Team 4

Navya Balaji, Peggie Chen, Ariana Decker, & Kendall Kopek

Introduction

Our research aims to investigate and comprehend the genes associated with ALS, using Drosophila melanogaster (commonly referred to as fruit flies) as our model organism. By altering the gene expression of drosophila, we aim to observe and understand how physiology and locomotor abilities are impacted over time.

This VIP course gives students comprehensive exposure to the process of conducting research and the opportunity to work in teams to work towards a common research goal.

Background

What is ALS?

ALS stands for Amyotrophic Lateral Sclerosis, which is a progressive neurodegenerative disease that affects nerve cells in the brain and the spinal cord. This degeneration leads to the deterioration of voluntary muscle movement, impacting functions such as walking, speaking, swallowing, and eventually breathing. As the disease progresses, individuals with ALS may become paralyzed and require assistance with daily activities. Currently, there is no cure for ALS, and treatments focus on managing symptoms and improving quality of life.

More About Our Flies

Why Drosophila; What do they look like?

Drosophila Melanogaster, a prevalent fly often located in proximity to both unripe and decaying fruit, has served as a prime subject for genetic and behavioral studies for more than a century. Thomas Hunt Morgan, a prominent biologist of the early 1900s, pioneered research on Drosophila, making groundbreaking discoveries such as sex-linkage and genetic recombination. These findings propelled the small fly to the forefront of genetic exploration. Thanks to its compact size, simple cultivation requirements, and rapid generational turnover, geneticists have continued to utilize Drosophila as a valuable model organism ever since.

Genes of Interest

Our group was assigned to make genetic crosses that express our genes of interest in the glial cells of drosophila. As such, each cross is done between the alrmGAL4 genotype and the gene of interest. The Luna gene in particular was chosen by using Google CoLab coding commands within data sets to find statistically significant genes impacted in individuals with ALS and find their corresponding fly ortholog.

alrmGAL4 x W1118

This cross is our negative control, as its progeny are wild type flies whose locomotor functioning is normal

alrmGAL4 x UAS-TDP43

This cross is our positive control, as it results in the overexpression of the TDP43 gene in glial cells to mimic the most severe locomotion issues in humans with ALS
The TDP43 gene contributes to the synaptic growth of motor neurons and glial wrapping

alrmGAL4 x UAS-LunaRNAi

The Luna gene is a transcription factor in drosophila and is particularly important in cell proliferation and development
The Luna gene enables sequence-specific DNA binding activity and RNA transcription regulation
RNAi allows for the transcription of the Luna gene, but interferes with the RNA such that it cannot be translated into proteins; this makes Luna a knockdown gene without completely removing it
Human gene: SP8
Luna is downregulated in ALS
Log 2FC: -0.39998
Adjusted p-value: 0.000963

Genetic Crosses Drawn Out

Behavior Testing

Our group chose to conduct behavior testing with the negative geotaxis design. This test measures locomotion and climbing ability by placing flies into a 250mL graduated cylinder, knocking them to the bottom, and observing the number of flies that climb up to/past the halfway mark (at 120mL). The behavior test is conducted over the course of two minutes.

The procedure done to conduct a negative geotaxis behavior test is as follows:

On a piece of paper, write down all relevant information about the cross of the flies you are testing. It will include the genotype of the flies, date of emergence, date of the test, number of flies, and team number.
Transfer files from their vial to the graduated cylinder through a funnel to make transfer smoother and no flies escape during the process.
While tapping the graduated cylinder on the table to ensure no flies escape, quickly place a square of Parafilm over the top of the graduated cylinder.
Allow the flies to adapt to the new environment for approximately one minute. While waiting, get a stopwatch ready and set up your filming device (a cell phone works fine, but make sure that it is stable and secure, and that the halfway mark on the graduated cylinder is clearly visible).
Start your filming device and show the piece of paper for a few seconds.
With moderate force, tap the graduated cylinder onto the table ten times to knock all of the flies to the bottom.
Start the stopwatch immediately after the tenth tap.
After 2 minutes, stop the filming device.
To transfer the flies back into their original vial, place the funnel into the vial, and while tapping the graduated cylinder on the table, quickly remove the Parafilm from the top before quickly flipping the flies back into the vial. You may need to tap the entire apparatus on the table in order to ensure that all flies make it back into the vial.

The process of analyzing the behavior videos requires recording how many flies reach/pass the tape mark every ten seconds.

Results

AlrmGAL4 x w118

AlrmGAL4 x UAS-TDP43

AlrmGAL4 x LunaRNAi

Conclusions

General observations across all 3 genotypes:

Based on the overall statistics and graphs generated with ANOVA, we found that there is a significant difference in the percentage of flies that climb the cylinder walls between week 1 and weeks 4-5. Flies that are one week old or younger are able to express stronger locomotor responses as compared to flies that are four to five weeks old. At around four weeks old, we observed weakened locomotion and climbing ability in the flies.

Observations of progeny of alrmGAL4 x W1118 cross:

The p-value of the wild-type progeny is 4.1 x 10^-7, which is significantly less than 0.05, making the data significant. Generally, the highest percentage of flies that climbed were Week 1 flies, and the lowest percentage of flies that climbed were Week 4 files, with slight overlap with Week 3 flies towards the end of the 2 minutes, as demonstrated by the graph.

Observations of progeny of alrmGAL4 x UAS-TDP43 cross:

The p-value of the UAS-TDP43 progeny is 0.0066, which is less than 0.05 and therefore statistically significant. Similarly, the Week 1 flies demonstrated the best climbing ability, followed by Week 2, Week 3, and Week 4, respectively. However, we see a dramatic decrease in the overall percentage of flies climbed in Week 4 flies. This makes sense intuitively considering that the UAS-TDP43 gene impairs locomotion as age progresses.

Observations of progeny of alrmGAL4 x UAS-LunaRNAi cross:

The p-value of the UAS-LunaRNAi progeny is 0.035, which is statistically significant, as it is less than 0.05, but not as dramatically significant as the data collected from the wild-type and UAS-TDP43 progeny. However, the trend is the same in that Week 1 flies demonstrate the best climbing ability, and Week 4 flies demonstrate the most impaired climbing ability.

Next Steps

Our next steps to create a better experiment:

1. Validation and Replication: Replicate our findings with a larger sample size and across multiple experiments to ensure the robustness of our results. This will increase confidence in the observed differences between age groups.

2. Longitudinal Study: Extend the duration of our experiment to track the decline in locomotor responses over a longer period. This will provide a more comprehensive understanding of the progression of muscle weakness with age.

Limitations of the Study:

1. Age-Specific Effects: The observed decline in locomotor responses may be influenced by factors other than age, such as environmental conditions. Further experiments controlling for these variables are needed to confirm age-related effects.

2. Generalizability: The findings from Drosophila melanogaster may not directly translate to humans. Consideration of species-specific differences and validation in mammalian models or human studies is necessary to generalize the results to ALS patients.

3. Gene-Environment Interactions: The impact of ALS-related genes on muscle function may vary depending on environmental factors. Future studies should explore gene-environment interactions to better understand disease mechanisms.

References

An introduction to fruit flies. The Berg Lab. (2017, July 11). https://depts.washington.edu/cberglab/wordpress/outreach/an-introduction-to-fruit-flies/

BCV for Als. Hayek Medical. (2022, August 2). https://hayekmedical.com/bcv-for-als/

The ALS Association. (2021, April 26). What is ALS? The ALS Association; The ALS Association. https://www.als.org/understanding-als/what-is-als

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