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

2. Need Parkinson’s Disease (PD)- 500,000 in U.S. $6,000,000,000 spent annually for PD treatment Figure 1: Number of People with PD in Europe between 2000-2008 http://www.parkinsonsdecisionaid.eu.com/images/ac/2008/incidence.gif

3. Need Cont. Figure 2: Coffee Consumption in 2000 http://www.e-importz.com/images/coffee_consumption.gif

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

5. Knowledge Base Cont. MPP + presence malfunctions enzyme complex I in respiratory chain → decreased ATP and increase in free radicals → destruction of dopaminergic neurons (Smith, 1987) Figure 5: Electron Transport Chain http://en.wikipedia.org/wiki/Electron_transport_chain

6. Literature Review Cont. 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 6: Caffeic acid

7. Literature Review Cont. Caenorhabditis elegans MPP + model used as PD model for testing anti-PD drugs (Braungart, et al. 2004) Figure 7: 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 8: GFP expression in dopaminergic neurons of untreated and MPP + treated worms

9. Literature Review Cont. Direct addition of dopamine-2-c did not aid to increasing noradrenaline presence in PD patients (Goodall and Alton, 1969) http://www.pubmedcentral.nih.gov/picrender.fcgi?artid=297487&blobtype=pdf Figure 9: % 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 MPP + at 1.5 mM N=30 Group 2 addition of MPP + caffeic acid-.5 mM N=30 Group 3 addition of MPP + caffeic acid- 1 mM 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 10: 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: MPP + Group 2: MPP + and Caffeic acid Group 3: MPP + and Caffeic acid Figure 11: 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 12: Sodium azide (NaN 3 ) Figure 13: 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 MPP +, 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 SigmaD048MPP+1100 mg$108.00 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 Total Cost$433.75

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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.