Spring 2023
Team 4
Introduction
Our team had the objective of determining if certain genes would affect the motor abilities of drosophilia. We were able to work towards this goal by setting up genetic crosses and doing locomotive tests frequently.
ALS and Our Genes
Background information
ALS
ALS or Amyotrophic Lateral Sclerosis is a motor disease where motor neurons are deteriorated over an extended period of time. Motor neurons are vital to transmitting messages from the brain to the spinal cord, and then to the muscles. When that pathway is disrupted that is where motor deficits can become more pronounced. The disease often begins with the twitching of the limb or even slurred speech. There are some identified risk factors such as sex, age, but more interestingly genetics. It is identified that 10 percent of people with ALS inherited it through a family member. Currently there is no treatment for this disease.
(The image to the left is an MRI of a patient who has ALS, you can noticed where the deterioration of the brain has begun)
Bridging off of the details of ALS in human models, we analyzed the possible expression of ALS genes in drosophila. Drosophila is a good model organism for this experiment because of the ability to express and repress genes, along with rapid regeneration time. With our selected model organism, next began the steps of identifying what gene expression might play a role in ALS motor deficits. We analyzed figures F and D within a research article in scientific reports. These figure specifically highlighted the different expression of genes that could possibly be identified as an ALS risk factor. With our prompt of analyzing genes that play a role in macroautophagy we select three different options, that being HDAC2, CRBN, and EXD2. After further analysis of each of our three chosesn genes we elected to experiment with CRBN and HDAC6 as they had consistant fly orthologs to drosophila, along with having significant evidence of the possibility of its expression regarding ALS.
(Image Listed to the right is Figure F within the research article Gene co‐expression network analysis in human spinal cord highlights mechanisms underlying amyotrophic lateral sclerosis susceptibility)
DROSOPHILA
HDAC6
HDAC is a gene that is involved in histone deacetylation. This essentially means that HDAC is an enzyme that propagate the removal of acetyl groups from both histone and non-histone proteins (Seto). We have been attempting to express this gene in both the UAS and RNAi lines of drosophilia. These genes can be both upregulating or down-regulation. This means we opted to get a UAS and RNAI strain of this speciifc gene.
CRBN
The official name for the CRBN gene is cereblon and it plays a role in encoding proteins, regulating the AMPK pathway, ion signaling, and influence apoptosis and cell proliferation. We are looking at this gene because it may play a role in the degradation of misfolded proteins. If CRBN is affected and does not degrade misfolded proteins, protein aggregation may occur.
Hypotheses
CRBN RNAI
If CRBN is expressed at a lower rate, then this could lead to lower rates of degradation of misfolded proteins, which would in turn lead to larger amounts of protein aggregation which is an indicator of ALS.
HDAC6 UAS
If HDAC contributes to synaptic strength an the regulation of misfolded proteins then the lack of expression of HDAC in neuron cells will lead to overall motor decline.
HDAC6 RNAI
If HDAC6 can alternatively play a role of neuroprotection in ALS then the expression of HDAC in neuron cells will lead to overall motor decline.
Methods
Locomotion Assays
Locomotion tests were the primary means of testing locomotion in drosophila. below is a list of stops for this process. The testing period is two minutes.
Make sure that the graduated cylinder is properly labeled at either the 110 mL or 190 mL line depending on the context of the trial being ran.
Quickly transfer the flies from their vial into the graduated cylinder and place piece of parafilm over the opening to ensure no flies escape
Let the graduated cylinder sit for one minute to help the flies assimilate to their surroundings.
After the minute has passed, tap the graduated cylinder 6 times in a disordered manner to ensure that all the flies have fell to the bottom, ensuring an equal starting point.
Place graduated cylinder in view of the camera recording and press start on a two minute timer, stop video after two minute testing period
Transfer flies back to original vial using funnel, then either dispose or put back for further testing.
Things we learned:
Use a funnel to transfer flies to cylinder more efficiently
If flies are particularly rambunctious, have another person assist in the transfer
To make sure that all the correct information is listed which is as follows DOB, number of flies in trial, the strain that is being tested
Typically we would not test flies after they reach an age older than 4 weeks, as their age would interfere with the accuracy of the data
Virgin Collection
For the purposes of our research, virgin collection was extremely important, especially for setting up crosses
To determine if a fly is a virgin we would often refer to the image shown
Drosophilia virgin females are often extremely light colored, possess a dark spot on the side of their abdomen, and are usually larger than non virgin females and males
It was also easy to distinguish if a fly was a male and demale based on the size and color of the abdomen
Males have dark genitals on the end of their abdomen and are often smaller than their female counterparts
Females (non-virgin) are typically larger and longer, and have a trail of dark squares in the middle of their abdomen
Figure:
Cross Setup
We set up a variety of crosses, however before physically achieving a cross, we first had to understand the genetic components to the cross
We used punnet squares as shown to create crosses and understand what result we could expect from having different flies procreate
After creating the punnett squares to determine the theoretical results of the cross, we could place virgins of one genetic target with males of the other and have them create offspring.
We then used physical markers to detemine if the crosses were successfull, an example of this would be seeing if the flies has curly or straight wings in accordance to the specific balancer used in the cross
Results
These graphs show the general trend in climbing data in groups of vglut GAL4 control flies. As shown by the data it is clear that as the flies' age progresses the climbing percentages significantly decrease. After analyzing the data we can see a drop in climbing effiency at around ten days old. All of these trials had at least twelve flies, which is a standard sample size for these tests.
Vglut; TDP 43/ScO male controls. We only have one trial due to them being born shortly before the conclusion of our data collection. This was a trial of 21 flies that were approximately 7-10 days old. These flies performed similarily to the Vglut GAL4/+ flies of the same age.
This graph shows the average fraction climbed across genotypes at 4 different time points. These averages included trials of at least 10 flies that were 6-10 days old at the time of the test. This data shows the general trend of the flies expressing UASHDAC and TP43 being less successful at the locomotion assay. However, we are unsure of the reliablity of this data, due to the limited amount of trials respresented.
Conclusion
We have not been able to find any definitive data to accurately link any of these genes to the progression and development of ALS symptoms. There is some data to suggest a correlation but its not enough and there is no way to determine the effects on a physiological scale with the methods we have used. We ran into a number of issues while carrying out this experiment, specifcally with out HDAC RNAi and CRBN genes. We were able to set up crosses for both, but the HDAC RNAi virgins kept mysteriously dying, so we were unable to carry out locomotion tests on that specific gene. In addition, the CRBN vials quickly accumulated mold, negatively impacting the health and reproduction ability of our cross. We were able to save the CRBN flies by flipping the vials frequently, however we did not have time to keep attempting to create progeny. We were able to learn how to set up genetic crosses and how to troubleshoot against the problems that occurred. We also noticed an anomally where our prodgeny from the HDAC UAS line where we were only seeing straight winged females and males. The problem with this is we were supposed to also observe some curly winged prodgeny. We checked the parents of our cross and they had the ocrrect genes expressed, so regarding that we dont know what caused this anomally. Overall, this semester was a learning experience and we can use what we learned to elevate our research next semester.
Further Implications
We hope to use the knowledge we have gained in setting up crosses and investigating the effects of different genes to expand our research in coming months. We hope to use coding technology to accurately determine the importance of certain genes for further analysis. We will use what we have learned to apply to a new set of genes and hopefully be able to run more physiological experiments, such as dissecting the brains of the drosophila or petri dish walking experiments.
In regard to our genes such as CRBN and the HDAC RNAI strain we were not able to have enough time to fully set up the crosses and run locomotive tests. We plan to start this process in the following semester. One idea that our group has reflected on was the development of our ability to tackle problems that would arise during lab. As it was stated in the conclusion we would consistantly run into problems that would essentially stunt out research. We believe that through this process we have a better idea of how research is conducted, and we believe that we will be able to learn and expand off of these issues we ran into.
We have hopes to expand off of our current research, and we know how to tackle problems that arise in lab a lot better. We have the framework of what we hope to do as a team, but also how each of us individually have used our critical thinking skills.
References
Mayo Foundation for Medical Education and Research. (2023, April 25). Amyotrophic lateral sclerosis (ALS). Mayo Clinic. Retrieved April 25, 2023, from https://www.mayoclinic.org/diseases-conditions/amyotrophic-lateral-sclerosis/symptoms-causes/syc-20354022
Seto, E., & Yoshida, M. (2014, April 1). Erasers of histone acetylation: The histone deacetylase enzymes. Cold Spring Harbor perspectives in biology. Retrieved April 25, 2023, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3970420/
Shi, Q., & Chen, L. (2017). Cereblon: A protein crucial to the multiple functions of immunomodulatory drugs as well as cell metabolism and disease generation. Journal of immunology research. Retrieved April 25, 2023, from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5574216/
Peer reviewed scientific video journal - methods and protocols. JoVE. (n.d.). Retrieved April 25, 2023, from https://app.jove.com/t/52741/quantitative-analysis-climbing-defects-drosophila-model
The power of drosophila genetics - umass. (n.d.). Retrieved April 25, 2023, from https://wahoo.nsm.umass.edu/sites/default/files/fly_module_course_guide_2016.pdf
A quick and simple introduction to drosophila melanogaster. Introduction to Drosophila. (n.d.). Retrieved April 28, 2023, from http://www.ceolas.org/fly/intro.html