identifying biomarkers in the exosomes of organisms modelling parkinson’s disease chai yi xuen...
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Identifying biomarkers in the exosomes of organisms
modelling Parkinson’s diseaseChai Yi Xuen (Leader) 4S302
Abraham Sui 4S317
Nanki Kaur (AOS)
Aditi Narvekar (AOS)
Group 1-51
Parkinson’s disease (PD)
Idiopathic neurodegenerative disease
Where?
(Oikawa et. al., 2002)
Adapted from Memory Disorders Project at Rutgers University
Causes
Neuronal cell death Aggregation of Lewy bodies
Adapted from http://www.mpg.de/
Effects
Loss of dopamine
Adapted from http://radicaislivres96.wordpress.com/
7-10 million
reported cases worldwide
Based on Parkinson’s Disease Foundation
most common neurodegenerative disorder
2nd Based on Parkinson’s Disease Foundation
Nonmotor Symptoms
Loss of sense of smell
Sleep Disorder
Mood Disorder
Adapted from Parkinson’s Disease Foundation
Motor Symptoms
Tremor
Bradykinesia
Rigidity
Adapted from Parkinson’s Disease Foundation
Cure
Levodopa
Current method of detection
SPECT scan (brain imaging)
$1100
Problem
Late Detection
Solution
Develop a cheap, non-invasive diagnostic kit for early detection of Parkinson’s disease
Solution
Develop a cheap, non-invasive diagnostic kit for early detection of Parkinson’s disease
Doing away with expensive machinery
Solution
Develop a cheap, non-invasive diagnostic kit for early detection of Parkinson’s disease
Does not require a spinal tap
Answer: Biomarkers
Our proposition: Exosomes
Exosomes
1. Nanoparticle-size vesicles secreted by all cells (Danzer, 2012)
2. Contains well-protected ‘cargo’ of RNA, miRNA and proteins (Rani et. al., 2011)
Theories of their purpose
1. Dispel waste
2. Cell to cell communication
Our Focus
“Trojan Horse” of neurodegeneration(Fevrier et. al., 2003)
WHY Exosomes?
‘Cargo’ is protected from degradation
(Simons et. al., 2009)
WHY Exosomes?
Pass through the Blood-Brain barrier (Seow et. al., 2011)
• Allows detection in blood
Our model organism…
C. elegans
WHY C. elegans?
35% of their genes are closely related to
human genes
Adapted from Worm Base
WHY C. elegans?
Table 1: PD-Associated Genes are Conserved in C. elegans (Springer, W. et. al)
The table depicts known human PD-associated genes and their homologous C. elegans genes
Homo sapiens Caenorhabditis elegans
α-synuclein no homolog
parkin pdr-1 (K08E3.7)
UCH-L1 F46E10.8, Y40G12A.1, Y40G12A.2
PINK1 EEED8.9
DJ-1 B0432.2, C49G7.11
Dardarin/LRRK2 lrk-1 (T27C10.7)
WHY C. elegans?
302
fully-mapped neurons
Adapted from Worm Base
WHY C. elegans?
8Dopaminergenic
Adapted from Worm Base
WHY C. elegans?
All models have been previously tested and
are proven to work
Adapted from Worm Base
Objective
Finding biomarkers by identifying differences in the quantities and
types of proteins in exosomes of C. elegans modelling Parkinson’s
disease.
Hypotheses
1. The types of proteins found in exosomes of organisms modelling Parkinson’s disease differ from the
control organisms
Hypotheses
2. The quantity of proteins found in exosomes of an organism modelling
Parkinson’s disease significantly differs from the control organisms
Apparatus
• Autoclave• Biological safety cabinet• Incubator• Centrifuge• Eppendorf Tubes• Dissecting
stereomicroscope equipped with a transmitted light source
• Scalpel • Cuvettes• Centrifuge tubes• Micropipettes• Pipettes• Petri dishes• Microtiter plates
Materials
• Enzyme-linked immunosorbent assay (ELISA) Kit• Ethanol• Sterile water• 6-OHDA• MPTP• M9 Buffer (3 g KH2PO4, 6
g Na2HPO4, 5 g NaCl, 1 ml 1 M MgSO4, H2O to 1 litre. Sterilize by autoclaving.)
• TAE Buffer:• 4.84 g Tris Base• 1.14 ml Glacial Acetic Acid• 2 ml 0.5M EDTA (pH 8.0)• LB agar:• Agar• Saline• Bacto-tryptone• Bacto-yeast
Materials
• NGM agar:• Agar• Saline• Peptone• 5 mg/ml cholesterol
in ethanol • 1 M KPO4 buffer pH
6.0 (108.3 g KH2PO4, 35.6 g K2HPO4, H2O to 1 litre)• 1M MgSO4
• Saline
• Wild type Caenorhabditis elegans
• Escherichia coli OP50• Plasmid construct
amplified from α-synuclein and human brain RNA (pRB454)
• Mutant human recombinant protein (A53T)
Variables
IndependentOrganism models
Parkinson’s disease
Dependent1. Types of proteins
found in exosomes2. Quantity of proteins
found in exosomes
Controlled 1. Quantity of food (E. coli)
2. Temperature3. Age of C.elegans
Methodology
Culturing of C. elegans
Enabling C. elegans to model
PD-Ensuring models
work
Isolation of exosomes
Quantifying A. synuclein
Characterising the types of
proteins found
Analysing the results
April AugustMay June July
Culturing of C. elegans and
testing of models
Preparing models, isolating
exosomes, characterising and
quantifying proteins
Analysis of results and
repeat tests if necessary
Consolidation of information with AOS side to draw
conclusions
FINALS
C. elegans Models
Model How it models PD
6-OHDA (hydroxydopamine) model
Degenerates dopaminergic neurons
MPTP model Degenerates dopaminergic neurons
Alpha synuclein model Degenerates dopaminergic neurons and causes aggregation of Lewy bodies
Preparation of E. coli stock culture
Standard procedure
Preparation of Nematode Growth Medium
Standard procedure
Preparation of MPTP/6-OHDA Models
Preparation of A. Syn Model
Heat Shock
Thrashing assay
Adapted from APS
Isolation of exosomes
Series of adding buffers and centrifuging
Adapted from SystemBio
Quantifying proteins
ELISABased on antibodies and antigens
Adapted from Innovative Research
Characterising proteins
Gel electrophoresis
Characterising proteins
Matrix-assisted laser desorption/ionization time of flight
(MALDI-tof)
OR
Analysis of results
For protein quantity, we will input the values in MiniTab to determine whether there is a significant change in quantity.
We will likely be using the Kruskal-Wallis K-Test and the ANOVA test with significance p value of 0.05
ReferencesMentalHelp. (Photograph). Retrieved from http://www.mentalhelp.net/images/root/depression1_id2374521.jpg
Hands [Web Photo]. Retrieved from http://storiesformuslimkids.files.wordpress.com/2014/01/hands.jpg
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Alvarez-Erviti, L., Seow, Y., Haifang, Y., Betts, C., Lakhal, S., & Wood, M. (2011). Delivery of siRNA to the mouse brain by systemic injection of targeted exosomes. Nature biotechnology, (29), 341-345. Retrieved from http://www.nature.com/nbt/journal/v29/n4/full/nbt.1807.html
Braungart, E., Gerlach, M., Riederer, P., Baumeister, R., & Hoener, M. (2004). Caenorhabditis elegans MPP+ model of parkinson’s disease for high-throughput drug screenings. Neurodegenerative diseases, 1, 175-183. Retrieved from http://www.karger.com/Article/Pdf/80983
ReferencesConcoran, C., Rani, S., O'Brien, K., Kelleher, F., Radomski, M., Crown, J., . . . Germano, S. (2011). Isolation of exosomes for subsequent mRNA, MicroRNA, and protein profiling. Methods molecular biology. Retrieved from http://www.ncbi.nlm.nih.gov/pubmed/21898221
Docherty, M., & Burn, D. (2010). Parkinson's disease dementia. Current neurology and neuroscience reports, 4(10), 292-298. Retrieved from http://link.springer.com/article/10.1007/s11910-010-0113-7
Dorsey, E., Constantinescu, R., Thompson, J., Biglan, K., Holloway , R., Kieburtz, K., . . . Marshall, F. (2007). Projected number of people with parkinson disease in the most populous nations, 2005 through 2030. Neurology, 68(5), 384-386. Retrieved from http://www.neurology.org/content/68/5/384
Fearnley, J., & Lees, A. (1991). Ageing and Parkinson's disease: Substantia nigra regional selectivity. Brain, 114(5), 2283-2301. Retrieved from http://brain.oxfordjournals.org/content/114/5/2283.short
Fevrier, B., Vilette, D., Archer, F., Loew, D., Faigle, W., Vidal, M., Laude, H., & Raposo, G. (2003). Cells release prions in association with exosomes. 101(26), 9683-9688. Retrieved from http://www.pnas.org/content/101/26/9683.full
Morimoto, R., & Nussbaum-Krammer, C. (2014). Caenorhabditis elegans as a model system for studying non-cellautonomous mechanisms in protein-misfolding diseases. Disease models and mechanisms, 7, 31-39. Retrieved from http://dmm.biologists.org/content/7/1/31.full.pdf+html
Oikawa, H., Sasaki, M., Ehara, S., & Tohyama, K. (2002). The substantia nigra in parkinson disease: Proton density-weighted spin-echo and fast short inversion time inversion-recovery mr findings. AJNR, 1(23), 1747-1756. Retrieved from http://www.ajnr.org/content/23/10/1747.full
Raposo, G., & Simons, M. (2009). Exosomes – vesicular carriers for intercellular communication. Current opinion in cell biology, (21), 575-581. Retrieved from http://users.ugent.be/~kraemdon/Exosomes-microvesicles/Simons%20and%20Raposo,%20Curr%20Opin.%20Cell%20Biol.,%202009_review%20exosomes.pdf
Thank you!
Culturing of C. elegans
Preparation of E. coli stock culture
1. 12.5g of Luria-Bertani (LB) broth powder measured using an electronic balance will be added to a 500cm3 blue-cap bottle.
2. 500cm3 of deionised water measured using a 500cm3 measuring cylinder and will be poured into the blue-cap bottle.
3. The LB broth will be autoclaved at a pressure of 15 pounds per square inch at 121°C for 2 hours.
4. The bottle will be cooled in 55°C water bath until further use.5. The LB broth will be poured into centrifuge tubes.6. Using sterile procedures, OP50 E. coli will be transferred into
the LB broth.7. The LB broth will be placed in a rotary shaker until further use.
Culturing of C. elegansPreparation of Nematode Growth Medium
1. 1.5g sodium chloride, 8.5g agar, 1.25g peptone was measured using an electronic balance into a 500ml blue-cap bottle.
2. 490ml deionised water measured using a 500cm3 measuring cylinder was added into the bottle.
3. The bottle was shaken until a homogeneous solution was obtained.4. The NGM was autoclaved at a pressure of 15 pounds per square
inch at 121°C for 2 hours.5. The bottle was cooled in a 55°C water bath until further use.6. Using a dropper, 1ml of 1M calcium chloride solution, 1ml 5mg/ml
cholesterol dissolved in ethanol, 1ml of 1M magnesium sulfate and 12.5ml of 1M potassium phosphate were added to the NGM.
7. Using sterile procedures, the NGM solution was dispensed into petri dishes.
8. The Petri dishes were left to set and solidify for about 30 minutes.9. A drop of E. coli OP50 culture (bacterial food source for C. elegans)
was transferred using a dropper and spread evenly at the center of the solidified NGM plates.
10. NGM was incubated at 36°C in an incubator for 1 day.
Preparation of models
Preparation of MPTP/6-OHDA Models1.Create 3 wells in each plate of the NGM plates that were
previously prepared
2.Micropipette 75 microlites of chemical into each well
3.Incubate overnight
Thrashing assay4.On the day of the assay, animals will be placed on to a 10-µL
drop of M9 buffer on a standard microscope slide and allowed to equilibrate for ~30 seconds.
5.Animals will be scored for the number of times the head crossed an axis drawn across the length of the body in 30 seconds
Preparation for Asyn. Model1. Put tubes with DNA and E. coli into water bath at 42 degrees
Celsius for 45 seconds.
2. Put tubes back on ice for 2 minutes to reduce damage done to E. coli.
3. Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37 degrees Celsius.
4. Spread about 100 microliters of resulting culture on LB plates (with Ampicillin added).
5. Grow overnight
6. Pick colonies about 12-16 hours later
Isolation of exosomes
Using the Exosome Isolation Kit1.Combine 500µl serum + 120 µl ExoQuick
2.Mix well by inversion three times
3.Place at 4ºC for 30 minutes (or up to 12 hours)
4.Centrifuge at 1500 × g for 30 minutes
5.Remove supernatant, keep exosome pellet
6.Centrifuge at 1500 × g for 5 minutes to remove all traces of fluid (take great care not to disturb the pellet)
7.Add 200 µl Exosome Binding buffer to exosome pellet and vortex 15 seconds
8.Incubate at 37 ºC temperature for 20 minutes to liberate exosome proteins
9.Centrifuge at 1500 × g for 5 minutes to remove all residual precipitation solution
10.Transfer supernatant to new centrifuge tube on ice
11.Exosome protein is now ready for immobilization onto micro-titer plate
Quantifying proteinsUsing the ELISA Kit
1. Pipette 100 µl of the Standard Diluent Buffer to the well(s) reserved for the standard blanks. Well(s) reserved for chromogen blank(s) should be left empty.
2. Pipette 100 µl of standards, controls, and diluted samples (typically >1:10 dilution for cell extract) to the appropriate microtiter wells. Tap gently on side of plate to thoroughly mix.
3. Cover wells with plate cover and incubate for 2 hours at room temperature.
4. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times.
5. Pipette 100 µl Streptavidin-conjugated HRP solution into each well except the chromogen blank(s). Tap gently on the side of the plate to mix.
6. Cover wells with plate cover and incubate for 1 hour at room temperature.
7. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times.
8. Pipette 100 µl streptavidin-HRP solution to each well except the chromogen blank(s).
9. Cover wells with the plate cover and incubate for 30 minutes at room temperature.
Quantifying proteinsUsing the ELISA Kit (cont.)
10. Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times.
11. Pipette 100 µl of Stabilized Chromogen to each well. The liquid in the wells will begin to turn blue.
12. Incubate for 30 minutes at room temperature and in the dark. Note: Do not cover the plate with aluminum foil or metalized mylar. The optical density (OD) values will be monitored and the substrate reaction stopped before the OD of the positive wells exceed the limits of the instrument. The OD values at 450 nm can only be read after the Stop Solution has been added to each well. If using a reader that records only to 3.0 OD, stopping the assay after 20 to 25 minutes is suggested.
13. Pipette 100 µl of Stop Solution to each well. Tap gently on the side of the plate to mix. The solution in the wells should change from blue to yellow.
14. Read the absorbance of each well at 450 nm having blanked the plate reader against a chromogen blank composed of 100 µl each of Stabilized Chromogen and Stop Solution. Read the plate within 2 hours after adding the Stop Solution.
15. Plot the absorbance of the standards against the standard concentration.
16. Multiply value(s) (protein concentration) obtained for sample(s) by the appropriate dilution factor to correct for the dilution with Standard Diluent Buffer.
Characterising proteinsRunning a gel electrophoresis
Add 6 l of 6X Sample Loading Buffer to each 25 l PCR reaction
• Record the order each sample will be loaded on the gel, including who prepared the sample, the DNA template - what organism the DNA came from, controls and ladder.
• Carefully pipette 20 l of each sample/Sample Loading Buffer mixture into separate wells in the gel and pipette 10 l of the DNA ladder standard into at least one well of each row on the gel.
• Connect the electrode wires to the power supply, making sure the positive (red) and negative (black) are correctly connected. (Remember – “Run to Red”)
• Turn on the power supply to about 100 volts. Maximum allowed voltage will vary depending on the size of the electrophoresis chamber – it should not exceed 5 volts/ cm between electrodes! .
• Check to make sure that the current is running in the correct direction by observing the movement of the blue loading dye – this will take a couple of minutes (it will run in the same direction as the DNA).
• Using gloves, carefully remove the tray and gel.
Characterising proteinsRunning a gel electrophoresis (cont.)
• Using gloves, remove the gel from the casting tray and place into the staining dish.
• Add warmed (50-55°) staining mix.
• Allow gel to stain for at least 25-30 minutes (the entire gel will become dark blue).
• Pour off the stain (the stain can be saved for future use).
• Rinse the gel and staining tray with water to remove residual stain.
• Fill the tray with warm tap water (50-55°). Change the water several times as it turns blue. Gradually the gel will become lighter, leaving only dark blue DNA bands. Destain completely overnight for best results.
• View the gel against a white light box or bright surface.
• Record the data while the gel is fresh, very light bands may be difficult to see with time.