What is ALS?

Amyotrophic lateral sclerosis, or ALS, is a neurological disease that primarily affects neurons in the brain as well as the spinal cord. Also referred to as Lou Gherig's disease, ALS is known to cause a loss of muscle control over time. 

Specific symptoms include immense weakness and clumsiness, which contributes to uncoordinated movements as well as difficulty participating in daily activities (i.e. walking, speech, swallowing, etc.). Feelings of weakness often begin in the extremities and spread towards to the rest of the body in individuals between athe ages of 50 to 70. However, ALS is not known to affect bladder control or the senses. Although uncommon, the disease may also impact cognition, such as language and behavior. 

While there is no initial pain in the earlier stages of ALS, patients tend to develop gradual pain as the disease progresses. 

Causes

Current research supports that ALS is a degradation of motor neurons, the cells that control voluntary muscle movements. Categorized by upper (brain and spinal cord) and lower (muscular) motor neurons, ALS deteriorates both groups of cells to an eventual death, which inhibits muscle functionality. 

Although the methods by which ALS impacts these cells is known, the exact reason for why this might be happening is still at the forefront of recent studies. For this project, we are exploring the theory of a connection between genes and the onset of ALS symptoms. Specifically, we aim to investigate how our gene of choice affects the cytoplasmic aggregation of TAR DNA binding protein 43 kDa, better known as simply TDP-43, which is believed to be a causal factor in both sporadic and familial cases of ALS symptoms. 

Mayo Clinic. (2019, August 6). Amyotrophic Lateral Sclerosis (ALS) - Symptoms and Causes. Mayo Clinic; Mayo Clinic Publishing. https://www.mayoclinic.org/diseases-conditions/amyotrophic-lateral-sclerosis/symptoms-causes/syc-20354022

Suk, T. R., & Rousseaux, M. W. C. (2020). The role of TDP-43 mislocalization in amyotrophic lateral sclerosis. Molecular Neurodegeneration, 15(1). https://doi.org/10.1186/s13024-020-00397-1

Gene of Choice: SOAT1

Fly Ortholog: CG8112

Upregulated in ALS

P-Value: 1.179733e-16

Function: 

• Initiates the formation of fatty acid-cholesterol esters from cholesterol to prevent an over-accumulation of free cholesterol at cellular membranes. 

• Involved in cholesterol metabolism.

Hypothesis #1

SOAT1 is indicated to be upregulated in ALS, which suggests that there are higher levels of cholesterol esters intracellularly. When there are high levels, SOAT1 comes in and converts them to cholesterol esters in lipid droplets. Higher amounts of CE and LD can cause toxicity in cells which might lead to the symptoms seen in ALS. 

Hypothesis #2

Our second hypothesis is that the upregulation of SOAT1 is a response to ALS symptoms rather than an underlying cause of the disease. We read that the acid bile branch of biosynthesis is found to be defective in ALS, this prevents the CNS from removing excess cholesterol. We hypothesis that SOAT1 might be upregulated in attempts to lower the amount of cholesterol in the CNS. 

Genotype of Parents

At the beginning of the semester we were focused on setting up control crosses, ie. alrmGAL4/+ and alrmGAL4; UAS-TDP43. We were not able to work with our SOAT1 flies until about the last month or so of class, because of issues with sick double balancer flies and trouble getting progeny. 

A few weeks ago we set up a cross with UAS-SOAT1 males and a vgluGAL4;TDP43 virgin female with all the double balancers, and we would have been able to experiment right away with these flies, but the female died and we never got offspring from the cross. 

Petri Dish Assay

After setting up productive crosses, we ran behavioral tests on offspring to assess movement. We used a petri dish assay as our behavioral test.

• Methods for Petri dish assay:

1. Collect a group of 3-4 flies.  

2. Transfer to an empty vial (one without food) and place in ice for approximately 30 seconds to 1 minute.  

3. When flies are asleep, place them into the shallow petri dish with ring insert. 

4. Cover with the black and white microscope disk. Ensure that white side is down (facing the flies) and secure with tape. 

5. Wait five minutes to begin behavior test to ensure flies adequately warm up. 

6. Meanwhile place the petri dish with flies on the ring stand (figure 1). 

7. On a peice of tape right the flies genotype, fly number, DOB, date of recording, team number, and fly group identifier (if applicable) 

8. Stick tape on the bottom of the ring stand where it will be in view in the recording (figure 1). 

9. Open Quicktime Player (only applicable to Mac users) on the computer. 

1. Click file in top left corner  

2. Select New Movie Recording 

3. Select drop down to the right of the red recording circle. Under camera select your iPhone 

4. Press red circle to begin recording 

1. This technique allows for use if backwards facing camera for clearer videos while also allowing for visualization of the flies 

10. After the five minutes, with tape still on ring stand, press record. Allow for a few seconds with the tape in view and then peel tape off.  

11. Continue recording for at least 2 minutes. 

12. After recording, stop the video and bring flies over to a functioning CO2 pad. 

13. Turn on microscope light and flip dish upside down in order to see flies. 

14. Tilt the petri dish up towards the light (to trigger climbing up towards light) (figure 2). 

15. Sneak in CO2 gun at the bottom of the petri dish and blast them until they sleep. 

1. If flies try to escape pull gun out and reset. 

16. Once flies are asleep, take top off and shake the flies onto CO2 pad. 

17. From there, sweep the flies back into labeled food vial. 

1. Remember to keep food vial sideways while they wake up so they are not asleep in the food (they will die) 

*Note: some members of our team used CO2 gun method for knocking the flies out at the end of the test. While we found this to be reliable, this method still comes with a risk of losing flies, particularly if the flies are very active.  Another method for knocking the flies out after the behavioral test is to put them back on the ice, which was a method used by the other members of our team. *

Video Analysis

All of the video analysis for this project was processed through the application "AnimalTA." The video rendering software is incompatible with fly groups larger than approximately 5 individuals. It becomes confused and unable to separate out the individual data. The data that we selected for our graphs and to report is data that came out with clear and uninterrupted tracking statistics; all with movement samples from fly groups comprised of between 1-5 individuals over the duration of 2 minutes. The software tracks the individuals as they move around in a petri dish, calculating their average speed and distance traveled. 

Due to the aforementioned difficulties securing testable progeny from the SOAT1 line, we were not able to conduct tests for that gene strain. We did collect valid data for our additional crosses; alrmGAL4/+ and alrmGAL4; UAS-TDP43. 

AlrmGAL4/+:  We were not able to collect behavior data for week 3, as this week fell during spring break. We did collect data for week 1, however we were still discovering which recording/testing methods resulted in valid statistics. Due to this, week 1 data was not admissible. Data collected for analysis spans week 2 and week 4. There is no statistically significant change between motor movement for flies from week 2 to week 4. We were unable to do ANOVA due to the incomplete nature of our collected data, resulting in our data being inconclusive. 

When looking at the overall averages for the flies in each week you can see a slight decrease from week 2 to week 4. It doesn't appear to be a large change and can possibly be explained by normal aging or external factors in the lab (ex. temperature or lighting).

alrmGAL4; UAS-TDP43: We were able to successfully gather data for all four weeks (and a fifth week) of alrmTDP43 flies. As our data shows there is obvious variation between the different flies in each week and the average speed of all the flies in each week. The data did not necessarily fit what we expected to occur, as the week 1 flies demonstrated a slower average speed when compared to the other analyzed weeks. 

Variations and inaccuracy can be explained by some weeks having less total flies analyzed than others. For example, our week 3 data is the average of less flies due to spring break. We had trouble utilizing ANOVA with our data, but if we were able to complete this step it would allow us to better comprehend the statistical variation and significance. 

Unfortunately, this semester we were not able to complete our final cross, which was the SOAT1 virgin females with another set of double balancer vglutGAL4; UAS-TDP43 males. Our cross was unsuccessful at producing progeny before the flies died or got sick. Without progeny from this final cross, we were unable to run the petri dish test with our SOAT1 flies and collect associated data. As this is the final semester of the three semester course, we will likely not be able to continue with our experimentation on the SOAT1 flies. Hopefully in the future sections of this course, students can start fresh with the SOAT1 gene and find better success than us. 

Even though we were not able to collect the SOAT1 data, our team was able to successfully master a "new" method at analyzing fly behavior--the petri dish test. With this new method, we were able to run several tests on both positive and negative control glia flies to contribute to the class data. 

Overall, the biggest limitations that we experienced as a group were time constraints, sick flies that were required for our SOAT1 crosses, and navigating a new form of behavior testing and video analysis (Animal TA). We also ran into issues with our crosses taking longer than 10 days to produce offspring, with some of them taking as long as a two weeks to even enclose pupal cases. As we were able to successfully work out the major issues with Animal TA and the petri dish assay, future classes that decide to continue with these methods will have an easier time with managing the associated data and possibly get further in their data collection. 

Other Genes of Interest 

• It's really important to keep up with your flies! Make sure you are flipping your stocks regularly and keeping your vials relatively clean.

• Keep your vial box organized and well-labeled! This will save miscommunication/misinterpretation from happening when coordinating group efforts throughout the week.

• Always stay on top of your group notebook! It is a great method of effective team communication.

• I started making checklists for our groups every week, and I would recommend it because it was a nice way to split up tasks and make sure everyone knew what they were supposed to be doing. 

• Don't be afraid of failure!! Research is hard but also so rewarding.