1. Group 1-51
Identifying biomarkers in the exosomes of organisms modelling Parkinson’s disease
Chai Yi Xuen (Leader) 4S302
Abraham Sui 4S317
Nanki Kaur (AOS)
Aditi Narvekar (AOS)

2. Parkinson’s disease (PD)
Idiopathic neurodegenerative disease
Cause of the disease is not known. Some percentage of people with the condition cannot be charaterised.

3. Adapted from Memory Disorders Project at Rutgers University
Where?
Substantia nigra is important for eye movement, motor planning, reward-seeking, learning and addiction.
within the A9-A11 region of the brain (Oikawa et. al., 2002)
(Oikawa et. al., 2002)
Adapted from Memory Disorders Project at Rutgers University

4. Adapted from http://www.mpg.de/
Causes
Neuronal cell death
Aggregation of Lewy bodies
Overexpression or misfolding of the protein Alpha synuclein,
Lewy Bodies, abnormal aggregates of proteins that develop inside nerve cells.
Adapted from

5. Adapted from http://radicaislivres96.wordpress.com/
Effects
Loss of dopamine
Loss of dopamine (impairs motor functions)
Adapted from

6. 7-10 million reported cases worldwide
Based on Parkinson’s Disease Foundation

7. 2nd most common neurodegenerative disorder
After Alzhiemer’s Disease
Based on Parkinson’s Disease Foundation

9. Adapted from Parkinson’s Disease Foundation
Nonmotor Symptoms
Loss of sense of smell Sleep Disorder Mood Disorder
Adapted from Parkinson’s Disease Foundation

10. Adapted from Parkinson’s Disease Foundation
Motor Symptoms
Tremor Bradykinesia Rigidity
Bradykinesia means “slow movement”
Rigidity means stiffness and inflexibility of limbs
Adapted from Parkinson’s Disease Foundation

11. Cure
Causes are not known

12. Levodopa
Levodopa is a medicine that the peripheral nervous system converts to dopamine. It makes up for the lack of dopamine but does not cure the disease.

13. Current method of detection
SPECT scan (brain imaging)
$1100
Single-photon emission computed tomography (SPECT). How it works: Detects reduced dopaminergic activity which mirrors the loss of dopaminergic neurons

14. Problem Late Detection
People will only go for SPECT scan when they detect the symptoms of Parkinson’s Disease. By then, disease is already at an advanced stage
Unless one experiences motor deficits, few would fork out $1100 to test if they have Parkinson’s disease. Some people cannot even afford the fee.

15. Solution
Develop a cheap, non-invasive diagnostic kit for early detection of Parkinson’s disease

16. Doing away with expensive machinery
Solution
Develop a cheap, non-invasive diagnostic kit for early detection of Parkinson’s disease
Doing away with expensive machinery

17. Solution
Develop a cheap, non-invasive diagnostic kit for early detection of Parkinson’s disease
Does not require a spinal tap

18. Answer: Biomarkers

19. Our proposition: Exosomes

20. Exosomes
Nanoparticle-size vesicles secreted by all cells (Danzer, 2012)
Contains well-protected ‘cargo’ of RNA, miRNA and proteins (Rani et. al., 2011)

21. Theories of their purpose
Dispel waste
Cell to cell communication

22. “Trojan Horse” of neurodegeneration
Our Focus
“Trojan Horse” of neurodegeneration
(Fevrier et. al., 2003)
Facilitates transport of toxic proteins such as Alpha synuclein in brain

23. ‘Cargo’ is protected from degradation
WHY Exosomes?
‘Cargo’ is protected from degradation
(Simons et. al., 2009)

24. WHY Exosomes?
Pass through the Blood-Brain barrier (Seow et. al., 2011)
Allows detection in blood

25. Our model organism…

26. C. elegans
Cell biology mirrors that of humans and has the mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism through exosomes

27. of their genes are closely related to human genes
WHY C. elegans?
35%
of their genes are closely related to human genes
Cell biology mirrors that of humans and has the mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism through exosomes
Adapted from Worm Base

28. WHY C. elegans? 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)
Cell biology mirrors that of humans and has the mechanisms to transmit certain classes of toxic proteins between tissues and a complex stress response that integrates and coordinates signals from single cells and tissues across the organism through exosomes
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

29. WHY C. elegans?
302
fully-mapped neurons
Adapted from Worm Base

30. WHY C. elegans? 8
Dopaminergenic
Adapted from Worm Base

31. All models have been previously tested and are proven to work
WHY C. elegans?
All models have been previously tested and are proven to work
Allowed in school lab
Adapted from Worm Base

32. Objective
Finding biomarkers by identifying differences in the quantities and types of proteins in exosomes of C. elegans modelling Parkinson’s disease.

33. Hypotheses
1. The types of proteins found in exosomes of organisms modelling Parkinson’s disease differ from the control organisms

34. Hypotheses
2. The quantity of proteins found in exosomes of an organism modelling Parkinson’s disease significantly differs from the control organisms

35. 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

36. 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

37. 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
Wild type Caenorhabditis elegans
Escherichia coli OP50
Plasmid construct amplified from α-synuclein and human brain RNA (pRB454)
Mutant human recombinant protein (A53T)

38. Variables Independent Dependent Controlled
Organism models Parkinson’s disease
Dependent
Types of proteins found in exosomes
Quantity of proteins found in exosomes
Controlled
Quantity of food (E. coli)
Temperature
Age of C.elegans

39. Methodology Culturing of C. elegans Isolation of exosomes
Enabling C. elegans to model PD
-Ensuring models work
Isolation of exosomes
Analysing the results
Characterising the types of proteins found
Quantifying A. synuclein

40. Preparing models, isolating exosomes, characterising and quantifying proteins
Consolidation of information with AOS side to draw conclusions
August
April
May
June
July
Culturing of C. elegans and testing of models
Analysis of results and repeat tests if necessary
FINALS

41. C. elegans Models Model How it models PD
6-OHDA (hydroxydopamine) model
Degenerates dopaminergic neurons
MPTP model
Alpha synuclein model
Degenerates dopaminergic neurons and causes aggregation of Lewy bodies
Mention that the first 2 simulate Parkinson’s by causing dopaminergic neuronal death and that last model works by first causing dopaminergic neuronal death and also by potentially causing aggregation of Lewy bodies

42. Preparation of E. coli stock culture
Standard procedure

43. Preparation of Nematode Growth Medium
Standard procedure

44. Preparation of MPTP/6-OHDA Models
We will be adding the chemicals into the well so that it can diffuse into the agar.

45. Preparation of A. Syn Model
Heat Shock
Through the use of plasmids acquired from Finnish researchers. Bacterial transformation into E. coli/Microinjection of plasmids into C. elegans. Ampicillin Selection to determine successful transformation

47. Thrashing assay Adapted from APS
A thrashing assay will be conducted to ensure that the C. elegans display parkinsonism
Adapted from APS

48. Isolation of exosomes Series of adding buffers and centrifuging
Adapted from SystemBio

49. Adapted from Innovative Research
Quantifying proteins
ELISA Based on antibodies and antigens
Specific to group types. So comparison of wild type and mutant strain will be made.
Adapted from Innovative Research

50. Characterising proteins
Gel electrophoresis

51. Characterising proteinsOR
Matrix-assisted laser desorption/ionization time of flight (MALDI-tof)

52. 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

53. References
MentalHelp. (Photograph). Retrieved from Hands [Web Photo]. Retrieved from WorldWide [Web Photo]. Retrieved from content/uploads/2014/02/Beautifeye-Worldwide-Delivery-Shipping.gif Head Brain [Web Photo]. Retrieved from Stock/brain in head man vector.jpg Pill Bottle [Web Photo]. Retrieved from CKdqkazqYIM/Tc1t1qenniI/AAAAAAAAAOE/NC-iuQm2RJ8/s1600/pill bottle.jpg SPECT-CT [Web Photo]. Retrieved from 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), Retrieved from 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, Retrieved from

54. References
Concoran, 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 Docherty, M., & Burn, D. (2010). Parkinson's disease dementia. Current neurology and neuroscience reports, 4(10), Retrieved from 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 Neurology, 68(5), Retrieved from Fearnley, J., & Lees, A. (1991). Ageing and Parkinson's disease: Substantia nigra regional selectivity. Brain, 114(5), Retrieved from 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), Retrieved from 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, Retrieved from 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), Retrieved from Raposo, G., & Simons, M. (2009). Exosomes – vesicular carriers for intercellular communication. Current opinion in cell biology, (21), Retrieved from microvesicles/Simons%20and%20Raposo,%20Curr%20Opin.%20Cell%20Biol.,%202009_review%20exos omes.pdf

55. Thank you!

56. Culturing of C. elegans Preparation of E. coli stock culture
12.5g of Luria-Bertani (LB) broth powder measured using an electronic balance will be added to a 500cm3 blue-cap bottle.
500cm3 of deionised water measured using a 500cm3 measuring cylinder and will be poured into the blue-cap bottle.
The LB broth will be autoclaved at a pressure of 15 pounds per square inch at 121°C for 2 hours.
The bottle will be cooled in 55°C water bath until further use.
The LB broth will be poured into centrifuge tubes.
Using sterile procedures, OP50 E. coli will be transferred into the LB broth.
The LB broth will be placed in a rotary shaker until further use.

57. Culturing of C. elegans Preparation of Nematode Growth Medium
1.5g sodium chloride, 8.5g agar, 1.25g peptone was measured using an electronic balance into a 500ml blue-cap bottle.
490ml deionised water measured using a 500cm3 measuring cylinder was added into the bottle.
The bottle was shaken until a homogeneous solution was obtained.
The NGM was autoclaved at a pressure of 15 pounds per square inch at 121°C for 2 hours.
The bottle was cooled in a 55°C water bath until further use.
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.
Using sterile procedures, the NGM solution was dispensed into petri dishes.
The Petri dishes were left to set and solidify for about 30 minutes.
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.
NGM was incubated at 36°C in an incubator for 1 day.

58. Preparation of models Preparation of MPTP/6-OHDA Models
Create 3 wells in each plate of the NGM plates that were previously prepared
Micropipette 75 microlites of chemical into each well
Incubate overnight
Thrashing assay
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.
Animals will be scored for the number of times the head crossed an axis drawn across the length of the body in 30 seconds

59. Preparation for Asyn. Model
Put tubes with DNA and E. coli into water bath at 42 degrees Celsius for 45 seconds.
Put tubes back on ice for 2 minutes to reduce damage done to E. coli.
Add 1 ml of LB (with no antibiotic added). Incubate tubes for 1 hour at 37 degrees Celsius.
Spread about 100 microliters of resulting culture on LB plates (with Ampicillin added).
Grow overnight
Pick colonies about hours later

60. Isolation of exosomes Using the Exosome Isolation Kit
Combine 500µl serum µl ExoQuick
Mix well by inversion three times
Place at 4ºC for 30 minutes (or up to 12 hours)
Centrifuge at 1500 × g for 30 minutes
Remove supernatant, keep exosome pellet
Centrifuge at 1500 × g for 5 minutes to remove all traces of fluid (take great care not to disturb the pellet)
Add 200 µl Exosome Binding buffer to exosome pellet and vortex 15 seconds
Incubate at 37 ºC temperature for 20 minutes to liberate exosome proteins
Centrifuge at 1500 × g for 5 minutes to remove all residual precipitation solution
Transfer supernatant to new centrifuge tube on ice
Exosome protein is now ready for immobilization onto micro-titer plate

61. Quantifying proteins Using the ELISA Kit
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.
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.
Cover wells with plate cover and incubate for 2 hours at room temperature.
Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times.
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.
Cover wells with plate cover and incubate for 1 hour at room temperature.
Pipette 100 µl streptavidin-HRP solution to each well except the chromogen blank(s).
Cover wells with the plate cover and incubate for 30 minutes at room temperature.

62. Quantifying proteins Using the ELISA Kit (cont.)
Thoroughly aspirate or decant solution from wells and discard the liquid. Wash wells 4 times.
Pipette 100 µl of Stabilized Chromogen to each well. The liquid in the wells will begin to turn blue.
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.
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.
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.
Plot the absorbance of the standards against the standard concentration.
Multiply value(s) (protein concentration) obtained for sample(s) by the appropriate dilution factor to correct for the dilution with Standard Diluent Buffer.

63. Characterising proteins
Running 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.

64. Characterising proteins
Running 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 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.