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

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

Need Cont. Figure 2: Coffee Consumption in

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

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

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) Figure 6: Caffeic acid

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.

Literature Review Cont. Green Florescent Protein (GFP) florescence shown in dopaminergic neurons of transgenic strains of C. elegans (Braungart, et al., 2004) Figure 8: GFP expression in dopaminergic neurons of untreated and MPP + treated worms

Literature Review Cont. Direct addition of dopamine-2-c did not aid to increasing noradrenaline presence in PD patients (Goodall and Alton, 1969) Figure 9: % 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 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.

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

Protocols Cont. 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

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 12: Sodium azide (NaN 3 ) Figure 13: 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 MPP +, 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$7.00 SigmaD048MPP+1100 mg$ 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$43.25 SigmaF0503fluorodeoxyurodine1100 mg$ Total Cost$433.75

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