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
 Lipofuscin is an auto-
fluorescent age
pigment which is found
in people with
neurodegenerative
diseases.
(Gray, et. al., 2005)
http://www.innovitaresearch.org/news/res/06042501_01.jpg
Figure 4 Lipofuscin in neurons of the human brain.
Knowledge Base
 Centrophenoxine is an
anti-aging medicine
which slows the
accumulation of
lipofuscin.
 Schneider, et. al. (1977)
http://commons.wikimedia.org/wiki/File:Centrophenoxine.svg
Figure 5 Molecular structure of centrophenoxine
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
 Sutphin, et. al. (2009)
Figure 8
Auto fluorescent pigments
present in Day 4 and Day 8 C.
elegans
Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009.
Literature Review
 Gerstbrein, et. al.
(2008)
Figure 9 Fluorescence of
the C. elegans using the
lipofuscin as a biomarker
for health span.
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 Fluorescence of
the C. elegans using the
lipofuscin as a biomarker
for health span.
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 the restriction of Parkinson’s
disease symptoms 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 the development of Parkinson’s disease in C.
elegans
C. elegans obtained from the Caenorhabditis
Genetics Center- N=80
Wild type C. elegans: N=40
Given
centrophenoxine
N=20
Control
N=20
ham-1(ot339) C. elegans: N=40
Given
centrophenoxine
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 pigmentlipofuscin- in C. elegans. GFP filter will also be
used.
Statistical analysis with SPSS
Control
N=20
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 12 Ampicillin
Figure 13 FUDR
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
Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009.
Figure 14 Age synchronization of C. elegans
Protocol
Picture by author
Figure 15 Process of centrophenoxine application and observation
amongst the four C. elegans groups
Protocol
http://upload.wikimedia.org/wikipedia/commons/7/7a/DAPI.png
Figure 16 4',6-diamidino-2phenylindole (DAPI)
http://www.wormbook.org/chapters/www_intromethodscellbiology/cellfig3.jpg
Figure 17 C. elegans as observed under DAPI
filter
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
SLC5377-25G Centrophenoxine Hydrochloride
Qty.
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
Total Cost
$545
Do-ability
Available for Purchase:
 The 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 purchaseable from Science Lab.com
Equipment already Acquired:
 The DAPI filter (excitation filter centered at 365 nm and 445/50 nm
emission band-pass filter), fluorescent microscope, UV lights
Bibliography
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Braungart, Evelyn; Gerlach, Manfred; Riederer; Peter, Baumeister, Ralf; and Hoener, Marius C. “Caenorhabditis elegans MPP+ Model of
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.
Hall, D. H.; Z. F. Altun. “C. elegans Atlas.” Genetics Research, 90 , pp 375-376. 2008.
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.
Kisiel, Marion J.; B. Zuckerman. “Effects of Centrophenoxine on the Nematode Caenorhabditis Briggsae” Age. Volume 1. Pp.17-20. January
1978.
Mc Naught, KS; P. Jenner. “Proteasomal function is impaired in substantia nigra in Parkinson's disease “ Neuroscience Letters. Volume 297. pp.
191-194. 2001.
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Volume 95. pp. 125-130. 1990.
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.