Science in Seattle

Our research started in Colorado where we worked for 9 weeks over the summer in our lab at Colorado College under Dr. Meredith Course. The purpose of our research was to develop fly models of beta-oxidation disorders and deficiencies. Our goal was to investigate the metabolic disruption that occurs in Drosophila Parkinson's disease lines. We did this by analyzing the flies' acylcarnitine profiles. Aceylcarnatines are important because they play an essential role in the transport of long-chain fatty acids to the mitochondria that are needed in beta-oxidation, a major cellular energy source.

Flies thrive in moist climates, so it can be challenging to have them prosper in dry Colorado. To combat this, we put extra water in their food so it doesn't dry out and keep them in temperature-controlled incubators.

We began this research by learning how to sex Drosophila. Once we could do this with confidence, we then collected a total of 15 vials (5 male vials, 5 female vials, and 5 mixed vials) with five flies in each, aged our wild-type stock for four days, and sent them to our collaborator Dr. Anna Scott who works in a lab at Seattle Children's Hospital in Seattle, WA. Dr. Scott then took our samples and ran them through the mass spectrometer to get the flies' acylcarnitine profile and calibrate the machine.

In order to prepare the samples for shipping, we had to extract the lysate from the flies. To do this, we followed the procedure below:

• 5 flies per sample were homogenized in 50ul of 3:1 methanol and acetonitrile

• Centrifuged to remove debris for 2 minutes at 10,00rpm

• Remove and save supernatant

• Send samples to Seattle

Once we received our new data, we were able to then compare male vs. female acylcarnitine profiles of the wild-type flies.

We analyzed 18 different acylcarnitines in total, but for concision and clarity, there are only three on this graph. The general trend across all graphs was that female flies had a higher acylcarnitine quantity than both the male sample groups and the mixed sample groups. This is because female flies are typically larger meaning there is more lysate per sample. Another finding from this preliminary analysis is that our flies are B12 deficient which means that we should supplement their food. However, we did not end up doing this, so we see this same deficiency in our later results.

After we had the baseline acylcarnitine profiles for the wild-type flies, we began to look at flies with known Parkinson's mutations. Specifically, we were looking at flies with the PINK1 B9 and PINK1 RV mutations.

In order to know which flies actually were carrying this mutation, the flies had a balancer chromosome, which works to preserve the deleterious mutation throughout generations. Balancer chromosomes are able to do this because they prevent recombination events in meiosis. The balancers carry phenotypic markers so you can easily tell which flies have the mutation. For example, we were looking at flies that had the bar-eye (FM7) balancer chromosome.

Our next step was to collect male flies that had the wild-type phenotype and age five vials for 1-2 days and another five vials for 14-15 days. We collected male flies because the balancer is on the X chromosome, insuring that only the males were full carriers of the deleterious mutation. We then followed the same procedure as we did for the wild-type flies, and sent our samples to Dr. Scott. This time, she waited for our arrival so that we could prepare our samples to be placed into the mass spectrometer.

The next week we flew to Seattle to get hands-on experience in Dr. Scott's lab.

The procedure we followed when to prepare them for acaeylcarnitine analysis is below:

• Included high, low, abnormal, and negative controls were included using 20 ul of sample - mass spec300 ul of isotope-labeled internal standards in acetonitrile and 0.4% formic acid were added to each sample

• Samples were vortexed and centrifuged for 5 minutes at 13,000rpm

• Supernatant was decanted into glass reaction vials and dried for at least 45 minutes under nitrogen

• Acylcarnitines were re-suspended and derivatized in 100ul of 3N HCL in butanol and incubated at 65ºC for 15 minutes

• Samples were dried again under nitrogen for at least 1 hour

• Samples were finally re-suspended in 200ul of 80% acetonitrile

• Samples were then ran in the mass spectrometer

Once the samples were processed, we were given data that we will then be able to analyze in the same fashion as the graph above. It will be interesting to compare our acylcarnitine analysis results of these mutagenic flies with the wild-type ones.

Our next steps for this project are to keep the same methodology but do this with flies that have different mutations associated with Parkinson's disease and then finally, with associated Alzheimer's disease mutations as well. This research is important because it allows us to learn more about neurodegenerative pathologies and lays the groundwork for future biomedical research.

We are incredibly grateful to the Keller Family that we were chosen to be recipients of the Venture Grant. We walked away from this experience feeling proud of all of our hard work this summer and confident in our abilities to succeed as young scientists. We truly cannot thank you enough for this valuable experience. Hands-on, immersive experiences are the most valuable to our development in the sciences, and they would not have been made possible without you. We would also like to thank our wonderful advisor, Dr. Meredith Course, for supporting and guiding us through this research. We are excited to continue working with her this spring.