The significantly increase their motility to cross

The
effects of Ascorbic Acid on motility of Drosophila flies that have Parkinson
Disease’s symptoms

NEU311L Section 004

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Instructors: Dr. Jennifer Taylor, Dr. Valerie Hedges
and Grant Kunzelman

Introduction

Parkinson’s disease is a common neurogenerative
disorder that affects up to 1 million people in the U.S. It
typically affects motor control movements and shows symptoms such as, facial expression
being immobile and rigid, speech slur, tremor of hands, fatigue, stiffness,
swallowing difficulties etc. The motor control defects occur
primarily due to loss of dopaminergic neurons in the substantia nigra of the
brain.

To test the effects of Ascorbic Acid, Drosophila flies
(of unknown sex) were considered the best subjects due to their similarity in
genes with humans. Parkinson Disease like symptoms are induced in the flies by
using the chemical Rotenone, a substance naturally found in South America and
Southeast Asia. Rotenone is supposed to be a natural electron transport inhibitor
thus, leading to electron transport defects which in turn cause Parkinson Disease
like symptoms.

Ascorbic Acid is found naturally in fruits and vegetables.
Another form of it is Vitamin C which is considered a good antioxidant and thus
this antioxidant feature is supposed to help in preventing Parkinson Disease
like symptoms. Also, the solubility nature of Ascorbic Acid in water makes it
convenient to be used in our experiment.

Our hypothesis is that exposing Drosophila flies to
high concentration of Ascorbic Acid will significantly increase their motility
to cross the threshold when compared to the flies that were exposed to only
Rotenone.

Materials
and Methods

The flies were bred in house at Michigan State
University. They were fed 62.5 µM of Rotenone and 45.42×10^-5 M of Ascorbic
Acid (Lot number: SLBL9227V) that was manufactured by Sigma-aldrich Corporation
located in St.Louis, MD.

There were 4 different conditions to be tested for the
flies. The first condition was Control, which meant that the flies were exposed
to neither compounds. The second condition was to expose the flies to only
Rotenone; the third condition had the flies exposed to both Rotenone and
Ascorbic Acid together and the fourth condition consisted of exposing the flies
to only Ascorbic Acid.

Ascorbic Acid was made by weighing out 44.03mg of the
same and dissolving it into 50ml of water. For the Rotenone, 0.005g of Rotenone
was used and dissolved in 200µl of DMSO. Then 10ml of water was added to both
the Control and Rotenone vials. Similarly, 10ml of water was added to the Ascorbic
Acid vial and Rotenone + Ascorbic Acid vial. 10µL of Rotenone was added to the
Rotenone and Rotenone + Ascorbic Acid vial. Food flakes were then added to these
vials. Then the vials were exposed CO2 to anesthetize. The flies
were then kept in these vials for 7 days, to be later conducted the trials upon
them.

The trials consisted of the containers with the flies
being tapped so that all the flies came to the bottom of the container and started
from the same place. Then the flies had 10 seconds to cross a certain threshold
of 8cm. After the first 3 trials of each condition were conducted, there was no
significance difference observed according to the statistical analysis done. The
flies were given an hour of rest, and then additional trials were conducted. To
test the results of our hypothesis, a one way between subjects ANOVA
statistical analysis was conducted, followed by an LSD post-hoc analysis.

Results

The flies were exposed to four different conditions: the
first condition was Control where the flies were exposed to neither compound;
the second condition consisted of the flies being exposed to only Rotenone; the
third condition had the flies exposed to both Rotenone and Ascorbic Acid and the
last condition had the flies exposed to Ascorbic Acid only. The
aim of our experiment was to test the effects of Rotenone and Ascorbic Acid on
Drosophila flies and compare it with the flies exposed to only Rotenone.  Likewise, the results showed that the flies
exposed to Rotenone had the least percentage of flies crossing the threshold whereas
the flies with the condition of both Rotenone and Ascorbic Acid had a
significant improvement in motility. The data presented a significant
difference as shown below:

 

Figure 1

Figure
1: The
effects of Ascorbic Acid on motility in drosophila exposed to rotenone. A
one-way Anova test revealed that there was a significant main effect of food
condition in motility (f (3) = 7.329, p<0.05).  LSD post hoc tests indicated that significantly fewer flies crossed the threshold in the rotenone condition compared to the Ascorbic Acid + Rotenone condition (*p<0.05). Compared to flies exposed to rotenone alone, motility was significantly improved in Rotenone + Ascorbic Acid (*p<0.05).  Discussion The results showed that the effects of Ascorbic Acid with Rotenone had significant effect on motility of the flies thus, confirming that Ascorbic Acid plays a positive role in delaying the effect of Parkinson Disease's motility. Like every research, there were a variety of limitations to our study. Not all our vials had the same number of flies, the Ascorbic Acid was only used in one concentration thus, we don't know the significance of various concentrations of Ascorbic Acid. Also, any foreign substance when exposed to Drosophila will have at least some type of effect on it, depending on the substance. After conducting our first 3 trials, the flies had an hour rest before we conducted the additional 3 trials as no significance effect was seen prior to the additional trials. Parkinson's Disease affects one in every 100 people, above the age of 60 with affecting at least one million people in the U.S. and about five million worldwide. People belonging to any ethnic regions have an equal chance of getting the disease; across genders, men are more prone to the disorder. Till this day, there is no cure available for Parkinson's Disease. There are several treatments available, but our experiment helps in taking us one step closer to better treatment options and possibly a cure.                               References: 1.         Anon (n.d.) Statistics of Parkinson's Disease (PD). Florida Hospital Available at: https://www.floridahospital.com/parkinsons-disease-pd/statistics-parkinsons-disease-pd Accessed December 8, 2017. 2.         Anon (n.d.) Parkinson's Disease Causes. The Michael J Fox Foundation for Parkinson's Research | Parkinson's Disease Available at: https://www.michaeljfox.org/understanding-parkinsons/living-with-pd/topic.php?causes&navid=causes Accessed December 8, 2017. 3.         Chagraoui A., Boukhzara L., Thibaut F., Anouar Y., Maltête D. (2017) The pathophysiological mechanisms of motivational deficits in Parkinson's disease. 4.         Dickson D. (2017), Neuropathology of Parkinson disease Parkinsonism and Related Disorders 46 (2018) S30-33 5.         National Center for Biotechnology Information. PubChem Compound Database; CID=54670067, https://pubchem.ncbi.nlm.nih.gov/compound/54670067 (accessed Dec 8, 2017). 6.         National Institute of Neurological Disorders and Stroke National Institutes of Health, Hope Through Research. Available: https://permanent.access.gpo.gov/gpo71661/parkinsons-disease-brochure.pdf. Accessed: December 8, 2017. 7.         Ott K Rotenone. A Brief Review of its Chemistry, Environmental Fate, and the Toxicity of Rotenone Formulations. Available: http://www.newmexicotu.org/Rotenone%20summary.pdf Accessed December 8, 2017. 8.         Safiya Khan, Smita Jyoti, Falaq Naz, Barkha Shakya, Rahul, Mohammad Afzal & Yasir Hasan Siddique (2012) Effect of L-Ascorbic Acid on the Climbing Ability and Protein Levels in the Brain of Drosophila Model of Parkinson's Disease, International Journal of Neuroscience, 122:12, 704-709, DOI: 10.3109/00207454.2012.709893 9.         Samii A., Nutt j., Ransom B. (2004) Parkinson's Disease 363: 1783-93.   10.    Service E (n.d.) The Wonderful Fruitfly. The Fruit Fly and Genetics Available at: http://www.unc.edu/depts/our/hhmi/hhmi-ft_learning_modules/fruitflymodule/ Accessed December 8, 2017. 11.    William Dauer, Serge Przedborski, Parkinson's Disease: Mechanisms and Models, In Neuron, Volume 39, Issue 6, 2003, Pages 889-909, ISSN 0896-6273, https://doi.org/10.1016/S0896-6273(03)00568-3. (http://www.sciencedirect.com/science/article/pii/S0896627303005683)