Quantitative comparison of pesticides and genes known to cause dopaminergic neuronal damage: Finding the optimal Drosophila melanogaster model for Parkinson’s disease (PD)

YPIE Regeneron Science Research 2024

Presented by Amy Kim

Introduction to Parkinson’s Disease

Definition

Neurodegenerative disease that affects movement, strength, and causes pain

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• Has caused 329,000 deaths since 2000 (15)

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• Incidence doubled since 2000 (15)

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• Incidence predicted to double again in the next 15 years (13)

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• Over 8.5 million individuals diagnosed in 2019 globally (15)

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• Second most common neurodegenerative disease in the United States (13)

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PD at the Cellular Level

Dark neurons of the substantia nigra (SN)

Dopamine neurotransmitter is produced

Healthy positive movement, sleep, motivation, attention, arousal, mood, etc.

Dopaminergic pathway in non-PD humans (5)

Dark neurons of the SN die (unknown cause)

Dopamine no longer produced efficiently

Physical and emotional symptoms of PD present themselves

Dopaminergic pathway in PD humans (1, 5)

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Review of Literature

Disease model organism for Parkinson disease: Drosophila melanogaster (4)

Brown, T. P., Rumsby, P. C., Capleton, A. C., Rushton, L., & Levy, L. S. (2006).

• Associated risk factors named: 20 genes, 3 pesticides
• Of the 20, 8 genes associated with causing dopaminergic neuronal loss in Drosophila melanogaster

Pesticides and Parkinson's disease--is there a link? (2)

Aryal, B., & Lee, Y. (2019).

• Correlation exists between pesticide exposure and PD
• No studies were found to independently associate pesticide exposure with PD
• Rotenone, paraquat, MPTP, and maneb are highly correlated with PD

Problem

Purpose

Hypothesis

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Testing is constantly being done in Drosophila to learn about PD, but models are highly inconsistent and possibly ineffective across studies. As a result, there is no standardization of experimentation and results are often incomparable.

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To find and establish the optimal genetic and environmental Drosophila melanogaster model of PD

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Drosophila exposed to the pesticides, most specifically MPTP, will experience forms of neurodegeneration most closely associated with PD.

Intended Methods

Prototype

11 groups of Drosophila melanogaster

Genetic experimental:

PARK1, PARK2, PARK5, PARK6, PARK8, PARK15

Environmental experimental:

Rotenone(ROT), paraquat (PQ), MPTP, maneb

In Vivo Testing

• Negative geotaxis (forced-climbing locomotion to observe motor ability)
• Courting and mating assay (to observe sensory and sexual ability)

In Vitro Testing

• Dissection, immunofluorescent staining, and microscopy (to analyze presence of dopaminergic neurons)

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Methods of Dissection (12)

• Anesthetize flies with CO₂
• Dewax cuticle
• Use dissecting microscope at 1.2X
• Follow steps A, B, C, and D

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Methods of Staining (12)

Day 1

1. Nutator (Fig. 1)
2. Rinse and wash samples
3. Incubate with primary antibodies and leave overnight

Day 2

1. Remove antibodies
2. Wash samples
3. Incubate with secondary antibodies
4. Wash and incubate samples
5. Incubate for 30 min in DNA dye
6. Rinse 3x
7. Dissection dish → separate brain

Figure 1. Nutator (7)

Methods of Microscopy (12)

1. Place samples in between coverslips
2. Remove excess liquid with fine pipette tip
3. Compound fluorescent microscope

Figure 2. Compound fluorescent microscope (8)

Example of what should be seen at 20X magnification (12)

Expected Results: Negative Geotaxis

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MPTP has the lowest average height climbed

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MPTP has the lowest % of climbers

Expected Results: Observed data

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Control will look most like image #1.

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Most gene groups will look like images #2 or #3. PARK1 and PARK2 will look most like image #3.

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Most pesticide groups will look like images #3 or #4. MPTP will look most like image #4.

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Interpretation:

PARK1 and PARK2 are the most effective gene groups. MPTP is the most effective overall. Generally, pesticide groups are more effective than gene groups.

Expected Results: Cellular data

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Pesticide groups have less TH+ neurons than gene groups and do not follow typical TH+ neuron pattern.

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MPTP overall has the least TH+ neurons left.

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PARK1 and PARK2 are the gene groups with the least TH+ neurons left.

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Interpretation:

Pesticide groups overall have most adverse and atypical effects. PARK1 and PARK2 have the most adverse effects of the gene groups. MPTP has the most adverse effects overall.

Cellular data: Dopaminergic clusters

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Significance of Results

MPTP Pesticide causes significant dopaminergic neuronal damage relative to other pesticides and genes associated with causing PD.

The MPTP pesticide group is the most accurate and effective group to model PD in Drosophila and should be used in future PD experiments on Drosophila.

• Standardize experimentalization to standardize results, thus increasing reliability and comparability in conclusions

Best genetic models of PD in Drosophila found: PARK1 and PARK2

Pesticide groups overall are much more reliable and accurate PD models for Drosophila and should be analyzed further in this realm.

Limitations

Future Research

1. Did not test all pesticides and genes associated with PD
2. Sample sizes were small due to budget limitations
3. Equipment was old, faulty, and had larger ranges of error
4. Did not count all dopaminergic neuron clusters in cellular results

• Repeat testing to reaffirm results, perhaps with additional experimental groups
• Repeated experiment in mice (changing gene groups as necessary)
• Give Drosophila under MPTP exposure different treatments to learn to better treat PD in humans

Conclusion and Summary

Results support my hypothesis.

• MPTP caused the most significant cellular and behavioral changes similar to that of PD compared to all other groups.
• Pesticide groups on average caused the most significant cellular and behavioral changes similar to that of PD compared to all gene groups.

Impact of conclusions are significant

• Drosophila PD experiments can now be standardized with the MPTP model so that their results and conclusions are comparable amongst different studies.

References

1. American Parkinson Disease Association. (2024, October 15). Symptoms of parkinson’s: APDA. American Parkinson Disease Association. https://www.apdaparkinson.org/what-is-parkinsons/symptoms/

2. Aryal, B., & Lee, Y. (2019). Disease model organism for Parkinson disease: Drosophila melanogaster. BMB reports, 52(4). https://doi.org/10.5483/BMBRep.2019.52.4.204

3. Blausen.com staff. (29 August 2014). Medical gallery of Blausen Medical 2014. WikiJournal of Medicine 1(2). https://doi.org/10.15347/WJM/2014.010.

4. Brown, T. P., Rumsby, P. C., Capleton, A. C., Rushton, L., & Levy, L. S. (2006). Pesticides and Parkinson's disease--is there a link?. Environmental health perspectives, 114(2), 156–164. https://doi.org/10.1289/ehp.8095

5. Cleveland Clinic. (2025, March 19). Dopamine: What it is, Function & Symptoms. Cleveland Clinic. https://my.clevelandclinic.org/health/articles/22581-dopamine

6. Devi, D., Biswas, S. K., & Purkayastha, B. (2021). Early Detection of Parkinson’s Disease: An Intelligent Diagnostic Approach. Research Anthology on Diagnosing and Treating Neurocognitive Disorders, 295–328. https://doi.org/10.4018/978-1-7998-3441-0.ch016

7. LabRepCo. 3D Nutator Mixer NS-01A. Crystal Technology & Industries. https://www.labrepco.com/product/3d-nutating-mixer-ns-01a/

8. Microscope.com. OMFL400 Fluorescence Compound Microscope. Omsno https://www.microscope.com/omano-omfl400-fluorescence-compound-microscope.html

9. Nagoshi, E. (2018). Drosophila Models of Sporadic Parkinson’s Disease. International Journal of Molecular Sciences, 19(11), 3343. https://doi.org/10.3390/ijms19113343

10. Philyaw, T. J., Rothenfluh, A., & Titos, I. (2022). The Use of Drosophila to Understand Psychostimulant Responses. Biomedicines, 10(1), 119. https://doi.org/10.3390/biomedicines10010119

11. The Northwest Parkinson’s Foundation. (2024, December 19). Overview of parkinson’s - NW parkinson’s foundation. NW Parkinson’s Foundation - Partnering With All People Impacted by Parkinson’s. https://nwpf.org/parkinsons-info/pd-overview/

12. Tito, A. J., Cheema, S., Jiang, M., & Zhang, S. (2016). A Simple One-step Dissection Protocol for Whole-mount Preparation of Adult Drosophila Brains. Journal of visualized experiments : JoVE, (118), 55128. https://doi.org/10.3791/55128

13. U.S. Department of Health and Human Services. (2025, February 25). Parkinson’s disease: Challenges, progress, and promise. National Institute of Neurological Disorders and Stroke. https://www.ninds.nih.gov/current-research/focus-disorders/parkinsons-disease-research/parkinsons-disease-challenges-progress-and-promise

14. Warner Gargano, J., Martin, I., Bhandari, P., & Grotewiel, M. S. (2005). Rapid iterative negative geotaxis (RING): a new method for assessing age-related locomotor decline in Drosophila. Experimental Gerontology, 40(5), 386–395. https://doi.org/https://doi.org/10.1016/j.exger.2005.02.005.

15. World Health Organization. (2023, August 9). Parkinson disease. World Health Organization. https://www.who.int/news-room/fact-sheets/detail/parkinson-disease

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