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

2. 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. 479-85 1991. Figure 1: Cost of PD treatment per person annually

3. http://www.parkinsonsdecisionaid.eu.com/images/ac/2008/incidence.gif http://www.e-importz.com/images/coffee_consumption.gif 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

4. Knowledge Base C. elegans biosynthesize dopamine in the corpus stratum (basil ganglia) (Braugart, 2004) Figure 4: http://en.wikipedia.org/wiki/Basal_ganglia Figure 5: 3-hydroxytyramine (Dopamine) http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=681&loc=ec_rcs

5. Knowledge Base Cont. Copper is a neurotoxin when consumed in excess amounts (Gaggelli et al., 2006) Figure 6: Copper oxide http://en.wikipedia.org/wiki/Copper(I)_oxide

6. 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) http://pubchem.ncbi.nlm.nih.gov/summary/summary.cgi?cid=689043&loc=ec_rcs Figure 7: Caffeic acid

7. 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. http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowPDF&ArtikelNr=80983&Ausgabe=230458&ProduktNr=229093&filename=80983.pdf

8. Literature Review Cont. Green Florescent Protein (GFP) florescence shown in dopaminergic neurons of transgenic strains of C. elegans (Braungart, et al., 2004) http://content.karger.com/ProdukteDB/produkte.asp?Aktion=ShowPDF&ArtikelNr=80983&Ausgabe=230458&ProduktNr=229093&filename=80983.pdf Figure 9: GFP expression in dopaminergic neurons of untreated and MPP + treated worms

9. Literature Review Cont. Direct addition of dopamine-2-c did not help increase noradrenaline presence in PD patients (Goodall and Alton, 1969) http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=297487&blobtype=pdf Figure 10: % noradrenaline recovery after dopamine-2-c infusion

10. 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

11. 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.

12. 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

13. Protocols Cont. http://www.scq.ubc.ca/wp-content/uploads/2006/07/wormcycle.gif 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

14. Protocols Cont. Data collect on scaled brightness of GFP expression (Braungart, et al., 2004) and area of florescence on Photoshop Data analysis using SPSS http://pubchem.ncbi.nlm.nih.gov/summary/summa ry.cgi?cid=33557&loc=ec_rcs Figure 13: Sodium azide (NaN 3 ) Figure 14: Florescent Microscope http://www.niams.nih.gov/Research/Ongoing_Research/ima ges/ost_images/leica_dmr_fluor_micro.jpg

15. Protocols

16. 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

17. Budget VendorCat #Pg.ItemQty.DescriptionUnit $Total $ Caenorhabditis Genetics CenterBZ555 C. elegans pdat- 1::gfp110$7.00 SigmaC0625Caffeic Acid15 g$53.30 Carolina Biological173520 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$117.00 Sigma208825copper125 g$28.30 Total Cost$354.05

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19. 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, 2009. 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. 2000.. Nass R, Blakely RD. 2003. 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. 9090-9095. August 1996. “Parkinson’s Disease: Glossary of Parkinson’s Disease Terms.” WebMD. Parkinson’s Disease Health. May 7, 2009.. “Parkinson's Disease: Hope Through Research”. National Institute of Neurological Disorders and Stroke. April 24, 2009. May 5, 2009. Shanam, Shai. “Worming into the cell: Viral reproduction in Caenorhabditis elegans.” Proceedings of the National Academy of Sciences. Vol. 103. n. 11. pgs. 3955-3956. 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.