ALS is a neurodegenerative disease that causes degeneration of motor neurons and results in impaired movement and breathing, leading to death 3-5 years from initial diagnosis. There are currently no treatments that extend lifespan more than a few months. To understand the genetic causes of ALS, many genome-wide association studies (GWAS) and RNA sequencing (RNAseq) experiments have identified changes in genes or transcripts that are correlated with an ALS diagnosis. However, very few of these genes have been tested to determine if the change seen in ALS contributes to motor functional decline. 

The objective of our project is to establish whether genes predicted to be altered in ALS directly affect motor function. To answer this question, our team manipulates the expression of genes of interest in neuronal or glial cells in a Drosophila melanogaster model of ALS caused by the overexpression of TDP43 to induce ALS-like symptoms. Using RNA interference (to reduce function) or UAS lines (to increase expression), we then assess the motor functions of experimental Drosophila using two behavioral assays, negative geotaxis (stimulated climbing in a graduated cylinder) or unprompted walking (in a petri dish), and digital movement tracking software. Through our approach, we aim to bridge the gap between genetic predictions and functional evaluation using a simple model organism, potentially advancing therapeutic strategies.

Conclusions

The data collected with the petri dish assay seems to corroborate the trends we have seen in earlier semesters. Older flies move at slower speeds on average compared to younger flies. The over-expression of TIMP did have a noticeable effect on the movement of the flies. However, with the data that we have we can not be sure that this case. To make this assertion, we would need to run enough trials of both genotypes over multiple weeks and perform a statistical analysis. 

Throughout this semester, we faced many challenges in trying to implement the new assay, as well as investigate the genes of interest we chose. We initially were planning to set up crosses with the gene VAMP8, however, the stock for that gene was not healthy and we were not able to finish our cross setup. Because we devoted a lot of time to setting up crosses for VAMP8, we were set back in our TIMP Trials. We also had issues with a lack of standardization in our protocols for the behavior assays. Many of our earlier trials had to be thrown out because the recording quality was not good enough to properly utilize animal TA. We also had organizational issues due to the amount of time it was taking to complete crosses.  All of these challenges led us to collect less data than we were anticipating. Some of the limitations of our data include: 

• Lack of trials

• Inability to perform age comparison with all 4 weeks

• Unreliability of Animal TA

• Confounding variables in behavior testing 

1. Further develop petri assay methods: With our current procedure, there are frequent fly mortalities and movements that cannot be analyzed with animal TA. The procedure should be fine-tuned or we could move to a different type of assay like the twelve-well plate.

2. Explore other analysis methods: Animal TA can be difficult to use and the results that it generates have varying accuracy. The accuracy of Animal TA highly depends on how well the video was taken and how the analysis was set up. It might be beneficial to explore using other types of behavior analysis software. 

3. Research new genes associated with ALS:  Many RNAseq and GWAS papers on ALS have been published since we chose these genes last Fall. We should consider looking for more genes to study and see how they compare expression-wise to the genes we currently study.