Team 2

Firass Aldahlawi - Heather Lambeth - Brooke Staples

Introduction and Purpose

We are studying drosophila to learn more about how various genes utilized in neurons and neuroglia that could possibly increase the ability for ALS become more likely. Our Goal was to determine genes that affected the locomotion of our model organism, drosophilia (fruit fly). We did this by setting up genetic crosses and performing locomotion tests

Background Information

ALS: What is it?

ALS is a fatal neurodegenerative disease in which motor neurons are degenerated and destroyed, leaving the afflicted without basic motor abilities, such as walking or even chewing. ALS stands for Amyotrophic Lateral Sclerosis, and it is also known as Lou Gehrig's disease. There is currently no cure for ALS, although the progression of the disease can be hindered by medication and therapy. ALS can be genetic, although this only correlates to 5-10% of all ALS cases. ALS is often fdeveloped spontaneously, with no genetic background.

Our genes:

CAMllK - This gene is responsible for regulating synaptic plasticity, excitability, and cytoskeletal interactions in neurons. It is down regulated in ALS.

DAB2 - An adapter protein coding gene that is utilized in modulating growth factor/Ras pathways. The gene is involved in the development of the central nervous system in mammals as well as endocytosis and protein binding. This gene is up-regulated in ALS and is normally turned off in drosophila.

CG5181 - A coding gene for nucleic acid binding protein; this is significant for DNA replication, recombination, and repair in flies and humans. It is upregulated in ALS. This could be because the gene causes the DNA in neurons with ALS to not be maintained as well, which would affect motor skills.

Hypotheses:

CG5181: If CG5181 plays a significant role in DNA replaicaiton, recombination, and repair, then we can expect an upregulation in ALS to lead to motor decline.

DAB2: If DAB2 modulates growth factor/Ras pathways, then we can expect an upregulation in this gene to lead to overall motor decline and ALS symptoms.

CAM11K: If CAMIIK is responsible for regulating synaptic plasticity, excitability, and cytoskeletal interactions in neurons, we can expect a downregulation in this gene to result in ALS symptoms and overall motor decline.

Model Organism: Dr0sophilia

Drosophilia, also known as the fruit fly, are perfect model organisms for ALS testing. They have a very similar genetic background to humans, and we can often find fly orthologs to match human genes, which makes them perfect for genetic testing

Tips and Tricks:

Make sure to use funnel to aid in smooth transfer of flies
Write as much information about the flies you are testing as possible including team name, genotype, date tested, birth data, and etc.
Avoid testing under 10 flies, as this may not be the best for data collection

Video analysis:

After recording of the video it is important to analyze the data properly

Open spreadsheet using data collection template provided
In tandem, have the video playing to simplify this task
Count the number of flies that cross the 110 mL line every 10 seconds, pausing after each time period
Double check that all 10 second time periods are filled in

Fly Crossing

We crossed flies with the hsp70flp gene and flies with the alrm-GAL4 gene to use the progeny in locomotion assays.

Vial Organization

We started to organize the behavior and cross vials by assigning them names unique names. This way communication is easier.

Methods:

Locomotion assays are very important in testing the locomotor abilities of flies. We have devised a series of steps to follow in order to properly collected data from both control and experimental flies. The testing period is two minutes.

Steps:

Determine if you have all needed materials

Video recorder
Graduated cylinder with tape at 110 mL
Test flies
Funnel
Parafilm to seal cylinder
Paper with fly information to show at beginning of video

Quickly transfer flies from vial into graduated cylinder using funnel, tapping graduated cylinder continuously to ensure no flies escape
Place parafilm over top of graduated cylinder after all flies have been moved
Allow the flies to rest for one minute in order to allow them to get used to their new surroundings
Start video and present information about the flies to the camera
Tap graduated cylinder in erratic and random pattern and then place in circle indicated on desk and start timer for two minutes
Observe fly climbing behaviors and stop timer after two minute period
Transfer flies back to their original vial using funnel and tapping motions

Flipping onto new food for stock maintenance

Make sure to flip flies onto new food at around every two weeks, as this ensures that the flies have enough food
This also ensures the progeny and parents are kept separate to avoid reproduction and skew results

Virgin collection is an essential part of this lab, and ensures the success of crosses

Tips:

Reference the photo above if not certain if a fly is a virgin female
Look for dark spot in side of the abdomen
Make sure the virgin is a female, as virgin males look similar

Results: Control - Alrm GAL4

Data Interpretation:

The data from the locomotion assays were separated by age, (WK1,WK2,WK3,WK4). After separation, the percent of flies climbed has then between multiple trials has been averaged, to create a second data set that shows that average for each week and created a line graph that represents it (Graph A). Following, we utilized stats.blue to conduct the statistical analysis, specifically one way ANOVA (Table A) and Tukey pairwise comparison (Table B). That has been done to allow us to obtain p-values and to determine if the difference between the ages of the flies is significant or not.

Graph A

Table A

Tukey Pairwise Results

Table B

Conclusions

Through the data collected on the alrm-GAL4 control group flies we can see a gradual decline in fly activity as they age each week. We can use the trend created from our data to compare RNAi line flies in the future. We were able to gain some data through locomotion assays on the CAMKII and Dab2 genotype flies, but not sufficient enough testing was done to draw any conclusions.

CG5181: This gene was successfully crossed, though we did have a hiccup when our original vial was lost. There were two RNAi strains of this gene that we examined. Although we were not able to garner any data from this genotype this semester, there are future implications for it next year.

Dab2: Dab2 was not able to reproduce fast enough for sufficient progeny collection and testing, however it will be prioritized in the next semester. We were able to test Dab2 two times, with a sufficient amount of flies.

CAMKII: CAMKII was not able to reproduce fast enough for sufficient progeny collection and testing, but it will be prioritized in the next semester. We were able to perform locomotion tests on these flies.

Something that was important to our team this semester was the importance of organization of the vials. Through trial and error we determined the best way to sort the vials and communicate what needs to be done every lab section

We found that labeling the vials with fun names was a great way to increase awareness of what each vial is and allowes communication between teammates, especially because not every lab section will have more than one person. Below is a photo of how this can be helpful, as you can see we referred to the vials as Lilly and Dave. The corresponding vials were labeled in colorful tape placed on the bottom.

Next Steps

We hope to use the knowledge we gained this semester to become further educated in genetics, fly pushing, and setting up successful crosses. Much was learned in terms of how to proceed in this course, especially when it came to data analysis, and introduction into coding, and how to set up genetic crosses to cause an ALS genetic background in our flies. In addition, we were able to work with three genes that all have further implications in ALS research and testing. CG5181, DAB2, CAMKII all will explored further as the course continues. We believe that these genes can be explored further, especially considering we were able to get far enough to perform locomotion tests on two of our strains. The loss of our CG5181 flies was unfortunate, however that is one more promising strain to study further in the coming semester.

Coding was a focus for this semester, however we were never able to figure out how to merge the datasets in order to determine which genes could be found in both. We are hoping that the general coding knowledge learned this semester can be parlayed into coding next semester and the kinks can be figured out as more students are working on this issue. Overall, learning how to extract data from large datasets is an invaluable skill that should be emphasized to the incoming students. The students in this course get better every semester

References

"What is ALS - Amyotrophic Lateral Sclerosis | The ALS Association." The ALS Association, https://www.als.org/understanding-als/what-is-als.

D'Erchia, A. M., Gallo, A., Manzari, C., Raho, S., Horner, D. S., Chiara, M., Valletti, A., Aiello, I., Mastropasqua, F., Ciaccia, L., Locatelli, F., Pisani, F., Nicchia, G. P., Svelto, M., Pesole, G., & Picardi, E. (2017). Massive transcriptome sequencing of human spinal cord tissues provides new insights into motor neuron degeneration in ALS. Scientific reports, 7(1), 10046. https://doi.org/10.1038/s41598-017-10488-7

Huang, C. H., Cheng, J. C., Chen, J. C., & Tseng, C. P. (2007). Evaluation of the role of Disabled-2 in nerve growth factor-mediated neurite outgrowth and cellular signalling. Cellular signalling, 19(6), 1339–1347. https://doi.org/10.1016/j.cellsig.2007.01.019

Kim, W., Kim, D. Y., & Lee, K. H. (2021). RNA-Binding Proteins and the Complex Pathophysiology of ALS. International journal of molecular sciences, 22(5), 2598. https://doi.org/10.3390/ijms22052598

Shen, K., & Meyer, T. (1999). Dynamic control of CaMKII translocation and localization in hippocampal neurons by NMDA receptor stimulation. Science (New York, N.Y.), 284(5411), 162–166. https://doi.org/10.1126/science.284.5411.162

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