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
http://www.wormatlas.org/handbook/fig.s/IntroFIG6.jpg
Figure 3 The life cycle of C. elegans
Knowledge Base
http://www.innovitaresearch.org/news/res/06042501_01.jpg
Figure 4 Lipofuscin in neurons of the human brain.
Knowledge Base
http://resources.metapress.com/pdf-preview.axd?code=w35465l87q324635&size=largest
Figure 5 structure of centrophenoxide and the products of
the hydrolysis- which are dimethylethanolamine (DMAE)
and p-chlorophenoxyacetic acid (PCPA)
Literature Review
 Caldwin, et. al. (2008)
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.000000. 2008.
Literature Review
 Gerstbrein, et. al.
(2008)
 Lipofuscin is a
marker of lifespan
in C. elegans
Figure 9 Auto fluorescent
lipofuscin pigments present in
Day 7 and Day 12 C. elegans.
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
 Nass, et. al., (2002)
 GFP is a usable method of
observing the dopamine
neurons in C. elegans.
Dopamine neurons are unaffected
Dopamine neurons are progressively dying
Figure 10 Degeneration of dopamine
neurons
http://docs.google.com/gview?a=v&q=cache%3AtdtxA_lhng0J%3Aweb.mac.com%2Frdblakely%
2FBlakelyLab%2FBlakely_Lab_News_files%2FWorm%2520research%2520yields%2520powerf
ul%2520model%2520for%2520Parkinson%27s%2520disease%25203.1.02.pdf+c+elegans+park
inson%27s+filetype%3Apdf&hl=en&gl=us
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
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.
The Effects of Centrophenoxine on Parkinson’s disease in Caenorhabditis elegans
Caenorhabditis elegans ham-1(ot339) obtained from the Caenorhabditis Genetics Center (CGC)
N=100
Given 6.8 mM centrophenoxine:
No centrophenoxine is applied:
N=50
N=50
C. elegans will be cultured in the Nematode Growth Media at 20 °C , and fed
Escherichia coli(op50). 6.8 mM centrophenoxine will be applied to the NGM
where the C. elegans are cultured and observed throughout lifespan.
Qualitative data will be collected on how strong
both the GFP and lipofuscin fluorescence is-using
a scale that ranges the strength of fluorescence.
Statistical analysis with SPSS
Protocol
 C. elegans are grown in
petri dishes at 20°C with
NGM and fed U.V. killed E.
coli.
 Both Ampicillin and 5-
Fluoro-2′-deoxyuridin
(FUDR) are added to the
plates
Picture by author
Figure 11 Culturing the C. elegans in
a petri dish
Protocol
E.coli + NGM with centrophenoxine + Amp + FUDR
using the DAPI and GFP filter to observe amount of
lipofuscin and GFP fluorescence present in both C.
elegans ham-1(ot339) groups
Protocol
Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009.
Figure 12 Age synchronization of C. elegans
Budget
Vendor
Cat#
Item
Caenorhabditis Genetics
Center
GS1214
ham-1(ot339) C. elegans
1
$7
$7
Caenorhabditis Genetics
Center
AB1
Wild type C. elegans
1
$7
$7
Sigma
D9542-5MG
DAPI
1
$51.60
$51.60
Sigma
S2002
Sodium azide
1
$21.20
$21.20
Sigma
F0503-100MG
FUDR
1
$117
$117
1
$97.82
$97.82
10
$6.45
$64.50
Sigma
Qty.
SLC5377-25G Centrophenoxine Hydrochloride
Unit $
Total $
Carolina Biological
741270 Petri dishes
Carolina Biological
216880 Ampicillin dry powder
1
$43.25
$43
Carolina Biological
173520 Nematode Growth Agar
2
$6.25
$12.50
Carolina Biological
OP50
E. coli
1
$7
$7
Invitrogen
D21490
DAPI
1
$116.00
$116.00
Nova-tech
1482FLi
Fluorescence Microscope
Tritech
MINJ-F-EFB
GFP filter set
1
$3,760
1
$3,760
$938.38
Total cost
$938
$5,243
Do-ability
Available for Purchase:
 ham-1(ot339) C. elegans strain from CGC
 DAPI and Sodium Azide from Sigma
 NGM and OP50 E.coli from Carolina Biological
 Centrophenoxine from Science Lab.com
Equipment already Acquired:
 DAPI filter, GFP filter, fluorescent microscope, UV lights
Bibliography
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Parkinson’s Disease for High-throughout Drug Screening.” Neurodegenerative Disease. 2004. Volume 1: pgs 175-183.
Caldwin, Guy A.; K.A. Caldwell. “Traversing a wormhole to Combat Parkinson’s disease.” Disease Models and Mechanisms. Volume 1. pp.000000. 2008.
Colleta, Susan. Introduction to C. elegans. Waksman Student Scholars.
<http://avery.rutgers.edu/WSSP/StudentScholars/project/introduction/worms.html>. 2009
Gerstbrein, Beate; G. Stamatas; N. Kollias; M. Driscoll. “In viv spectrofluorimetry reveals endogenous biomarkers that report healthspan and
dietary restriction in Caenorhabditis elegans.
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Hunt, Sara S. The Aging Process. Washington D.C. April 2004.
Kenyon, Cynthia. “Environmental Factors and Gene Activities That Influence Life Span” C. elegans II. Cold Spring Harbor Press. 1997.
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Nass, Richard; R. Blakely; D. Miller. “Worm yields powerful research model for Parkinson’s disease.” The Reporter. 2002.
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Rajput AH, Offord KP, Beard CM, Kurland LT. Epidemiology of parkinsonism: incidence, classification, and mortality. Ann Neurol. 1984;16:278282.
Schneider, Howard F.; C. Nandy. “Effects of Centrophenoxine on Lipofuscin Formation in Neuroblastoma Cells in Culture” Journal of
Gerontology. Volume 32. Pp. 132-139. 1997.
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.
Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009.
“What is Parkinson’s?” American Parkinson Disease Association West Coast Office. <“http://www.apdawest.org/WhatIsParkinsons.html#2”>.
2009.