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:

 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

The Locomotor Tests

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

Note:  If a fly falls back down past the line subtract that fly from the count

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.