Caffeic Acid and C. elegans’ Protection Against Toxin Induced Neurodegeneration By: Michelle Chyn

Degree of PD in PatientsDMUS $pounds Average person3,9202,5901,690 Fluctuations in performance6,0103,9702,580 More stable condition2,7001,7801,160 Hoehn and Yahr scale stage I2,2301,4709,60 Hoehn and Yahr scale stage V11,8707,8305,100 Need Parkinson’s Disease (PD)- 500,000 people in U.S. $6,000,000,000 spent annually for PD treatment National Institute of Neurological Disorders and Stroke Dodel RC, Pepperl S, Köhne-Volland R, Szucs T, Werhahn KJ, Noachtar S, Oertel WH. “Costs of drug treatment of neurologic diseases: Parkinson disease, dystonia, epilepsy.” European Journal of Neurology. Vol. 7. Pp Figure 1: Cost of PD treatment per person annually

Coffee consumption in North America and Europe = 1/3 of the tap water intake Need Cont. Figure 2: PD in European Countries Figure 3: Coffee Consumption

Knowledge Base C. elegans biosynthesize dopamine in the corpus stratum (basil ganglia) (Braugart, 2004) Figure 4: Figure 5: 3-hydroxytyramine (Dopamine)

Knowledge Base Cont. Copper is a neurotoxin when consumed in excess amounts (Gaggelli et al., 2006) Figure 6: Copper oxide

Literature Review Antioxidants in coffee decreases risk of development of age- related diseases (Eskelinen et. al, 2009) Caffeine intake doesn’t show health benefits (Lopez-Garcia, et. al, 2008) Figure 7: Caffeic acid

Literature Review Cont. Caenorhabditis elegans MPP + model used as PD model for testing anti-PD drugs (Braungart, et al. 2004) Figure 8: Dose- response effect of MPP+ treatment on the mobility of wild-type C. elegans animals.

Literature Review Cont. Green Florescent Protein (GFP) florescence shown in dopaminergic neurons of transgenic strains of C. elegans (Braungart, et al., 2004) Figure 9: GFP expression in dopaminergic neurons of untreated and MPP + treated worms

Literature Review Cont. Direct addition of dopamine-2-c did not help increase noradrenaline presence in PD patients (Goodall and Alton, 1969) Figure 10: % noradrenaline recovery after dopamine-2-c infusion

Purpose Observing the effect of caffeic acid on neurotoxin- induced degeneration in C. elegans H 0 - caffeic acid will not affect the degeneration rate in the C. elegans H A - caffeic acid will decrease degeneration in the C. elegans. Hypothesis

Methodology Compounds in Coffee and C. elegans Protection Against Parkinson’s Disease Caenorhabditis elegans egIs1[Pdat-1::GFP] strain from Caenorhabditis Genetics Center (CGC) N=90 C. elegans cultured at 20°C in NGM plates in normal oxygen environment. Caffeic acid and MMP + concentrations inserted in food of E. coli for 3 days at L1 stage. Organisms then moved out into normal NGM plates. Control addition of copper oxide, concentratin according to LD 50 N=30 Group 2 addition of copper oxide caffeic acid- lower concentration according to LD 50 N=30 Group 3 addition of copper oxide caffeic acid- higher concentration according to LD 50 N=30 Statistical analysis using SPSS and ANOVA Data collected through stereo florescent microscope viewing of GFP florescence on a scale of normal, weak, and none, and area through Adobe Photoshop on the 3 rd day.

Protocols Eggs laid overnight at L4 stage on fresh NGM plates (Mehta, et al, 2009) Picture drawn by Victoria Wei Figure 11: Culturing of C. elegans on a petri dish

Protocols Cont. N=30 per group, total N=90 Group 1: Copper oxide Group 2: Copper oxide and Caffeic acid Group 3: Copper oxide and Caffeic acid Figure 12: C. elegans growth stages

Protocols Cont. Data collect on scaled brightness of GFP expression (Braungart, et al., 2004) and area of florescence on Photoshop Data analysis using SPSS ry.cgi?cid=33557&loc=ec_rcs Figure 13: Sodium azide (NaN 3 ) Figure 14: Florescent Microscope ges/ost_images/leica_dmr_fluor_micro.jpg

Protocols

Do-Ability Equipment available: GFP filter and florescent microscope UV lamp Petri dishes Photoshop Equipment needed: C. elegans pdat-1::GFP strain from CGC E. coli OP50 strain from CGC Copper oxide, sodium azide, and caffeic acid from Sigma NGM from Carolina Biological

Budget VendorCat #Pg.ItemQty.DescriptionUnit $Total $ Caenorhabditis Genetics CenterBZ555 C. elegans pdat- 1::gfp110$7.00 SigmaC0625Caffeic Acid15 g$53.30 Carolina Biological Nematode Growth Agar2135 mL$6.25$12.50 Caenorhabditis Genetics CenterE. coli OP50$7.00 Carolina Biological741270petri dishes10100 x 15 mm$6.45$64.50 SigmaS2002Sodium azide125 g$21.20 Carolina Biological216880ampicillin15 g$43.25 SigmaF0503fluorodeoxyurodine1100 mg$ Sigma208825copper125 g$28.30 Total Cost$354.05

Bibliography American College of Physicians. “Coffee Drinkers Have Slightly Lower Daeth Rates, Study Finds.” ScienceDaily 17 June May Biology The Dynamics of Life. New York: McGraw-Hill Glencoe, Braungart, Evelyn; Gerlach, Manfred; Riederer, Peter; Baumeister, Ralf; and Hoener, Marius C. “ Caenorhabditis elegans MPP + Model of Parkinson’s Disease for High-Throughput Drug Screening.” Neurodegenerative Diseases Vol. 1: pgs Brown, Marishka K.; Evans, Joseph L.; Yuan, Luo. “Beneficial effects of natural antioxidants EGCG and α-lipoic acid on life span and age-dependent behavioral declines in Caenorhabditis elegans” Elsevier Science. Vol. 85. pp Colleta, Susan. Introduction to C. elegans. Waksman Student Scholars “Flavonoids.” Phytochemicals May Goodall, McC. and Alton, Harold. “Dopamine (3-Hydroxytyramine) Metabolism in Parkinsonism.” The Journal of Clinical Investigation. Volume 48. pp Halliwell, Barry and Gutterridge, John M.C. “Free Radicals in Biology and Medicine.” Third Edition. Oxford Science Publications. Oxford Unitersity Press Hoch, Daniel B.; Zieve, David. “Parkinson’s Disease.” U.S. National Library of Medicine. January 21, May 6, Jessica C. Greene; Whitworth, Alexander J.; Kuo, Isabella; Andrews, Laurie A.; Feany, Mel B.; Pallanck, Leo J. “Mitochondrial pathology and apoptotic muscle degeneration in Drosophila parkin mutants.” Davidson College Review Papers Karolinska Institute. “Midlife Coffee And Tea Drinking May Protect Against Late-life Dementia”. ScienceDaily 15 January May

Bibliography Cont. Kiontke, Karin and Sudhaus, Walter. “Ecology of Caenorhabditis species.” WormBook The online review of C. elegans Biology. Lagasse, Paul. Columbia Encyclopedia. “Coffee”. Columbia University Press. May 2, Lopez-Garcia, Esther; van Dam, Rob M.; Li, Tricia Y.; Rodriguez-Artalejo, Fernando; and Hu, Frank B. “The Relationship of Coffee Consumption with Mortality.” Annals of Internal Medicine, June 16, 2008 Margreet R. Olthof, Peter C.H. Hollman, and Martijn B. Katan. “Chlorogenic Acid and Caffeic Acid are Absorbed in Humans.” Human Nutrition and Metabolism Nass R, Blakely RD The Caenorhabditis Elegans Dopaminergic System: Opportunities for Insights into Dopamine Transport and Neurodegeneration. Annual Review of Pharmacology and Toxicology 43: 521 ‐ 544 Natarajan, K.; Singh, Sanjaya; Burke, Terrence R. Jr.; Grunberger, Dezider. “Caffeic acid phenethyl ester is a potent and specific inhibitor of activation of nuclear transcription factor NF- κB”. Immunology. Vol 93, pp August “Parkinson’s Disease: Glossary of Parkinson’s Disease Terms.” WebMD. Parkinson’s Disease Health. May 7, “Parkinson's Disease: Hope Through Research”. National Institute of Neurological Disorders and Stroke. April 24, May 5, Shanam, Shai. “Worming into the cell: Viral reproduction in Caenorhabditis elegans.” Proceedings of the National Academy of Sciences. Vol n. 11. pgs Sutphin GL, Kaeberlein M (2009). Measuring Caenorhabditis elegans Life Span on Solid Media. JoVE. 27. Tipton KF, Singer TP. “Advances in our understanding of the mechanisms of the neurotoxicity of MPTP and related compounds.” J Neurochem 1993;61:1191–1206.