Effect of celastrol on C. elegans with
increased beta-amyloid production due
to an increase in local cholesterol
Project Proposal
Lim Eu Gene 3A315
Jarel Teo Shao Hean 3P310
Hwa Chong Institution
Mentor: Mrs Har Hui Peng
Introduction
Background
Alzheimer's Disease (AD) is a neurodegenerative disease characterized by the loss of
neural tissue. It is also a type of dementia that impairs memory, thought, and behavior.
Victims of AD experience problems with language, decision-making ability, judgment, and
personality.
The cause of AD is not known, but closely tied to the onset of the disease is the
accumulation of extracellular amyloid-β plaques and neurofibrillary tangles. These plaques
and tangles lead to the degeneration of neurons via apoptosis.
Aβ proteins are formed by cleavage of the amyloid precursor protein. The amyloid
precursor protein (APP) is regularly processed at the cell surface of neurons and is also
processed intracellularly after being trafficked into the cell by lipid rafts. Lipid rafts are
specialized microdomains within plasma membranes which are rich in cholesterol and
shingolipids. The existence of lipid rafts is still debated, since they cannot be conventionally
imaged due to their small size (~20 nm). Protein distribution in lipid rafts is therefore
observed biochemically (e.g. detergent-resistant membrane (DRM) isolation). The role of
lipid rafts is to regulate signal transduction and trafficking. After APP is brought into a cell
by lipid rafts, it is cleaved by β-secretase and γ-secretase (Marquer et al., 2011). This
cleavage produces a soluble N-terminal fragment (sAPPβ) and an amino acid amyloid βpeptide (Aβ).
Research investigating the effect of an increase in local cholesterol on lipid-raft
endocytosis discovered that an increase in cholesterol resulted in rapid endocytosis, as well as
the clustering of β-secretase enzymes and APP in lipid rafts. This indicated that increased
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cholesterol levels may not directly cause an increase in the production of Aβ, but be an
indirect cause by bringing the β-secretase enzyme and amyloid precursor protein in close
proximity. Also, an increase in intracellular APP leads to a greater rate of production of the
Aβ peptide, which then accumulates extracellularly to form insoluble, neurotoxic plaques.
(Marquer et al., 2011)
Celastrol, a chemical derived from the Chinese “Thunder of God Vine” is a potent
antioxidant known to have anti-cancer effects. Research has shown that celastrol greatly
inhibited beta-amyloid plaque formation by reducing the beta-cleavage of amyloid precursor
protein. It is also known for its ability to cross the blood brain barrier and offer
neuroprotection in animal models of Parkinson’s disease and Huntington’s disease. Previous
work has revealed that celastrol can prevent neurodegeneration and extend the life span of a
transgenic mouse model of amyotrophic lateral sclerosis. Celastrol displays potent antiinflammatory activities in vivo and has been shown, for example, to be beneficial against
allergy-induced asthma as well as rheumatoid arthritis. (Paris et al., 2010)
Resveratrol, a polyphenol that occurs in abundance in grapes and red wine, is
suspected to afford antioxidant and neuroprotective properties and therefore to contribute to
the beneficial effect of wine consumption on the neurodegenerative process. (Jang & Surh,
2003) Researchers (Marambaud, Zhao, & Davies, 2005) have reported that resveratrol has a
potent anti-amyloidogenic activity by reducing the levels of Aβ produced from different cell
lines. They also found that resveratrol acts by promoting the intracellular degradation of Aβ
by a mechanism that implicates the proteasome.
Since celastrol and resveratrol has recently emerged as a new possibility of a natural
neuroprotective agent, our team has decided to study the effects of both celastrol and
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resveratrol so as to identify it as a potential cure for Alzheimer's Disease and even other
neurological diseases.
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Rationale
In our project, we will be using celastrol extracted from the “Thunder God of Vine”
plant. Paris et al. (2010) has discovered that celastrol is a potent Aβ lowering compound in
transgenic mice models of AD and is possible that celastrol can also improve cognition and
tau pathology in these transgenic mice models. We will also be using resveratrol which is
found in grapes and red wine. Marambaud, Zhao, & Davies (2005) have concluded that
resveratrol does not inhibit Aβ production, because it has no effect on the Aβ-producing
enzymes β- and γ-secretases, but promotes instead intracellular degradation of Aβ via a
mechanism that involves the proteasome.
Caenorhabditis elegans (or in short C. elegans) will be used as the test system in this
study. It shares many similar biological traits and genes with humans, thus the results from
this study could be examined for application in humans. In addition, it has a short lifespan
that allows results to be obtained within a short duration.
Increased local cholesterol will serve as a mutagen in this project. According to
Marquer et al. (2011), an increase in cholesterol level will increase the rate of lipid raft
endocytosis, which indirectly leads to the increase in production of Aβ, which then
accumulates extracellularly to form insoluble, neurotoxic plaques.
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Hypothesis
If local cholesterol of wild C. elegans is increased, the beta-amyloid plaque
accumulated would increase. Movements of the wild C. elegans should be slower or at the
same speed of the mutated C. elegans. Under an increase of beta-amyloid plaque due to
increase in local cholesterol, if the concentration of celastrol and resveratrol of wild C.
elegans is increased, the beta-amyloid plaque accumulated would reduce. Movements of wild
C. elegans should be faster as compared to mutated C. elegans.
If the concentration of celastrol and resveratrol on mutated C. elegans is increased,
movements of the mutated C. elegans should be faster.
Objective
The objectives of this project are:
1. To study the effects of celastrol and resveratrol on the beta-amyloid plaque
concentration, the movement and protein profile of both wild and mutated C. elegans
2. To determine possible sources of neuroprotective agents which can prevent or cure
neurological diseases
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Methodology
List of Variables
Independent Variables

Concentration of celastrol (µM/l)

Concentration of resveratrol (µM/l)

Concentration of local cholesterol (g/ml)
Dependent Variables

Beta-amyloid plaque accumulation

Movement of C. elegans

Thrashing of C. elegans

Protein profiles of C. elegans
Controlled Variables

Culture conditions

Types of C. elegans used

Number of C. elegans per agar plate
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Procedures
Outline of Procedures
C. elegans (wild and
mutated) + increased
cholesterol
C. elegans (wild and
mutated)
Celastrol
Celastrol +
Resveratrol
Aβ production
SDS-PAGE
C. elegans
Movement
Diagram 1
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Resveratrol
Experimental Procedure
I)
Experimental Setup
A. Preparation of NGM Agar Culture
Materials
Sodium Chloride (NaCl), Peptone, Agar, 5mg/ml Cholesterol in Ethanol (Not Autoclaved),
1M MgSO4, 1M KPO4 Buffer pH 6.0, Deionised Water
Apparatus
Petri Dish, Conical Flask, 500 ml Glass Bottle, Measuring Cylinder, Electronic Weighing
Scale, Beaker
Procedure
1. Add 3g NaCl, 17g NGM agar and 2.5M NaOH into a flask.
2. Mix well.
3. Add 970ml of deionised water using a measuring cylinder.
4. Add 1ml 1M CaCl2, 1ml 5mg/ml cholesterol in ethanol, 1ml 1M MgSO4 and 25ml
1M KPO4 buffer.
5. Pour the NGM agar into the plates until the plates are ⅔ full. This would obtain 1 litre
of NGM agar culture
6. Leave the plates at room temperature for 2 days.
B. Preparation of Increased Cholesterol Setups
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Materials
NGM Agar Culture, 5mg/ml Cholesterol in Ethanol (Not Autoclaved)
Apparatus
Electronic Beam Balance
Procedure
1. Increase the amount of 5mg/ml cholesterol in ethanol solution to be added into the
NGM agar mixture according to Table 1 below.
2. Decrease the amount of deionized water added to the NGM agar mixture to retain the
total volume of NGM agar culture at 1l
5.0mg
7.5mg
10.0mg
Cholesterol
Cholesterol
Cholesterol
1.0
1.5
2.0
999.0
998.5
998.0
1000.0
1000.0
1000.0
5mg/ml Cholesterol in Ethanol
/ml
Remaining NGM Agar
Contents /ml
Total /ml
Table 1
C. Preparation of Celastrol Setups
Materials
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Celastrol Crystalline, Dimethyl Sulfoxide (DMSO) Solution, NGM Agar Culture
Apparatus
50ml Falcon Tube, Electronic Beam Balance, Micro Pipette
Procedure
1. Pour 20ml of 100% dimethyl sulfoxide (DMSO) concentration into a Falcon tube.
2. Weigh 0.04506g of celastrol crystalline solid and put them in the DMSO solution.
This would obtain 20ml of 5mM/l celastrol stock solution.
3. Add the amount of 5mM/l celastrol stock solution and NGM agar culture into the
Petri dish according to Table 2 below.
5mM/l Celastrol
NGM Agar Culture
Stock Solution /ml
/ml
1µM/l Setup
0.0024
11.9976
12.0
2µM/l Setup
0.0048
11.9952
12.0
3µM/l Setup
0.0072
11.9928
12.0
4µM/l Setup
0.0096
11.9904
12.0
5µM/l Setup
0.012
11.9880
12.0
Table 2
D. Preparation of Resveratrol Setups
Materials
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Total /ml
Resveratrol Crystalline, Dimethyl Sulfoxide (DMSO) Solution, NGM Agar Culture
Apparatus
50ml Falcon Tube, Electronic Beam Balance, Micro Pipette
Procedure
1. Pour 20ml of 100% dimethyl sulfoxide (DMSO) concentration into a Falcon tube.
2. Weigh 0.02282g of resveratrol crystalline solid and put them in the DMSO solution.
This would obtain 20ml of 5mM/l resveratrol stock solution
3. Add the amount of 5mM/l resveratrol stock solution and NGM agar culture into the
Petri dish according to Table 3 below.
5mM/l Resveratrol
NGM Agar Culture
Stock Solution /ml
/ml
1µM/l Setup
0.0024
11.9976
12.0
2µM/l Setup
0.0048
11.9952
12.0
3µM/l Setup
0.0072
11.9928
12.0
4µM/l Setup
0.0096
11.9904
12.0
5µM/l Setup
0.012
11.9880
12.0
Table 3
E. Preparation of Celastrol + Resveratrol Setups
Materials
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Total /ml
5mM/l Celastrol Stock Solution, 5mM/l Resveratrol Stock Solution, NGM Agar Culture
Apparatus
Micro Pipette
Procedure
1. Add the amount of 5mM/l resveratrol stock solution, 5mM/l celastrol stock solution
and NGM agar culture into the Petri dish according to Table 4 below.
2µM/l
5mM/l Celestrol
5mM/l Resveratrol
NGM Agar
Total
Stock Solution /ml
Stock Solution /ml
Culture /ml
/ml
0.0024
0.0024
11.9952
12.0
0.0048
0.0048
11.9904
12.0
0.0072
0.0072
11.9856
12.0
Setup
4µM/l
Setup
6µM/l
Setup
Table 4
F. Preparation of Bacteria Food Source
Materials
Nutrient Agar Powder, Deionised Water, E. coli OP50 Starter Culture
Apparatus
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500ml Glass Bottle, Micro Pipette, Incubator
Procedure
1. Using a starter culture of E. coli OP50, isolate a single colony on Petri dish of nutrient
agar.
2. Allow the inoculated cultures to grow overnight at 37°C.
G. Seeding of NGM Agar Plates
Materials
E. coli OP50 Liquid Culture, NGM Agar Plates
Apparatus
Micropipette, Glass Rod
Procedure
1. Apply 50µl of E. coli OP50 liquid culture onto the one end of the NGM agar plates
using a micro pipette.
H. Chunking of C. elegans
Materials
C. elegans Culture Plate, Seeded NGM Agar Plates, NaOH, Household Bleach
Apparatus
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Sterilised Scalpel, 50ml Falcon Tube
Procedure
1. Pour 20ml 5M NaOH and an equal amount of household bleach into a 50ml Falcon
tube. Mix well.
2. A 5µl drop of the solution should be placed on the other side of the NGM agar plate.
3. Using a sterilised scalpel, cut out a 3mm x 3mm x 3mm cube from the C. elegans
culture plate.
4. Place the cube face down onto the drop of bleach solution.
5. Incubate the plate for 4 days at 27°C.
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II)
Data Collection
A. Plaque Formation (Herndon et al., 2002)
Materials
5-fluorescein Isothiocynate, Dimethylformamide, M9 Buffer, Seeded Growth Medium Plate,
Apparatus
Fluorescence Microscope
Procedure
1. Mix 20mg/ml of 5-fluorescein isothiocyanate (FITC) in dimethylformamide.
2. Mix 50µl of the resulting stock dye solution with 200µl M9 Buffer
3. Apply the resulting solution to the seeded growth medium plate 2-12 hours before
moving to step 4.
4. Observe the seeded growth medium plate using the fluorescence microscope mounted
with FITC filter.
B. Worm Movement
Materials
C. elegans Petri Dish
Apparatus
Paper, USB Microscope, Computer Screen, Transparency Sheet
Procedure
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1. Place a Petri dish containing C. elegans on a piece of paper
2. Observe the C. elegans with a USB microscope on a computer screen
3. Place a piece of transparency sheet over the computer screen
4. Trace the path of the worm for 30 seconds
5. Obtain the actual distance and the speed of travel of the worm
C. Thrashing Assay (Nazir, Sammi, Singh, & Tripathi, 2010)
Materials
C. elegans Petri Dish, M9 Buffer, Deionised Water
Apparatus
USB Microscope, Glass Slide
Procedure
1. Wash off the C. elegans from their parent plates using the M9 buffer 2-3 times.
2. Place a drop of M9 buffer on a clean glass slide.
3. Place a single worm into the buffer.
4. Allow the sample to stabilise for 30 seconds.
5.
Count the number of body bends for 30 seconds using the USB microscope.
D. Protein Profile (Neo, Tan, Morris, & Ferguson, 2011)
Materials
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Crushed C. elegans Suspension, Polyacrylamide Gel, Coomassie Blue Stain,
Apparatus
SDS-PAGE Kit, Eppendorf Tubes
Procedure
1. Mix 10µl of C. elegans Suspension and 5µl of loading dye into an Eppendorf tube.
2. Place the Eppendorf tubes into a hot water bath at approximately 50°C.
3. Load the Polyacrylamide Gel into the electrophoresis kit.
4. Load 10µl of protein ladder into the first well.
5. Load the subsequent wells with the C. elegans suspension mixed with loading dye.
6. Set the electrophoresis kit to 25mA.
7. Allow the gel to run until the protein column runs to the end of the gel.
8.
Stain the gel with Coomassie Blue.
Safety Precautions
During experimentation, latex gloves will be worn when handling the C. elegans
samples. All cultures will be decontaminated by autoclaving before disposal
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Timeline
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Bibliography
Herndon, L. A., Schmeissner, P. J., Dudaronek, J. M., Brown, P. A., Listner, K. M., Yuko, S.,
et al. (2002). Stochastic and genetic factors influence tissue-specific decline in ageing C.
elegans. Nature Journal, 419, 808-814.
Jang, J.-H., & Surh, Y.-J. (2003). Protective effect of resveratrol on β-amyloid-induced
oxidative PC12 cell death. Free Radical Biology and Medicine, 34(8), 2003.
Marambaud, P., Zhao, H., & Davies, P. (2005). Resveratrol Promotes Clearance of
Alzheimer’s Disease Amyloid-β Peptides. The Journal of Biological Chemistry, 280(45),
37377-37382.
Marquer, C., Devauges, V., Cossec, J., Liot, G., Lécart, S., Saudou, F., et al. (2011). Local
cholesterol increase triggers amyloid precursor protein-Bace1 clustering in lipid rafts and
rapid endocytosis. FASEB Journal, 25(4), 1295-1305.
Nazir, A., Sammi, S. R., Singh, P., & Tripathi, R. K. (2010). Trans-Cellular Introduction of
HIV-1 Protein Nef Induces Pathogenic Response in Caenorhabditis elegans. PLoS ONE,
5(12).
Neo, A., Tan, D., Morris, C., & Ferguson, A. (2011). Experiment. Retrieved Nov 11, 2011,
from Investigating the Effects of St. John's Wort on Drosophila and Cancer:
http://projectsday.hci.edu.sg/2011/15-FinalsWeb/Cat-01/1-084/exp.html
Paris, D., Ganey, N., Laporte, V., Patel, N., Beaulieu-Abdelahad, D., Bachmeier, C., et al.
(2010). Reduction of beta-amyloid pathology by celastrol in a transgenic mouse model of
Alzheimer's disease. Journal of Neuroinflammation, 7(17), 1-15.
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