TEMPLATE DESIGN © 2008

www.PosterPresentations.com

The Effects of Changing Circadian Rhythm on Drosophilia With Parkinson’s Tam Thanitcul & Henry Sanford

Background

Parkinson’s disease is a neurodegenerative disease characterized by motor symptoms such as resting tremor and postural instability, and pathologically by the degeneration of multiple neuronal types, including, dopaminergic (DA) neurons in the substantia nigra (1). Since there is a striking similarity of deep homology of human’s basal ganglia and the arthropod’s central complex, we hope to study the behavioral effects of the disease by conducting experiments on Drosophila melanogaster. S-methyl-L-cysteine (SMLC) is a substance found abundantly in garlic, cabbage, and turnips. It has been shown that dietary supplementation with SMLC inhibits the locomotor and circadian rhythm defects caused by ectopic expression of human α-Synuclein in Drosophila (5). Circadian rhythms are genetically determined biological oscillations with a period of close to 24 hours. They are present in the physiology and behavior of most organisms. In Drosophila, since the circadian rhythm controls the dopamine receptor responsiveness, the disruption of circadian rhythms would hypothetically lead to the progression of PD. These observable PD-like symptoms include negative geotaxis, reduction of licking activity, defective climbing activity. The PD-expressed that has been fed with SMLC group will behave in a fashion closer to that of the control group than the PD-expressed group. The flies that have PD expressing genes require a Gal4 cross for the pathogenesis of PD to occur. The crossing will be very similar to the Gal4 UAS Channelrhodopsin crosses that have done in the past. When the crosses have taken place, the PD genes will be expressed in the flies brain, specifically in the dopaminergic terminals in the β-lobes. As of now it is believed that the effect will be felt throughout the flies brain, similar to the area that a human PD patient experiences. The buildup of Alpha-Synuclein and subsequently Lewy Bodies will cause cell death. At this stage the symptoms of PD should be observable in the flies.

Methods

Materials

●  Fish tank ●  Tin foil ●  Arduino microcontroller ●  breadboard ●  1 potentiometer ●  1 green LED ●  3 white LEDs ●  wires ●  duct tapes ●  electrical tapes ●  220 ohms resistor ●  Around 5 vials with food ●  Strains of Drosophila: ●  P{UAS-Hsap\SNCA.A30P}: FBti0040566 ●  P{GAL4-elav.L}2/CyO: FBst0008765 ●  Oregon-R (wild type) : FBab0002009 ●  S-methyl-L-cysteine (SMLC): Solution of SMLC will

be mixed with food to a final concentration of 10 mM

Results

Graphs

Conclusion & Discussion

References

By doing the Chi Square analysis of the data, we conclude that the null hypothesis is false. Therefore the initial hypothesis is valid. The data indicates that inverting a PD expressed fly’s circadian rhythm leads to an earlier onset of PD symptoms, or at least a lesser ability ability to climb, compared to several control groups. While collecting the data, there were several factors that could affect the outcome we have reported. The fly strains we have cultivated and used are assumed to have been bred correctly and all the aspects that we expect them to express (ie. PD symptoms) were expressed. Several other factors also could contribute to these observations. The first is that the vial in which the flies are being tested in is different than their normal vials. The testing vial has no food, no dead flies, and no larvae. It is observed that in vials that have food, larvae, and dead flies the mature flies tend to be closer to the top of the vial. The difference in the contents of the vial could contribute to different behavioral habits. The second aspect that could hinder the OregonR flies from climbing is the anesthetization that they go through when they are transferred from their food vial to the testing vial. The time between anesthetization and testing has not been recorded, but it would seem logical that the closer the anesthetization and testing are, the less active the flies will be. The third thing that could affect the results is the “tapping” method that is used to get all the flies to the bottom of the vial before they are timed. The sudden impact of the vial on the counter could stun the flies or condition them to stay at the bottom of the vial. Further observations are needed to identify all the aspects that contribute to the behavior of flies when they are placed into the testing chamber.

1 "What Have We Learned from Drosophila Models of Parkinson's Disease?"National Center for Biotechnology Information. U.S. National Library of Medicine, 2010. Web. 28 Feb. 2015. 2 "Circadian Rhythms Fact Sheet." National Institute of General Medical Sciences. National Institute of Health, Nov. 2012. Web. 19 Feb. 2015. 3 Andretic, R. "Circadian Modulation of Dopamine Receptor Responsiveness in Drosophila Melanogaster." Proceedings of the National Academy of Sciences 97.4 (2000): 1873-878. Web. 4 Paricio, Nuria. "Drosophila Models of Parkinson's Disease: Discovering Relevant Pathways and Novel Therapeutic Strategies." Drosophila Models of Parkinson's Disease: Discovering Relevant Pathways and Novel Therapeutic Strategies. Hindawi, 7 Jan. 2011. Web. 28 Feb. 2015. 5 "Methionine Sulfoxide Reductase A and a Dietary Supplement S-methyl-L-cysteine Prevent Parkinson's-like Symptoms." National Center for Biotechnology Information. U.S. National Library of Medicine, 21 Nov. 2007. Web. 28 Feb. 2015.

We have taken a rectangular fish tank and covered it in one layer of newspaper and one layer of tinfoil. We have wired and programed a system of LED lights that light on for 12 hours and off for 12 hours. Daily observation of the flies and their behaviors will be recorded. The climbing ability of the flies will be the best indicator of a flies condition. Six groups of flies will be used to test our hypothesis. Set 1: Inverted Circadian Rhythm

Group 1: PD expressed, Gal4 crossed, SMLC fed Group 2: PD expressed, Gal4 crossed Group 3: Oregon-R (Control Group)

Set 2: Normal Circadian Rhythms (Regular Light) Group 1: PD expressed, Gal4 crossed, SMLC fed Group 2: PD expressed, Gal4 crossed Group 3: Oregon-R (Control Group)

After the Set 1 flies have become acclimated to their new circadian rhythms, flies from individual vials will be tested in a climbing test vial to quantify their physical and motor health. Data from these tests will be collected and measured over time.

Figure 1: Parkinson’s disease is a hypokinetic disorder that is the result of basal ganglia dysfunction. Patients with Parkinson’s disease exhibit several key signs of motor dysfunction. The motor symptoms result from the death of dopamine generating cells in the Substantia nigra, a region of the midbrain. Figure 2: Cell death may be caused by α-synuclein aggregation and reduced mitochondrial activity. Gene mutations are associated with this mechanism. Figure 3: If functioning correctly, the Substantia nigra has the responsibility of facilitating intended movement and inhibiting unintended movement. It does so by exciting the direct pathway and inhibiting the indirect pathway through the release of the neurotransmitter dopamine. Striatal neurons of the direct pathway have D1 receptors which are excited by dopamine, whereas Striatal neurons of the indirect pathway have D2 receptors which are inhibited by dopamine. This death of neurons in the Substantia nigra means that they can no longer release dopamine and excite the Striatal neurons in the direct pathway and inhibit the Striatal neurons in the indirect pathway. The excitatory and inhibitory pathways in different regions of the brain associated with Parkinson’s disease are also illustrated in the figure above.

Figures: The Circadian Rhythm Inverter is made from a fish tank covered with newspaper and tin foil. I use Arduino circuit board to control the on-off cycle of the LEDs inside.

Figure: The vial above is the vial we use to do the climbing assay. I use the smooth-surfaced cotton so that the “ceiling” for the flies to climb up to would be flat. I also marked the 4-cm line and 8-cm line. When doing the experiment, ten flies are put in the vial. In 30 seconds, we count the number of flies that have crossed the 4-cm line.

Day PD-GAL4 flies PD-GAL4 flies fed with SMLC

Oregon-R flies

Day 1 3 % 60 % 23 % Day 2 60 % 37 % 20 % Day 3 30 % 52 % 30 % Day 4 20 % 17 % 66 % Day 5 26 % 22 % 3 % Day 6 46 % - 80 %

Quantification of the climbing ability of our Inverted Circadian Rhythm flies over a time period of 6 days. Table 1

Quantification of the climbing ability of our normal Circadian Rhythm Flies over a time period of 6 days. Table 2

Day PD-GAL4 flies PD-GAL4 flies fed with SMLC

Oregon-R flies

Day 1 23 % 63 % 40 % Day 2 3 % 10 % 63 % Day 3 46 % 15 % 40 % Day 4 33 % 79 % 53 % Day 5 20 % 48 % 53 % Day 6 20 % - 37 %

Graphs of the quantified climbing ability data

Hypothesis Hypothesis/Alternative Hypothesis: Part 1: By inverting the circadian rhythms in flies susceptible to Parkinsons disease, it is hypothesized that early onset behavioral disorders will occur. Part 2: By treating the flies food with SMLC, the early onset of Parkinsonism with be slowed or halted. Null Hypothesis: Part 1: Early onset behavioral disorders will not occur if the circadian rhythms are inverted. Part 2: By treating the flies food with SMLC, the early onset of Parkinsonism will not be stopped.