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

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SPECT-CT [Web Photo]. Retrieved from http://www.whatisnuclearmedicine.com/upload/SPECT-CT.jpg

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.