Team 3
Introduction
Image courtesy of https://www.nature.com/articles/srep13416
ALS Background
Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease that affects nerve cells in the brain and spinal cord. Specific genetic mutations have been tied to familial cases of ALS and some sporadic ALS cases; however, much is still unknown about the genetic causes of this disease.
Drosophila Background
Drosophila melanogaster are useful in experimental research for multiple reasons. They share approximately 60% of the human genome, and over 65% of human disease associated genes have a homologue in Drosophila. Additionally, they have a short life cycle during which many offspring can be produced. At 25 degrees Celcius, adult flies can develop in 9-12 days; however, flies can also be kept at 18 degrees Celcius in order to slow down development and extend lifespan.
When working with the flies, we use carbon dioxide to anesthetize them, and then we examine them through the microscopes while they are knocked out on the fly pads.
https://vishnukarthigeyansrp.wordpress.com/2017/02/27/fly-crosses/
Pictured below: drosophila female (left) and male (right)
Pictured below: normal male (left) vs curly phenotype (right)
+Ly, -CyO, +IF
+Ly, +CyO, +IF
What is our experiment about?
We are looking to test 2 genes (PEX19 and DEF8) that have been previously indicated to be associated with ALS for their involvement in motor function. For reference, the scientific article that we used to choose our genes from is linked here: www.nature.com/articles/s41598-021-85061-4
(Wang, J.C., Ramaswami, G. & Geschwind, D.H. Gene co-expression network analysis in human spinal cord highlights mechanisms underlying amyotrophic lateral sclerosis susceptibility. Sci Rep 11, 5748 (2021). https://doi.org/10.1038/s41598-021-85061-4)
Genes in Use:
PEX19 (Bloomington stock #50702)
PEX19 is a gene involved in the organization and function of peroxisomes. Mutations in genes that regulate the functionality of peroxisomes are known to cause degeneration of nerves as seen in peroxisome biogenesis disorders. [1]
Peroxisomes are a vital organelle found in all eukaryotic cells. Their role is to produce hydrogen peroxide and carry out oxidative reactions, and they are important for lipid synthesis and breakdown.
While ALS is characterized by buildups of proteins in motor neurons, buildups of long-chain fatty acids can also cause damage with symptoms similar to ALS. Since peroxisomes are crucial to the breaking down of long-chain fatty acids, it would make sense that a lack of peroxisome function could lead to neurodegenerative disease.
Furthermore, there are some proposed links between dysregulated lipid metabolism and ALS [2].
DEF8 (Bloomington stock #34384)
DEF8 is a gene predicted to enable metal ion bonding, positive regulation of bone resorption, and most important to us, is integral in lysosome localization
Lysosome localization is an important process in autophagy, which is a cell’s way of breaking down old, damaged, and abnormal proteins.
Current studies suggest that suppression of DEF8 results in defects of lysosome positioning [3] which we expect to cause disturbances in autophagy. Disturbances in autophagy are often early indicators of neurodegenerative diseases (ex. Alzheimer's).
DEF8, a protein proposed to act at the final step of the autophagy/endolysosomal pathway, is differentially (reduced) expressed in PBMCs of Alzheimer's patients. These results suggest a potential role for DEF8 in the pathophysiology of AD. Differential expression of DEF8 disturbed autophagy systems [4].
ALS is characterized by the aggregation of proteins in motor neurons. Buildup of protein TDP-43 is observable in 97% of all ALS cases [5]. We believe that since DEF8 is integral in lysosome localization, it plays a role in autophagy, and that downregulation of DEF8 would leave cells unable to break down harmful proteins like TDP-43, thus increasing susceptibility to ALS.
Moreover, DEF8 has a direct ortholog in humans; DEF8 in drosophila corresponds to DEF8 in humans.
Sources:
https://www.ncbi.nlm.nih.gov/gene/5824
https://pubmed.ncbi.nlm.nih.gov/31406145/
https://pubmed.ncbi.nlm.nih.gov/27777970/
https://pubmed.ncbi.nlm.nih.gov/33612542/
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3661910/
PEX19 Hypothesis
We expect that downregulation of PEX19 would result in less peroxisome functionality, which could cause symptoms of neurodegenerative disease similar to ALS (such as motor impairment) due to the buildup of long-chain fatty acids
DEF8 Hypothesis
We expect that differential (down) expression of DEF8 in motor neurons will lead to interrupted autophagy function, causing abnormal protein buildup and motor neuron death, leading to development of ALS.
Methods
The following has been replicated off of The University of Alberta’s research guidelines.
Fly Collection
Collect 20 flies of a given genotype by anesthetizing them with Carbon Dioxide gas. Place them in a 25 by 99mm vial with food.
Label vial with genotype, the number of flies contained, and the date they were born
Incubate these flies at 22°C and 45% humidity. The incubator should be set with a 12 hour day/night cycle.
The Locomotor Tests
In the morning, transfer 20 flies of a genotype into a 250mL graduated cylinder-- it is crucial that these tests are repeated at a similar time each day (NOTE: Surrounding conditions should be constant for each trial. This includes light, temperature, and humidity).
Place a piece of tape near the 110mL line to use as a reference point
Barricade the top of the cylinder with paraffin wax to prevent escape
Set up a camera, focused on the 110mL line of the graduated cylinder.
Give flies around 1 minute to rest prior to beginning any experiment.
Create a card indicating the genotype, fly birth date, date of test, and total number of flies in vial. These are crucial for data analysis.
Begin recording on camera, show the card, and tap graduated cylinder against the table 6-8 times in a non rhythmic pattern, using enough force to move and disrupt the flies.
Start a timer as soon as the last tap is done.
Allow the trial to run for two minutes.
Record any dead flies as mortalities.
At the end of the test, return flies back into their collection vials using a funnel. Remove any dead flies, and update the total number of living flies on the vial label.
Note: We stop testing the flies once they reach an age greater than 4 weeks, when this time comes dump all of the flies straight into the fly morgue (ethanol)
ANALYSIS, DISPOSAL
Analysis
Organize the videos by genotype
Each genotype will have its own spreadsheet for analysis
Record the date born, date of test, total number of flies, and age of flies
Open a video. Every ten seconds after the final tap, record the total number of flies past the 110mL mark
Note: If a fly falls back down past the line subtract that fly from the count
Enter this data into the appropriate spreadsheet
Plot each data point as a percentage of total flies in trial
Now organize the data to view relationships between performance and time. Typically, compare the total number of flies that complete the trial at t=120 seconds, for different age intervals (eg. 1 week, 2 weeks, etc)
Compare this data with other genotypes and note any differences
Other useful data pursuits: Slope of each graph for fly movement and average out flies at each interval per age.
The Crosses
This semester we set up 4 crosses, and next semester we will have to complete one more cross before we are able to cross our genes into the flies. We had to set up so many crosses because we had an issue with our selected genes being on chromosome 3 which lead us to have to create multiple double balancer lines. When tracking fly genetics it is crucial to have placeholders (aka balancers) on each chromosome or else you risk "loosing" the genotype because you are not able to track it in future crosses. We discovered this early in the process, however the addition of having to create the double balancers set us back and limited what we were able to get done this semester. We labeled and planned out our crosses on the google doc linked below.
docs.google.com/document/d/1sxLWmDy1i3DAa-lmnriEuzhRhMuH7zIUmiRIylLS8Tg/edit
Data
Please note: Our data collection methods were not fully set in until about halfway through the semester. Consequently, many analyzed runs had to be discarded as they lacked information regarding the total amount of flies present, or the age of the flies in each trial. As a result, the presence of outliers is much more noticeable in some of these graphs, as there is not a large body of data to be collected to combat this. Moving forward, this problem can be easily fixed, as we have now established explicit criteria on all the points of data we must collect when analyzing our locomotor runs
AlrmGAL4/+ Trials
Performance in locomotor tests per time interval. Age of the flies used is reflected by the color of the lines
Total percent of flies climbed at the end of each trial, split up by age in weeks
Control Data - Alrm/TDP43
This chart illustrates the results of our locomotor tests as a line graph. Each point along the line reflects what percent of flies had crossed the 110mL line at each time interval
This chart shows the total amount of flies that were able to cross the 110mL line by the end of the trial, which was at 120 seconds, or two minutes. Age is measured in days. Each bar represents a single trial.
With this [Alrm/TDP] data in mind, we can see that progress consistently declines with age. As the flies get older, not only do fewer flies end up crossing the line by the end of the trial, but we also see that they do not make it up as quickly. The slope of the lines from t=0 to t=20 decreases noticeable as they age.
DEF8 Protein
Image taken from https://www.genecards.org/cgi-bin/carddisp.pl?gene=DEF8
Expected Results
PEX19
We believe that the flies with downregulated PEX19 will exhibit symptoms similar to ALS. This is because the PEX19 gene regulates the formation and function of peroxisomes, which play a vital role in the breakdown of lipids. Without fully functional peroxisomes, lipids can build up and cause damage, leading to neurodegeneration. Thus, these flies would likely have observable motor deficits and possibly reduced lifespans.
DEF8
We expect the downregulation of DEF8 to would lead to a higher rate of and more rapid development of ALS. This is due to the nature of DEF8 assisting in autophagy. If a cell cannot complete autophagy as it should, it will lose mechanisms to combat ALS development. In data, this would look similar to our ALRM/TDP runs, but the decline with age would be more prominent and would be noticeable at earlier time. It could also be expected to see less total flies climbing to the top, as well as slower rates of movement over time.
Conclusion
While we were unable to cross our chosen genes into the flies during this semester, we were successful in setting up our double balancer stocks which will be helpful in future semesters. We were able to put together graphs using the available data from the alrmGAl4/+ and alrm/TDP43 trials, however the alrmGAL4 /+ data is incomplete and thus unreliable because of the lack of trials that could be included in the data. One of the biggest takeaways from this semester is that it is so important to include all information about the flies that are being tested in the locomotor tests.
Next Steps
The good thing about starting again next semester is that we already have our double balancer stocks made. When we come back from summer, we will need to collect virgins from our UAS-TDP43CyO;+TM6b,SbTb x ifCyO;LyTM6b,SbTb and +CyO;alrm-GAL4TM6b,SbTbx ifCyO;LyTM6b,SbTb stocks. The progeny from those crosses will then be crossed with each other, and then crossed into our genes of choice. We can then conduct locomotor tests on those offspring and analyze them for motor defects. Then we can compare those results to the control flies and see what the differences are.