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.

http://www.spinstudios.co.uk/sa/pa3.jpg

Figure 2 The effects of Parkinson’s disease

Knowledge Base

Figure 3 The life cycle of C. eleganshttp://www.wormatlas.org/handbook/fig.s/IntroFIG6.jpg

Knowledge Base

http://www.vhl.org/gifs/hif-1.jpg

Figure 4 The hypoxic response in normal and low oxygen environments

Knowledge BaseLipofuscin is an auto-

fluorescent age pigment which is found in people with neurodegenerative diseases. (Gray, et. al., 2005)

Figure 5 Lipofuscin in neurons of the human brain.

http://www.innovitaresearch.org/news/res/06042501_01.jpg

Literature Review“Caenorhabditis elegans MPP+ Model of Parkinson’s

Disease for High-Throughput Drug Screenings”Braungart, et. al. (2004)

Figure 6 The C. elegans with and without MPP+

Taken from 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.

Literature Review“Measuring Caenorhabditis elegans lifespan using

solid media” Sutphin, et. al. (2009)

Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009.

Figure 7Auto fluorescent pigments present in Day 4 and Day 8 C. elegans

Literature Review“In vivo spectrofluorimetry reveals endogenous biomarkers that

report healthspan and dietary restriction in Caenorhabditis elegans”

Gerstbrein, et. al. (2008)

Figure 8 Fluorescence of the C. elegans using the lipofuscin as a biomarker for healthspan.

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“Proteasomal Regulation of the Hypoxic Response

Modulates Aging in C. elegans” Mehta, Ranjama.

Mehta, Ranjama; K.A. Steinkraus; G. L. Sutphin, F. J. Ramos, L. S. Shamieh;A. Huh; C. Davis; D. Chandler-Brown; M. Kaeberlein. “Proteasomal Regulation of the Hypoxic Response Modulates Aging in C. elegans.” Science. 2009.

Figure 9 Mutation of VHL-1 reduces accumulation of auto-fluorescent age pigments

PurposeThe purpose of the experiment is to observe the effects

of a hypoxic response in anoxia on the amount of lipofuscin present in C .elegans

HypothesisNull- the amount of lipofuscin present during the hypoxic response in

anoxia will be the same as the amount present without the hypoxic response in anoxia.

Alternate- the amount of lipofuscin present during the hypoxic response in anoxia will be less than the amount present without the hypoxic response in anoxia.

The Effects of the Hypoxic Response in Anoxia on the Amount of Lipofuscin Present in C. elegans

C. elegans obtained from the Caenorhabditis Genetics Center- N=80

Wild type C. elegans: N=40 VHL-1(ok161) C. elegans strain: (vhl-1 deletion) Constitutive HIF-1 in normoxia; slow growth and reduced brood size: N=40

2 hour timed egg laying for C. elegans. During the L4 stage, the C. elegans will be transferred to the Nematode Growth Media *(NGM) at 20°C. The C. elegans are fed U.V. killed E. coli stored at room temperature and grown overnight at 37°C. C. elegans are grown in liquid media at 16°C. 1.5 mM of 1-methyl-4-phenylpyridinium (MPP+) after the L1 stage is applied in the food.

Use of 4',6-diamidino-2-phenylindole (DAPI) to observe the amount of auto fluorescent pigment- lipofuscin- in C. elegans- scale will be used to measure the amounts of lipofuscin.

Statistical analysis using SPSS and T-test

Given MPP+N=20

Control VHL-1N=20

Given MPP+N=20

Control Wild TypeN=20

ProtocolC. elegans are grown

in petri dishes containing Nematode Growth Media (NGM) from Carolina Biological and fed U.V. killed Escherichia coli.

Figure 10 Culturing the C. elegans in Petri dishes

Picture by author

ProtocolBoth 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.svghttp://www.sigmaaldrich.com/structureimages/30/mfcd00006530.gif

Figure 11 Ampicillin Figure 12 FUDR

Protocol

http://upload.wikimedia.org/wikipedia/commons/a/a1/MPP%2B.svg

Figure 13 1-methyl-4-phenlypyridinium (MPP+)

Protocol

Figure 14 4',6-diamidino-2-phenylindole (DAPI)

http://upload.wikimedia.org/wikipedia/commons/7/7a/DAPI.png

Protocol

Sutphin, George; M. Kaeberlein. “Measuring Caenorhabditis elegans Life Span on Solid Media” JOVE. 2009.

Figure 15 Age synchronization of C. elegans

Protocol

Figure 16 Process of MPP+ application and observation amongst the four C. elegans groups

Picture by author

BudgetVendor Cat# Pg. Item Qty. Unit $ Total $  

Caenorhabditis Genetics Center CB5602   VHL-1(ok161) C. elegans 1 $7 $7  

Caenorhabditis Genetics Center AB1   Wild type C. elegans 1 $7 $7  

Sigma D048   MPP+ 1 108 108  

Sigma D9542-5MG   DAPI 1 51.6 51.6  

Sigma S2002   Sodium azide 1 21.2 21.2  

Sigma F0503-100MG   FUDR 1 117 117  

Carolina Biological 741270   Petri dishes 10 6.45 64.5  

Carolina Biological 216880   Ampicillin dry powder 1 43.25 $43  

Carolina Biological 173520   Nematode Growth Agar 2 6.25 12.5  

Carolina Biological OP50   E. coli 1 $7 $7  

$439 Total Cost

Do-abilityAvailable for Purchase:

The VHL-1 and wild type C. elegans strains from CGC DAPI, MPP+, and Sodium Azide from SigmaNGM and OP50 E.coli from Carolina Biological

Equipment already Acquired:The DAPI filter (excitation filter centered at 365 nm and 445/50 nm

emission band-pass filter), fluorescent microscope, UV lights, and petri dishes

Bibliography "About Parkinson Disease." National Parkinson Foundation. <”http://www.parkinson.org/Page.aspx?pid=225”>. 1996-2007. 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. 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. Longo, V. “Oxygen? No thanks, I’m on a Diet” Science, Aging Knowledge Environment. Volume 2002. Pp. 10. 19 June 2002. Mc Naught, KS; P. Jenner. “Proteasomal function is impaired in substantia nigra in Parkinson's disease “ Neuroscience Letters. Volume

297. pp. 191-194. 2001. O'Riordan ; A.M. Burnell. Intermediary metabolism in the dauer larva. II. The glyoxylate cycle and fatty acid oxidation. Comp. Biochem.

Physiol. Volume 95. pp. 125-130. 1990. Rajput AH, Offord KP, Beard CM, Kurland LT. Epidemiology of parkinsonism: incidence, classification, and mortality. Ann Neurol.

1984;16:278-282. Shen, Chuan; Daniel Nettleton; Min Jiang; Stuart K. Kim; Jo Anne Powell-Coffman. “Roles of the HIF-1 Hypoxia Inducible Factor during

Hypoxia Response in Caenorhabditis elegans” The Journal of Biological Chemistry. Volume 280. pp.20580-20588. 2005. 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.