By Sophia Nelson
One of the world’s leading neurodegenerative diseases, Amyotrophic Lateral Sclerosis (ALS), causes its destruction of the nervous system in largely unknown ways. Recent research has shown that a hexanucleotide repeat, GGGGCC in intron one of c9orf72, is the most frequent genetic cause of ALS, with the number of repeats in affected patients ranging anywhere from hundreds to thousands. While these repeat sequences are found in the non-coding region of the gene, they still appear to contribute to toxicity through repeat associated non-ATG translation, which can begin at any point without the presence of the start codon, ATG. This unconventional translation allows repeats to be translated into five different dipeptide repeat proteins (DPRs): GA, GP, GR, AP, and PR. One of these proteins, GA, forms paranuclear amyloidogenic aggregates which have been found in the brains of human ALS patients. However, the direct role of GA—or any DPR— in disease pathology and toxicity is not yet known, particularly because the toxicity of GA varies heavily based on the model system in use.
As a Velay Scholar this past summer, I got the chance to work with professors Robert Fairman and Roshan Jain to investigate the protein GA through a comparative study characterizing the aggregation and toxicity of GA in three model systems: worms (C. elegans), zebrafish (D. rerio), and fruit flies (D. melanogaster). The bulk of my work was in worms and flies, as my fish have not yet grown large enough for testing! I expressed GA in the neurons— ALS is known to attack motor neurons, so neuronal expression is important to study— in each of the two model systems and then performed behavioral and confocal imaging analysis for comparison. In the imaging studies, I was looking for multiple things: firstly, the large, paranuclear puncta that are hallmark of GA aggregation; and second, the localization pattern within each organism. Behaviorally, I was attempting to understand whether or not GA expression within neurons was toxic by comparing organism performance in simple behavioral assays both with and without GA. For worms, this behavior was thrashing; for flies, it was the ability of larvae to crawl. I learned so much throughout the summer! I dissected fruit flies (both larvae and adults) and removed and imaged their brains, which are barely the size of a poppyseed. I learned behavioral testing across all three organisms, as well as PCR genotyping, staining and imaging techniques, confocal microscopy, and the beginning stages of biochemical assays as well, such as lysate preparation.
The dissection of fruit fly larvae and brains showed that GA was heavily concentrated within their developing brains, particularly in the progenitor of the neural column and the neuronal ganglia that develop and associate with the eyes. The confocal images of my fly brains were likely the coolest result of my whole summer! In worms, GA puncta is found throughout the body; heavily concentrated in the brain and along both the ventral and dorsal cord. Behavioral assays indicated that the presence of GA had a negative effect on C. elegans thrashing. Larval crawling data was collected and differences between controls and GA positive larvae were found, but this data is still being followed up on to conclusively determine any effects. Biochemical results, obtained through SDD-AGE, will be gathered this spring. Overall, this study indicates that the form of GA aggregation is consistent across species despite slight differences in localization, and the presence of GA appears to have a negative effect on behavior, indicating it may have a role in disease toxicity and should be tested further.
Performing these experiments was an incredible experience! My mentors were both amazing and taught me so much. It was my first time working in a research lab, and as a biology major with minors in neuroscience minor and health studies, it has only confirmed my interests further. After graduation, I now plan on going to medical school and graduate school for an M.D.-Ph.D so that I can continue to perform research while also gain a clinical perspective and directly help patients. I am so excited to be able to apply the knowledge I gained this summer (and will continue to gain in my last year at Haverford) in my future career and work with a topic that has the potential to have a direct impact on many people’s lives.