The Effect of Centrophenoxine on Parkinson’s Disease Victoria Wei

Need Taken from Rajput AH, Offord KP, Beard CM, Kurland LT. Epidemiology of parkinsonism: incidence, classification, and mortality. Ann Neurol. 1984;16:278-282. Figure 1 The amount of Parkinson’s disease cases per 100,000 people in the United States as age increases

Knowledge Base Parkinson’s disease is a brain disorder involving the nerves. Figure 2 The effects of Parkinson’s disease http://www.spinstudios.co.uk/sa/pa3.jpg

Knowledge Base Figure 3 The life cycle of C. elegans http://www.wormatlas.org/handbook/fig.s/IntroFIG6.jpg Figure 3 The life cycle of C. elegans

Knowledge Base Figure 4 Lipofuscin in neurons of the human brain. http://www.innovitaresearch.org/news/res/06042501_01.jpg Figure 4 Lipofuscin in neurons of the human brain.

Knowledge Base Figure 5 Molecular structure of centrophenoxine http://commons.wikimedia.org/wiki/File:Centrophenoxine.svg Figure 5 Molecular structure of centrophenoxine

Literature Review Caldwin, et. al. (2008) Figure 7 Figure 6 Notes that I will need about this slide Figure 6 Figure 7 Both images taken from Caldwin, Guy A.; K.A. Caldwell. “Traversing a wormhole to Combat Parkinson’s disease.” Disease Models and Mechanisms. Volume 1. pp.000-000. 2008.

Literature Review Sutphin, et. al. (2009) Figure 8 Auto fluorescent pigments present in Day 4 and Day 8 C. elegans Notes that I will need about this slide Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009.

Literature Review Gerstbrein, et. al. (2008) Figure 9 Auto fluorescent pigments present in Day 7 and Day 12 C. elegans. Notes that I will need about this slide Gerstbrein, Beate; G. Stamatas; N. Kollias; M. Driscoll. “In viv spectrofluorimetry reveals endogenous biomarkers that report healthspan and dietary restriction in Caenorhabditis elegans.

Literature Review Application of centrophenoxine to the C. elegans decreased the rate of lipofuscin accumulation by an average of 41.3%. (Shulkin, et. al., 1978) Figure 10 Table displaying effect of centrophenoxine on lipofuscin in C. elegans Notes that I will need about this slide Shulkin, D.J.; B.M. Zuckerman. “Spectrofluorometric analysis of the effect of centrophenoxine on lipofuscin accumulation in the nematode C. elegans.” Age. Volume 5. Pp. 50-53. 1982.

Purpose The purpose of the experiment is to observe the effects of centrophenoxine on Parkinson’s disease in C. elegans Hypothesis Null- the symptoms of Parkinson’s disease will remain the same with or without the application of centrophenoxine. Alternate- the symptoms of Parkinson’s disease will lessen with the application of centrophenoxine.

Methodology The Effects of Centrophenoxine on the development of Parkinson’s disease in C. elegans Wild type C. elegans and ham-1 (ot339) C. elegans obtained from the Caenorhabditis Genetics Center N=80 Wild type C. elegans: N=40 ham-1(ot339) C. elegans: N=40 Given 6.8 mM centrophenoxine N=20 Control N=20 Given 6.8 mM centrophenoxine N=20 Control N=20 6.8 mM centrophenoxine will be applied to the Nematode Growth Media for 21 days. Use of 4',6-diamidino-2-phenylindole (DAPI) to observe the amount of auto fluorescent pigment- lipofuscin- in C. elegans. GFP filters may also be used to observe whether the C. elegans DA neurons will be affected by the application of centrophenoxine Statistical analysis

Protocol C. elegans are grown in petri dishes containing Nematode Growth Media (NGM) from Carolina Biological and fed U.V. killed Escherichia coli. Picture by author Figure 11 Culturing the C. elegans in Petri dishes

Protocol Both Ampicillin and 5-Fluoro-2′-deoxyuridin will be used with NGM in the petri dishes with C. elegans http://upload.wikimedia.org/wikipedia/commons/b/b6/Ampicillin_structure.svg Figure 13 FUDR Figure 12 Ampicillin http://www.sigmaaldrich.com/structureimages/30/mfcd00006530.gif E.coli + NGM with centrophenoxine + Ampicillin + FUDR  using the DAPI and GFP filter to observe amount of lipofuscin and fluorescence present in both C. elegans groups

Protocol Figure 14 Age synchronization of C. elegans Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009. Figure 14 Age synchronization of C. elegans

Protocol Figure 15 4',6-diamidino-2-phenylindole (DAPI) http://upload.wikimedia.org/wikipedia/commons/7/7a/DAPI.png Figure 15 4',6-diamidino-2-phenylindole (DAPI) http://www.ion.ucl.ac.uk/images/Fluorescence-Microscope.jpg http://www.bcgsc.ca/people/msleumer/htdocs/fact_sheet/image004.jpg Figure 17 GFP expressed in C. elegans Figure 16 Fluorescence microscope

Budget Vendor Cat# Item Qty. Unit $ Total $ Caenorhabditis Genetics Center GS1214 ham-1(ot339) C. elegans 1 $7 AB1 Wild type C. elegans Sigma D9542-5MG DAPI $51.60 S2002 Sodium azide $21.20 F0503-100MG FUDR $117 SLC5377-25G Centrophenoxine Hydrochloride $97.82 Carolina Biological 741270 Petri dishes 10 $6.45 $64.50 216880 Ampicillin dry powder $43.25 $43 173520 Nematode Growth Agar 2 $6.25 $12.50 OP50 E. coli Invitrogen D21490 $116.00 Nova-tech 1482FLi Fluorescence Microscope $3,760 Tritech MINJ-F-EFB GFP filter set $938.38 $938   Total cost $5,243

Do-ability Available for Purchase: Equipment already Acquired: ham-1(ot339) and wild type C. elegans strains from CGC DAPI and Sodium Azide from Sigma NGM and OP50 E.coli from Carolina Biological Centrophenoxine purchasable from Science Lab.com Equipment already Acquired: DAPI filter, GFP filter, fluorescent microscope

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