Research
iPLA2-6A Knockout Gene Partially Rescued Using UAS-iPLA2-6A Transgene in the Muscle Cells of Drosophila melanogaster
By: Yosef Scher
Abstract
Muscle cells play a critical role in the locomotor ability of an organism. While neurons send electrical signals via neurotransmitters to an organism’s muscle cells to tell that muscle to move, it is ultimately the muscle cells themselves that conduct the movement through contraction and relaxation. Since many neurodegenerative diseases, such as Parkinson’s disease, present with locomotor problems, muscle cells were specifically scrutinized. More specifically, iPLA2-6A functionality in muscle cells of Drosophila melanogaster to maintain aging health was investigated. In order to determine this, the wild-type allele was placed in mutant fly groups through the transgene, UAS-iPLA2-6A, to ascertain if the wild-type allele was sufficient to rescue motor decline in knockout iPLA2-6A flies. Climbing assays were performed to test if locomotor ability in the flies improved with the wild-type allele present. The 15-day climbing assay results suggested that there was a partial rescue of the iPLA2-6A gene in the mutant fly groups. However, this conclusion could not be definitively made, as p = 0.084 was slightly higher than an acceptable p-value. Unlike the 15-day climbing assay data, the 20-day climbing assay results revealed that partial rescue of the iPLA2-6A gene in the mutant fly groups was accomplished. The results were statistically significant, as p = 0.020. A comparison of the 15 and 20-day climbing assay results displayed a decrease in climbing index scores for both the control and mutant fly groups. That being said, the control fly group had a more significant reduction in climbing index scores than the mutant fly groups as the flies aged. Thus, partial rescue of the iPLA2-6A gene in the mutant fly groups was more apparent with time.
Introduction
Neurodegenerative diseases are a classification of diseases characterized by progressive dysfunction of neurons, synapses, glial cells, and their networks. 1 Although there are over a hundred documented neurodegenerative diseases known to date, including Alzheimer’s disease, Huntington’s disease, and amyotrophic lateral sclerosis (ALS), Parkinson’s disease has become the most prevalent and fastest-growing neurodegenerative disease in the world. 2, 3 From 1990 to 2015, 6.2 million people were diagnosed with Parkinson’s disease––a 118% increase in global Parkinson’s disease cases. 4 As global populations continue to age, the primary factor that has been proven to be a cause of neurodegenerative disease, Parkinson’s cases are predicted to increase exponentially. 2, 5 By 2040, researchers predict that the number of Parkinson’s cases will double to 12 million documented cases. 2
Even though most Parkinson’s cases are sporadic, believed to be caused by a combination of genetic and environmental factors, the less common inherited familial parkinsonism has allowed researchers to identify the various loci attributed to Parkinson’s disease when the loci are perturbed. 6 By gaining a more advanced understanding of the underlying molecular and cellular mechanisms that lead to the rarer inherited familial parkinsonism, scientists hope to apply their findings to the more frequent sporadic cases of Parkinson’s. In order to investigate human familial parkinsonism, orthologous genes to human familial parkinsonism loci were mutated in Drosophila melanogaster. One such orthologous gene, iPLA2-6A, has been shown to be a disease locus for various neurodegenerative diseases, including a rare inherited familial parkinsonism called autosomal recessive dystonia-parkinsonism. 7
By examining the molecular effects of the iPLA2-6A mutation, researchers have garnered a better etiological understanding of Parkinson’s disease. In a recent journal article by Mori et al., two novel conclusions revealed how the iPLA2-6A mutation affected molecular mechanisms in organisms. 8 First, Mori et al. discovered that a lack of iPLA2-6A activity resulted in the shortening of phospholipid acyl chains. 8 This led to endoplasmic reticulum stress that affected neuronal activity in the flies. Second, the researchers found that 𝛼-Synuclein, a presynaptic neuronal protein that is linked neuropathologically and genetically to Parkinson’s disease, is aided by phospholipids with shorter acyl chains. 8, 9 With these two pieces of information, Mori et al. proposed a hypothesis as to how neurodegeneration occurs in flies with the loss of the iPLA2-6A gene and affects their brains at the molecular level (Figure 1). When the iPLA2-6A gene was removed, phospholipid acyl chains in the brain were shortened. As a result, the shortened phospholipid acyl chains had altered curvature, membrane fluidity, and lipid packing abilities. One of the effects of the altered phospholipid properties was 𝛼-Synuclein aggregation, which was caused by 𝛼-Synuclein’s decreased ability to bind to the synaptic membrane effectively. Additional effects of the altered phospholipid properties were stress placed onto the endoplasmic reticulum and abnormal functionality of neurotransmitters.
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Figure 1. Mori et al. Hypothesis of the Molecular Effects of the iPLA2-6A Gene Removal. 8
When the iPLA2-6A gene was removed, phospholipid acyl chains in the brain were shortened. This caused the shortened phospholipid acyl chains to have altered curvature, membrane fluidity, and lipid packing abilities. One of the effects of the altered phospholipid properties was 𝛼-Synuclein aggregation, which was caused by 𝛼-Synuclein’s decreased ability to bind to the synaptic membrane effectively.
Previous research investigating the effects of knocking out the iPLA2-6A gene has demonstrated that neurodegeneration occurs in flies and that rescue of the iPLA2-6A gene in tissue-specific cells is possible. In 2021, Steinhauer et al. published a paper that illustrated that Drosophila melanogaster null iPLA2-6A mutants exhibited a progressive loss of locomotor ability with age, consistent with neurodegeneration. 10 Using an RNAi knockdown in specific neuronal subsets and muscle cells, the locomotor defect was phenocopied. Control and mutant flies performed standard climbing assays to test for locomotor defects at fifteen and twenty days after eclosion. It was found that certain subsets of cells, such as muscle and pan-neuronal cells, were rescued with the DJ667-GAL4 and elav-GAL4 phenocopies. A critical takeaway from the research was that iPLA2-6A is required in muscles and neurons in order for the flies to maintain normal locomotor skills as they age. 10
While extensive research has been conducted on the effects of removing the iPLA2-6A gene from specialized cells, such as neurons and muscle cells, no known research has been conducted to investigate in which specific subset of cells iPLA2-6A functions to maintain aging health. To elucidate an answer, wild-type iPLA2-6A expression in neurons, specific subsets of neurons, and muscle cells were used to examine if the wild-type allele was sufficient to rescue motor decline seen in PLA2G6 Associated Neurodegeneration (PLAN) disorders, such as autosomal recessive dystonia-parkinsonism.
Methods
Fly Crosses
Fly strains were provided by outside researchers unbeknownst to the experimenter. In order to establish fly strains that enabled gene expression only in muscle cells, a GAL4-UAS system was required. GAL4 is a transcriptional activator that binds to a UAS enhancer sequence found in an organism’s DNA. 11 The GAL4-UAS system recruits transcription machinery to a specific locus for gene expression to occur. This causes any genes downstream of the UAS sequence to just be expressed when GAL4 is expressed. Therefore, a fly strain expressing GAL4 with a particular promoter can cause tissue-specific expression. 11 To restore the wild-type UAS-iPLA2-6A only in muscle cells, a specific GAL4 driver, GAL4-DJ667, was needed. The fly crosses made for the muscle cell group, representing the parental generation, are written out in Punnett Squares 1 and 2.
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* Note: The crosses were maintained at 26 ºC. The highlighted cells represent the genotypes of the flies that needed to be collected. The phenotype of these flies was non-curly wings and non-stubble hairs located on the thorax of the fly. Additionally, only male flies were collected to ensure that no F2 progeny arose from matings between F1 flies.
Collecting and Sorting the F1 Progeny
After the initial crosses were made, ten to twelve days passed before the F1 flies eclosed and were ready to be sorted and collected. The flies were first gassed with a small amount of carbon dioxide so that they could be transferred to a platform emitting carbon dioxide. Using a microscope, flies with the desired phenotype of male, non-curly, and non-stubble were sorted and collected every two to three days for approximately two weeks. A group of 6–13 flies with the desired phenotype was placed into a vial until the 15 and 20-day climbing assays were conducted. Each new group of flies for the control group was marked Cx and Ex for the experimental group, where “x” represents the group number used in the climbing assay. From October 24th to November 28th, 15 control groups and 14 experimental groups were created every 2–3 days, holding 6–13 F1 flies in a vial. While the number of flies varied in each vial, 115 flies from the control group and 108 flies from the experimental group were collected over the approximate month time period.
Passing the Flies
In addition to collecting and sorting the flies every 2–3 days, the F1 flies placed in vials to age needed to be passed 3–5 days either because they required more food or the food was receding from the wall. In order to pass the flies, two techniques were utilized. One technique used small amounts of carbon dioxide to gas the F1 flies so that they could be transferred to a new, fresh vial of food. The other technique involved transferring the flies without the use of carbon dioxide; the old food vial with a cotton stopper in it would be tapped on a mouse pad continuously to ensure that no flies escaped while the cotton stopper was removed, allowing the new food vial to be placed on top of it. The food vial would be flipped, and the flies would be transferred to the new vial of fresh food.
Climbing Assays
At the 15 and 20-day mark of the F1 flies being in their respective vials, climbing assays were performed. Climbing assays are tests that measure the locomotor ability of the flies. After the flies were transferred to a new food vial, a second glass vial was taped to the food vial containing the flies. A ruler measured 6 cm from the edge of the food, acting as the threshold line that the flies needed to pass to be counted as having sufficient locomotor ability. When the climbing assay apparatus was all setup, the climbing assay test began (Figure 2).
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Figure 2. Climbing Assay Apparatus (Adapted from 12). The climbing assay apparatus consisted of two plastic vials stacked on top of each other. 6 cm from the edge of the food (not shown) was measured with a ruler. A marker was used to mark the 6 cm threshold line. A cotton stopper was placed at the top of the climbing apparatus so the flies would not escape.
One person tapped the apparatus on a mouse pad for 1–2 seconds before letting go of it, as the second person started the stopwatch to see how many flies crossed the 6 cm threshold in 20 seconds. The climbing assay test was performed five times per fly vial. Each time, the number of flies that crossed the 6 cm threshold was recorded. When the five trials for a group were completed, the climbing index was calculated. The climbing index was calculated by summing the total score of all the flies from the five trials that crossed the 6 cm threshold and dividing that by the number of flies in a vial. The minimum score a vial could receive was 0.0, and the maximum was 5.0. Climbing indices were averaged for the 15 and 20-day climbing assays for the control and experimental groups. The climbing indices were plotted with standard deviations. Climbing indices for each condition were verified for normal distribution around the average. Statistical comparisons were performed using unpaired t-tests.
Statistical Tests
An unpaired t-test was used to compare the average between the two independent groups, i.e., control and mutant fly groups, to test whether a significant difference was present between the two groups. Several assumptions were made when performing the unpaired t-test. 13 First, it was assumed that there was equal variance between the control and mutant fly groups. If equal variance exists between the two groups, then the data from the two groups should have the same standard deviation. Second, two independent groups were needed in order to conduct the unpaired t-test. Finally, observations between the control and mutant fly groups were required to be sampled independently.
Results
Raw Climbing Index Data
The 15 and 20-day climbing assay data for the control group collected from October 24th to November 28th are shown below in Figures 4 and 5, respectively. Five climbing assay trials were performed for each F1 fly control group, which were then used to calculate the climbing indices of each group. A total of 115 male flies that varied in number between the 15 control groups had an average climbing index of 3.25 with a standard deviation of 1.50 for the 15-day climbing assay and an average climbing index of 1.88 with a standard deviation of 1.36 for the 20-day climbing assay (Figure 3).
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Figure 3. Graphical Representation of the Mean Climbing Index Plotted with the Standard Deviation. The results for the 15 and 20-day climbing assays are illustrated in the graph. The blue bars represent the average climbing index score for the control male F1 flies, and the orange bars represent the average climbing index score for the mutant male F1 flies. The error bars represent the standard deviation of the datasets. It should be noted that 8 out of the 58 climbing assays performed were done 1–2 days before or after the planned climbing assay day, which could have potentially skewed the average climbing index.
Figure 4. Raw Data for 15-day Climbing Indices (CI) of the Control Group. A total of 115 male flies comprised the 15 groups of F1 control flies. Climbing indices for the 15 groups of F1 control flies had anywhere from 5–14 flies in a group, represented by “N Flies,” were calculated. “T#X,” where “X” represents a number 1–5, was the trial number of how many flies crossed the 6 cm threshold to be counted as a successful climb. It should be noted that group C10’s climbing assay was conducted on day 19 instead of day 15. The mean climbing index was determined to be approximately 3.25, with a standard deviation of approximately 1.50.
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Figure 5. Raw Data for 20-day Climbing Indices (CI) of the Control Group. A total of 112 male flies comprised the 15 groups of F1 control flies. Fewer flies performed climbing assays because some flies were lost, transferring the flies to fresh food vials. Climbing indices for the 15 groups of F1 control flies had anywhere from 5–14 flies in a group, represented by “N Flies,” were calculated. “T#X,” where “X” represents a number 1–5, was the trial number of how many flies crossed the 6 cm threshold to be counted as a successful climb. It should be noted that groups C9 and C10’s climbing assays were conducted on day 22 instead of day 20. Additionally, group C11’s climbing assay was conducted on day 18 instead of day 20. The mean climbing index was determined to be approximately 1.88, with a standard deviation of approximately 1.36.
The 15 and 20-day climbing assay data for the experimental group collected from October 24th to November 28th are shown below in Figures 6 and 7, respectively. Five climbing assay trials were performed for each F1 fly experimental group, which were then used to calculate the climbing indices of each group. A total of 108 male flies that varied in number between the 14 experimental groups had an average climbing index of 4.08 with a standard deviation of 0.907 for the 15-day climbing assay and an average climbing index of 3.08 with a standard deviation of 1.21 for the 20-day climbing assay.
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Figure 6. Raw Data for 15-day Climbing Indices (CI) of the Experimental Group. A total of 108 male flies comprised the 14 groups of F1 experimental flies. Climbing indices for the 14 groups of F1 experimental flies had anywhere from 6–10 flies in a group, represented by “N Flies,” were calculated. “T#X,” where “X” represents a number 1–5, was the trial number of how many flies crossed the 6 cm threshold to be counted as a successful climb. The mean climbing index was determined to be approximately 4.08, with a standard deviation of approximately 0.907.
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Figure 7. Raw Data for 20-day Climbing Indices (CI) of the Experimental Group. A total of 102 male flies comprised the 14 groups of F1 experimental flies. Fewer flies performed climbing assays because some flies were lost, transferring the flies to fresh food vials. Climbing indices for the 14 groups of F1 experimental flies had anywhere from 5–10 flies in a group, represented by “N Flies,” were calculated. “T#X,” where “X” represents a number 1–5, was the trial number of how many flies crossed the 6 cm threshold to be counted as a successful climb. The mean climbing index was determined to be approximately 3.08, with a standard deviation of approximately 1.21.
Unpaired T-test Results
The results of the unpaired t-tests for the control and experimental groups are shown in Table 1. The 15-day climbing assay unpaired t-test revealed that the results were not statistically significant, indicated by the p-value being greater than 0.05. In contrast, the 20-day climbing assay unpaired t-test demonstrated that the results were statistically significant. This was proven by the p-value being less than 0.05.
Discussion
15-Day Climbing Assay Data Analysis
An analysis of the data amassed for the 15-day climbing assay suggested that rescue of the iPLA2-6A gene occurred, but this conclusion could not be corroborated since the p-value for the unpaired t-test was not statistically significant. That being said, the p-value was near the standard p < 0.05 value for the results from this data to be considered significant and not due to chance. It is likely that with a larger sample size and additional 15-day climbing assays performed on new fly groups, the results would yield a statistically significant value. 14 Further research must be conducted to test this hypothesis. Not surprisingly, the mutant male F1 flies had a higher climbing index than the control F1 male fly group. This was expected since rescue was more able to occur in the mutant male flies, helping them potentially be more successful at climbing than in the control fly group. The standard deviation was much higher than expected. Possible explanations for why the data varied so much from the mean include the time of day the climbing assays were conducted and unequal tapping strength applied to the climbing assay apparatus by different members in the lab group, leading to variable results. Reiger et al. found that fruit flies were most active at early dawn and late dusk. 15 At the beginning of the little over a month time period spent collecting climbing index data, many of the climbing assays performed were conducted at late dusk. Interestingly, the highest climbing indices were recorded at that time of day. Therefore, climbing assays performed at times not at early dawn or late dusk may be the cause for the variable data, leading to a high standard deviation. However, once again, increasing the sample size should alleviate the high standard deviation, but further testing is needed to validate this claim. 16
20-Day Climbing Assay Data Analysis
The data collected for the 20-day climbing assay was determined to be statistically significant, as the p-value for the unpaired t-test was 0.020. The mutant F1 flies had a significantly better climbing index score than the F1 control fly groups. This was expected since rescue was more able to occur in the mutant male flies, helping them potentially be more successful at climbing than in the control fly groups. As expected, the climbing index score for both the control and mutant flies of the 20-day climbing assays was lower than the climbing index score for the control and mutant flies of the 15-day climbing assays. Since more time passed, the 20-day control and male mutant flies had less locomotor ability than they did 5 days prior. Furthermore, as predicted, the control fly groups from the 15 and 20-day climbing assays had a more profound loss of successful climbs compared to the male mutant flies. As a result, the average climbing indices for the control fly groups had a 42.1% reduction in successful climbs (Calculation 1). Compared to the control fly groups, the average climbing indices for the male mutant fly groups were only reduced by 24.4% (Calculation 2).
Like the 15-day climbing assay data, the standard deviation for the 20-day climbing assay data was higher than expected. This could be due to the reasons proposed in the 15-day climbing assay data analysis section or some other reasons yet to be determined. In any case, to make the data less varied from the mean, further research should be conducted where the sample size of the flies is larger than the one used here. 16
Examining the Molecular Mechanisms that Led to Rescue in the Mutant Fly Groups
A thorough explanation of the molecular mechanisms helps one to understand why the wild-type iPLA2-6A allele was able to be more rescued in the muscle cells of the mutant male fly groups than the control fly groups. As mentioned earlier, a specific GAL4 driver, GAL4-DJ667, was required to restore the wild-type allele for the iPLA2-6A gene only in muscle cells. In order to accomplish this, GAL4-DJ667 was bound with UAS-iPLA2-6A (Figure 8).
Figure 8. Molecular Representation of How Gene Expression Only Occurred in the Muscle Cells of the Mutant Flies (Adapted from 17). In order to express the iPLA2-6A gene only in the muscle cells, a fly with DJ667-GAL4 was mated to a fly with the UAS-iPLA2-6A transgene. The F1 progeny’s genotype thus became DJ667-GAL4/UAS-iPLA2-6A. A GAL4 protein that acts as a transcriptional activator binds to the UAS-iPLA2-6A transgene. When RNA polymerase binds to the GAL4 protein, Phospholipase A2, an enzyme that helps with lipid metabolism and maintaining the integrity of the cell membrane, is produced and expressed. 18 Any genes that are encoded downstream of the UAS-iPLA2-6A transgene will only be expressed when GAL4 is expressed. 11
The DJ667-GAL4/UAS-iPLA2-6A system expressed the wild-type allele of the UAS-iPLA2-6A transgene in the mutant fly groups but not in the control fly groups since the control fly groups lacked the UAS-iPLA2-6A transgene. As such, Phospholipase A2, the gene product of the UAS-iPLA2-6A transgene, was not able to be produced and expressed. Phospholipase A2 is an important enzyme that assists with lipid metabolism and maintaining the integrity of the cell membrane. 18 As Mori et al. hypothesized, the lack of integrity of the cell membrane caused 𝛼-Synuclein to aggregate in the form of Lewy bodies and Lewy neurites. 8, 19 An overabundance of 𝛼-Synuclein has been shown to lead to neuronal death, a defining feature of neurodegenerative diseases like Parkinson’s disease. 9 Specifically related to Parkinson’s disease, 𝛼-Synuclein has also been proven to regulate dopamine production. 9 When 𝛼-Synuclein accumulates, dopamine production is inhibited. Since dopamine normally inhibits 𝛼-Synuclein aggregation, Lewy bodies and Lewy neurites, large clumps of 𝛼-Synuclein, continue to accumulate in an organism’s brain.
When a loss of function mutation occurred in the iPLA2-6A gene of the control group flies, Phospholipase A2 was not produced, and so the integrity of the cell membrane was not able to be maintained. This led to an accumulation of 𝛼-Synuclein in the brains of the flies, which resulted in them developing Parkinson’s disease. The locomotor ability of the control fly group was hindered, as seen by the lower climbing index scores compared to the mutant fly groups. Like the control fly groups, the mutant fly groups initially also had a loss of function mutation in the iPLA2-6A gene. This caused the flies to have an accumulation of 𝛼-Synuclein in their brains, which led to Parkinson’s disease. However, after establishing crosses that yielded F1 fly progeny with the UAS-iPLA2-6A transgene, Phospholipase A2 was able to be produced once again. Consequently, the mutant fly groups performed significantly better in the climbing assay tests than the control fly groups.
The results of this experiment have proven that muscle cells are affected by Parkinson’s disease, especially as time passes, and that rescue can occur in tissue-specific cells. Future research should focus on investigating if tissue-specific rescue can be achieved in humans who have developed Parkinson’s disease.
Future Experimentation: Adaptation of FlyVRena to Further Investigate Rescue of the iPLA2-6A Gene, Leading to Better Locomotor Ability
FlyVRena, a device created by Cruz et al., can be adapted to examine how rescue of the iPLA2-6A gene can lead to better locomotor ability in flies. 20 The analyzed results from the FlyVRena experiment will be compared to the results of the flies’ locomotor ability during climbing assays.
FlyVRena is a virtual reality system that allows insects to walk freely in an arena, all the while sensors and cameras collect and analyze data on the fly’s movement (Figure 9).
Figure 9. FlyVRena 20. A fruit fly is placed in the arena and is allowed to roam the arena floor. Cameras and sensors track the movement of the fly and collect and analyze data based on the fly’s movement. The data that is collected and analyzed is sent to a computer, where the data can be further analyzed with specific software. While the figure does not show it, the top of the arena is enclosed with a plate of glass covered in sigmacote, a chemical that the flies do not like. This deters the flies from walking on the ceiling.
While the original experiment conducted by Cruz et al. used virtual reality to make objects appear as the flies roamed the arena floor, the use of virtual reality will not be required in this future experiment. 20 As such, the projector and mirror parts of FlyVRena would not be used. However, the other components of FlyVRena, including the arena, stand to hold the arena, cover of the arena coated with a layer of sigmacote, camera, sensors, and a computer to further analyze the data, would be used.
The climbing assays performed in this experiment tested one form of locomotion that the flies could perform: flying. However, flies have other locomotor abilities, such as walking. By using FlyVRena, the walking abilities of the flies would be examined. New control and mutant fly groups that had the iPLA2-6A gene knocked out would be used. The same procedure in terms of passing, collecting, and sorting the flies from both groups would be followed. When the F1 flies from the control and mutant fly groups reached 15 and 20 days old, a single fly from the control and mutant groups would be placed in a FlyVRena apparatus. In order to ensure that the flies would walk only on the floor of the arena, certain modifications would need to be made to the apparatus. To start, since flies have been shown to be phobiocentric, the circumference of the arena would need to be heated to a temperature of 40 ºC. This would ensure that the flies stayed toward the middle of the arena. Additionally, to ensure that the flies stay on the floor of the arena and not roam on the ceiling, a cover with a thin layer of sigmacote, a chemical that the flies are averse to, would be placed on top of the arena. With these modifications in place, the fly would be forced to walk on the floor of the arena.
The flies would have a 10-minute exploring period so that they could acclimate to their new environment. When the 10 minutes passed, the fly would be allowed to walk the arena for 30 minutes. Cameras and sensors would track the movement of the fly and send this information to the computer, where software can be used to analyze the data. This procedure would be done for five trials. The walking index, which is the total distance covered by all the flies in a group divided by the total number of flies in a group, would then be calculated. It is predicted that the control fly groups would explore a lot less of the arena than the mutant fly groups since locomotor ability in the control fly groups should be more impeded as rescue of the iPLA2-6A gene is not occurring (Figure 10). This would be shown statistically by the control fly groups having a lower walking index than the mutant fly groups. The data from the FlyVRena experiment would be compared to the data from the climbing assay experiment to examine if certain forms of locomotion seem to have better rescue than the other forms of locomotion.
Figure 10. Predicted Walking Patterns of the Control Fly Groups (Left) vs. the Mutant Fly Groups (Right) 20. A fly would be placed in the FlyVRena and then be given a 10-minute exploratory period to acclimate the fly to its environment. Then, the fly would be given 30 minutes to walk around the arena. The movement of the fly would be tracked by cameras and sensors, and that data would be sent to a computer for analysis using software programs. It is predicted that the control fly groups would have a similar walking pattern to the picture on the left, as no rescue of the iPLA2-6A gene is occurring in those flies. As a result, Phospholipase A2 is not being produced, so the flies would accumulate 𝛼-Synuclein in their brains. This would lead to Parkinson’s disease, which presents with reduced locomotor ability. In contrast, the mutant fly groups are predicted to have walking patterns similar to the picture on the right since rescue of the iPLA2-6A gene is occurring in only those flies. Phospholipase A2 is being produced, so the flies are not accumulating a large amount of 𝛼-Synuclein in their brains. This should lead to not as severe locomotor impediments as the control fly groups are predicted to have.
This experiment investigated if iPLA2-6A functions in muscle cells of Drosophila melanogaster to maintain aging health. In order to determine this, the wild-type allele was placed in mutant fly groups through the transgene, UAS-iPLA2-6A, to ascertain if the wild-type allele was sufficient to rescue motor decline in knockout iPLA2-6A flies. Climbing assays were performed to test if locomotor ability in the flies improved with the wild-type allele present. Although the 15-day climbing assay results suggested that there was a partial rescue of the iPLA2-6A gene in the mutant fly groups, this conclusion could not be definitively stated, as the p-value was slightly higher than an acceptable p-value. Unlike the 15-day climbing assay data, the 20-day climbing assay p-value revealed that partial rescue of the iPLA2-6A gene in the mutant fly groups was accomplished. A comparison of the 15 and 20-day climbing assay results displayed a decrease in climbing index scores for both the control and mutant fly groups. That being said, as the flies aged, the control fly groups had a more significant reduction in climbing index scores than the mutant fly groups. Thus, partial rescue of the iPLA2-6A gene in the mutant fly groups was more apparent with time. Future research should focus on investigating if tissue-specific rescue can be achieved in humans who have developed Parkinson’s disease.
Acknowledgments
All fly data was conducted in Professor Josefa Steinhauer’s laboratory, an associate professor of biology at Yeshiva University. I would like to thank her for her continuous support and assistance over the course of my research. Additionally, I would like to thank my laboratory partners, Aaron Lubat, Alexander Siegman, and Shlomo Shaulian, for their help with collecting data for this research project.
References