 1197.2 ± 21.8 811.1 ± 12.0* 0.68 ns * Methionine medium NIST 1517.2 15.7 ± 4.3 12.2 ± 2.5 0.77 17.1 ± 4.7 11.4 ± 0.8 0.67 Trihydroxybutyric acid medium NIST 1576.4 25.9 ± 3.7 27.6 ± 0.3 ns 1.06 24.0 ± 3.0 28.0 ± 1.8* 1.17 * ns Mannose medium NIST 1881.4 7.0 ± 3.0 3.3 ± 0.6 0.48 4.8 ± 2.1 2.2 ± 2.0 0.45 116 Fructose Mannitol Lactate Oxalic acid Norleucine Hydroxylamine Valine Serine Niacin Glycerol Threonine Uracil Itaconic acid Aspartic acid Malic acid N-acetylglutamate Threitol 2,3,4-Trihydroxybutyric acid 2-Hydroxyglutaric acid Ribitol Citric acid Myristic acid Fructose Myo-Inositol Arachidonic acid medium medium lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST 1927.7 2004.1 1059.8 1121.3 1149.8 1152.5 1227.0 1256.4 1272.9 1294.7 1297.8 1330.7 1336.6 1415.2 1501.6 1522.1 1547.9 1576.4 1584.4 1796.7 1844.0 1844.7 1928.1 2155.8 2352.5 278.2 ± 26.3 78.1 ± 6.1 0.8 ± 0.1 9.0 ± 1.5 92.0 ± 6.9 3.3 ± 0.3 59.9 ± 17.7 51.3 ± 4.1 1.2 ± 0.3 32.6 ± 9.2 133.9 ± 7.0 1.5 ± 0.3 2.9 ± 0.4 91.7 ± 8.6 20.5 ± 1.5 ns 1282.9 ± 90.8 4.3 ± 0.4 0.3 ± 0.1 4.9 ± 0.7 3.4 ± 0.5 7.5 ± 0.9 11.5 ± 1.4 16.0 ± 2.4 22.1 ± 2.0 3.0 ± 0.3 117 152.2 ± 20.1* 79.0 ± 2.7 ns 1.3 ± 0.2* 13.9 ± 3.4* 95.7 ± 3.0 ns 3.3 ± 1.1 ns 70.7 ± 26.0 ns 26.3 ± 1.3* 2.5 ± 0.5* 32.8 ± 2.3 ns 134.0 ± 6.1 ns 1.5 ± 0.6 ns 9.3 ± 1.1* 56.3 ± 3.8* 21.6 ± 1.3* 815.2 ± 70.0* 6.1 ± 0.6* 0.6 ± 0.1* 10.7 ± 1.1* 4.1 ± 0.7 ns 8.4 ± 3.3* 13.6 ± 0.8* 22.0 ± 2.7* 34.6 ± 3.4* 3.5 ± 0.4* 0.55 1.01 1.62 1.54 1.04 1.01 1.18 0.51 2.10 1.01 1.00 1.01 3.25 0.61 1.05 0.64 1.42 2.05 2.17 1.21 1.12 1.19 1.37 1.57 1.17 273.7 ± 22.6 73.0 ± 6.6 0.8 ± 0.2 10.8 ± 1.5 89.0 ± 2.0 3.6 ± 0.4 78.0 ± 4.4 56.6 ± 2.3 1.3 ± 0.2 28.9 ± 2.0 144.0 ± 3.2 1.7 ± 0.2 2.6 ± 0.2 87.4 ± 5.5 16.6 ± 0.9 1068.2 ± 95.0 4.9 ±0.2 0.4 ± 0.0 4.9 ± 0.6 2.7 ± 0.3 16.6 ± 1.5 13.9 ± 1.2 23.4 ± 2.1 25.5 ± 2.0 3.2 ± 0.1 146.2 ± 27.8* 81.1 ± 5.1* 1.6 ± 0.2* 16.4 ± 3.9* 101.2 ± 6.1* 3.0 ± 0.2* 94.8 ± 8.9* 29.5 ± 1.7* 2.2 ± 0.4* 32.4 ± 1.8* 158.9 ± 6.7* 2.1 ± 0.2* 9.4 ± 0.7* 47.9 ± 3.5* 25.3 ± 1.7* 764.1 ± 74.6 * 7.8 ± 0.2* 0.7 ± 0.0* 12.2 ± 0.1* 3.4 ± 0.3* 29.6 ± 2.4* 16.6 ± 1.2* 31.6 ± 1.3* 41.5 ± 2.3* 2.7 ± 0.3* 0.53 1.11 1.89 1.52 1.14 0.85 1.22 0.52 1.77 1.12 1.10 1.19 3.62 0.55 1.52 0.72 1.60 1.89 2.49 1.29 1.99 1.19 1.35 1.63 0.85 11-Eicosenoic acid Piceatannol Docosahexaenoic acid 2-Monopalmitin 1-Monopalmitin 1-Monooleoylglycerol Stearic acid Uridine monophosphate Cholesta-3,5-diene Eicosanoic acid Zymosterol lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate lysate NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST NIST 2407.5 2521.1 2552.3 2575.8 2599.4 2765.2 2792.5 2836.0 2885.6 2983.4 3202.0 2.1 ± 0.2 0.7 ± 0.1 3.2 ± 1.4 22.8 ± 1.4 48.5 ± 3.5 0.2 ± 0.1 72.7 ± 4.4 0.3 ± 0.1 3.0 ± 0.3 2.3 ± 0.2 4.3 ± 0.4 a 2.2 ± 0.4 ns 2.2 ± 0.4* 2.5 ± 0.7* 19.5 ± 2.2* 42.3 ± 4.3* 0.3 ± 0.0* 62.4 ± 4.8* 0.8 ± 0.3* 3.1 ± 0.4 ns 1.9 ± 0.3* 7.6 ± 0.9* 1.04 3.14 0.78 0.85 0.87 1.39 0.86 2.71 1.05 0.83 1.77 2.3 ± 0.1 0.5 ± 0.1 3.5 ± 0.2 26.3 ± 0.7 53.5 ± 3.5 0.2 ± 0.0 77.3 ± 6.6 0.4 ± 0.1 3.0 ± 0.4 2.7 ± 0.3 4.1 ± 0.3 2.1 ± 0.1* 1.7 ± 0.2* 4.5 ± 0.3* 20.5 ± 0.6* 41.9 ± 1.4* 0.2 ± 0.0* 59.5 ± 3.8* 0.7 ± 0.2* 1.5 ± 0.5* 2.0 ± 0.1* 6.6 ± 0.7* 0.90 3.44 1.29 0.78 0.78 1.27 0.77 1.63 0.52 0.73 1.59 Metabolite identification using standard compound or NIST library search. Kovats RI refers to Kovats retention index c Normalized peak area values expressed as mean ± S.E.M. d Fold change (∆): CHO-AβPP695 (treatment) / CHO-wt (treatment). * p[...]... the development and availability of sensitive and reproducible experimental platforms for monitoring biomarkers and drug responses, for drug screening process and to evaluate the efficacy and mechanism of the tested agents in AD Amongst the different ‘omics’ based approaches, metabonomics provides holistic understanding of the disease process by allowing simultaneous identification and quantification... of selective and potent GSK-3 inhibitors for use as AD therapeutics However, no effective therapeutic outcomes have emerged from using GSK-3 inhibitors, mainly due to the limited specificity and high toxicity of these agents Therefore, there is a need for identifying additional pathway regulators for modifying and enhancing the therapeutic efficacy of currently available GSK-3 inhibitors for AD therapeutics... microglial and astrocyte cells, stimulating them to release different inflammatory cytokines and cytotoxic substances, leading to establishment of chronic inflammation and neurodegeneration in AD brain In addition, increased expression of inflammatory cytokines and oxidative stress mediators has been shown to induce Aβ deposition and senile plaque formation in neuronal cells [21,22] and transform non-aggregated... sporadic and familial forms of AD [37-40] The two isoforms of GSK-3, GSK-3α and GSK-3β, are important for normal development, neuronal growth and differentiation, metabolic homeostasis, cell polarity, cell fate Aberrant activation of GSK-3 enzymes has been proven to: 1) form multi-protein complexes with PS-1 that inactivates Wnt/β-catenin signaling pathway causing degradation of βcatenin protein and leading... increase in its incidence, potential novel targets for effective AD therapeutics are urgently needed While the precise molecular mechanisms underlying the disease pathology is poorly understood, several approaches have been proposed to mitigate the effects of this devastating disorder The overall goal of this thesis is thus to identify and explore potential novel targets for therapeutic intervention in... to address this gap by evaluating the novel interaction between folate metabolism and 9 GSK-3 signaling pathway to enhance the neuroprotective and anti-inflammatory effects of a GSK-3 inhibitor and it is discussed further in Chapter 3 1.4 Alternative treatment approaches Many clinical and experimental studies are ongoing for developing disease- modifying agents for AD However, the disappointing results... significantly to the achievement of this goal and is discussed in detail in the following section Metabonomics platform: An overview Over the years, the ‘omics’ technology using genomics, proteomics and metabonomics platforms has gained rapid popularity for conducting high throughput identification and quantification of large groups of targets, namely genes, proteins and metabolites, respectively Although... this technology in neurodegenerative diseases is relatively premature, it is quickly gaining interest owing to the large data-processing capacity, sensitivity and robustness of the platform Particularly for a multifactorial disease like AD, these platforms provide excellent opportunity to gather high-density biological information related to different physiological and pathological processes much efficiently... 52 xix Figure 4.3: Model validation for CHO-wt and CHO-APP695 cells and effect of DHA on Aβ40 release 54 Figure 4.4: Overlay of GC/TOFMS chromatograms 55 Figure 4.5: PLS-DA score plot and validation plots .56 Figure 4.6: PLS-DA score plot and validation plot for lysate samples 58 Figure 4.7: PLS-DA score plot and validation plot for medium samples .60 Figure 4.8: DHA... million by 2050 [3] Before 2011, AD was classified mainly into three stages: mild, moderate and severe AD However, with the implementation of new criteria and guidelines for early detection and diagnosis of AD by the National Institute of Ageing (NIA) and the Alzheimer’s Association in 2011 [1], AD is now classified as preclinical AD, mild cognitive impairment (MCI) due to AD and dementia due to AD . TOWARDS IDENTIFYING NOVEL MODULATORS AND TARGETS FOR ALZHEIMER’S DISEASE THERAPY BAHETY PRITI BALDEODAS (B. Pharm, Nirma University) A THESIS SUBMITTED FOR THE DEGREE. helped me to move forward and fulfil this dream of mine. Foremost, I would like to express my sincerest gratitude to my supervisor Dr Ee Pui Lai Rachel for her constant guidance and supervision. unwavering support and encouragement and unreserved nature gave me countless opportunities to learn and try new things, to explore different projects outside our own lab and truly understand the science