Effect of Neuroprotective Substances
on beta-Amyloid Production in C.
elegans
Project Proposal
Lim Eu Gene 4A315
Jarel Teo Shao Hean 4P309
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 behaviour.
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
sphingolipids. 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 God of 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 organism 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 (WormClassroom, 2012). 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 type C. elegans is increased, the beta-amyloid plaque
accumulated would increase. Movements of the wild C. elegans should be slower or have at
the same speed of the mutated C. elegans. If the concentration of celastrol and resveratrol of
wild type C. elegans is increased, there would be a reduction of beta-amyloid plaque
production. Movements of wild type C. elegans should be faster as compared to mutated C.
elegans.
The movement of mutated C. elegans can be sped up by treating it with increasing
concentrations of celastrol and resveratrol
Objective
The objective of this project is:
1. To study the effects of celastrol and resveratrol on the beta-amyloid plaque production,
the movement and protein profile in both wild type and mutated C. elegans
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Methodology
List of Variables
Independent Variables

Concentration of celastrol

Concentration of resveratrol

Concentration of local cholesterol
Dependent Variables

Beta-amyloid plaque accumulation

Speed of movement of C. elegans

Frequency of thrashing of C. elegans

Protein profiles of C. elegans
Controlled Variables

Culture conditions

Types of C. elegans used

Age of C. elegans
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Procedures
Outline of Procedure
C. elegans (wild type
and mutated)
Celastrol
Treatment
C. elegans (wild type
and mutated) +
increased cholesterol
Celastrol +
Resveratrol
Treatments
Resveratrol
Treatment
Observations
Aβ production
C. elegans
Movement
SDS-PAGE
Diagram 1
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C. elegans (wild type
and mutated) with &
w/o cholesterol
control setups
Experimental Procedure
I)
Experimental Setup
A. Preparation of NGM Agar Culture (Stiernagle, 2006)
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. Place 3g NaCl, 17g NGM agar and 2.5g peptone into a flask. Mix well.
2. Place 970ml of deionised water using a measuring cylinder.and add 1ml 1M CaCl2,
1ml 5mg/ml cholesterol in ethanol, 1ml 1M MgSO4 and 25ml 1M KPO4 buffer.
3. Pour the NGM agar into the plates until the plates are ⅔ full.
4. Leave the plates at room temperature for 2 days.
B. Preparation of Cholesterol Setups Containing Excess Cholesterol
Materials
NGM Agar Culture, 5mg/ml Cholesterol in Ethanol (Not Autoclaved)
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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
Celastrol Crystalline, Dimethyl Sulfoxide (DMSO) Solution, NGM Agar Culture
Apparatus
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50ml Centrifuge Tube, Electronic Beam Balance, Micropipette
Procedure
1. Pour 20ml of 100% dimethyl sulfoxide (DMSO) concentration into a 50ml centrifuge
tube and add 0.04506g of celastrol crystalline solid. This would obtain 20ml of 5mM/l
celastrol stock solution.
2. Add the stated 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
0.024
119.976
120.0
2µM/l Setup
0.048
119.952
120.0
3µM/l Setup
0.072
119.928
120.0
4µM/l Setup
0.096
119.904
120.0
5µM/l Setup
0.12
119.880
120.0
Total /ml
Table 2
D. Preparation of Resveratrol Setups
Materials
Resveratrol Crystalline, Dimethyl Sulfoxide (DMSO) Solution, NGM Agar Culture
Apparatus
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50ml Centrifuge Tube, Electronic Beam Balance, Micropipette
Procedure
1. Pour 20ml of 100% dimethyl sulfoxide (DMSO) concentration into a 50ml centrifuge
tube and add 0.02282g of resveratrol crystalline solid. This would obtain 20ml of
5mM/l resveratrol stock solution
2. Add the stated 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
0.024
119.976
120.0
2µM/l Setup
0.048
119.952
120.0
3µM/l Setup
0.072
119.928
120.0
4µM/l Setup
0.096
119.904
120.0
5µM/l Setup
0.12
119.880
120.0
Total /ml
Table 3
E. Preparation of Celastrol + Resveratrol Setups
Materials
5mM/l Celastrol Stock Solution, 5mM/l Resveratrol Stock Solution, NGM Agar Culture
Apparatus
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Micropipette
Procedure
1. Determine the optimum concentration of celastrol and resveratrol solutions from the
celastrol and resveratrol trials in (C.) and (D.) and place both concentrations into the
Petri dish
F. Preparation of Bacteria Food Source
Materials
Luria Broth, E. coli OP50 Starter Culture
Apparatus
50ml Centrifuge Tube, Incubator
Procedure
1. Using a starter culture of E. coli OP50, isolate a few colonies in 20ml of Luria Broth.
2. Allow the inoculated cultures to grow overnight at 37°C.
G. Seeding of NGM Agar Plates (Stiernagle, 2006)
Materials
E. coli OP50 Liquid Culture, NGM Agar Plates
Apparatus
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Micropipette, Glass Rod
Procedure
1. Apply 100µl of E. coli OP50 liquid culture onto the one end of the NGM agar plates
using a micropipette.
H. Chunking of C. elegans (Tan et al., 2010)
Materials
C. elegans Culture Plate, Seeded NGM Agar Plates
Apparatus
Sterilised Scalpel, 50ml Centrifuge Tube
Procedure
1. Pour 20ml 5M NaOH and an equal amount of household bleach into a 50ml Falcon
tube. Mix well.
2. A 15µl drop of the solution should be placed on the other side of the NGM agar plate.
3. Using a sterilised scalpel, cut out a 1cm x 1cm x 1cm cube from the C. elegans
culture plate.
4. Place the cube face down onto the drop of bleach solution.
5. Incubate the plate for 5 days at 27°C.
I. C. elegans Setup Treatment
Materials
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Wild Type and Mutated (FX776) C. elegans Culture Plate, Seeded NGM Agar Plates with or
without Neuroprotective Substances.
Apparatus
Sterilised Scalpel, 50ml Centrifuge Tube
Procedure
1. Using a sterilised scalpel, cut out one quarter from the C. elegans culture plate.
2. Place the quarter of C. elegans culture face down onto the OP50 seeded NGM agar
plate with or without neuroprotective substances, seeded with 200µl of OP50 culture.
3. Incubate the plates for 1 day at 27°C.
4. Conduct fluorescence staining, protein profiling and observe the movement of the C.
elegans.
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5. Data Collection
A. Plaque Formation (Herndon et al., 2002)
Materials
PBS, Paraformaldehyde, β-mercaptoethanol, Triton X-100, Tris, Thioflavin S
Apparatus
Fluorescence Microscope
Procedure
1. Wash off the C. elegans from their parent plates.
2. Fix the worms in 4% paraformaldehyde/PBS, pH 7.4, for 1 day at 4°C
3. Permeabilize the worms in 5% fresh β-mercaptoethanol, 1% Triton X-100, 125mM
Tris, pH 7.4, in a 37°C incubator for 1 day.
4. Stain the worms with 0.125% thioflavin S in 50% ethanol for 2 minutes.
5. Destain and mount the worms onto glass slides and observe under microscope.
6. Take a photograph of the stained C. elegans worm and quantify the intensity of the
plaque using ImageJ software.
B. Worm Movement (Tan et al., 2010)
Materials
C. elegans Petri Dish
Apparatus
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Paper, USB Microscope, Computer Screen, Transparency Sheet
Procedure
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 and measure its length
5. Calculate the speed of travel of the worm based on the data collected in (4).
C. Thrashing Assay (Nazir et al., 2010)
Materials
C. elegans Petri Dish, M9 Buffer
Apparatus
USB Microscope
Procedure
1. Place 1ml of M9 buffer on a C. elegans petri dish.
2. Allow the sample to stabilise for 30 seconds.
3. Observe one worm under the microscope.
4.
Count the frequency of body bends for 30 seconds using the USB microscope.
D. Protein Profile (Neo et al., 2011)
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Materials
Crushed C. elegans Suspension, Polyacrylamide Gel, Coomassie Blue Stain,
Apparatus
SDS-PAGE Kit, Microfuge Tubes
Procedure
1. Mix 10µl of C. elegans Suspension and 5µl of loading dye into a microfuge tube.
2. Place the microfuge 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.
9. Compare the protein components between the varied C. elegans setups and the control
setups.
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
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