I had the great opportunity of continuing my lab work under Dr. Kelly Jordan-Sciutto working on “Investigating the role of BACE1 Substrates on Neurite Regrowth following Induced Injury.” The CURF Jumpstart for Juniors grant allowed me to continue my research here while at the same time establishing a more independent research project. The grant and applying for the grant allowed me to develop a more personal relationship with my PI and my post doc who helped guide me through our experiments and work together.
A main focus of the Kelly Scuitto lab is BACE I, (β-site amyloid precursor protein-cleaving enzyme), which is critical for generating Aβ by cleaving Amyloid Precursor Protein (APP) into Aβ, which leads to oligomerization and ultimately plaque formations that are observed in neurodegenerative diseases, including Alzheimer's Disease. We used this focus of the lab, and looked at another mechanism of interest, the ER stress response and inflammation associated with neurodegeneration and traumatic brain injury. With this in mind, a substrate cleaved by BACE1 that may have direct involvement in recovery of neurons from injury is L1/CHL1. L1, a transmembrane protein member of the L1 protein family, is an adhesion molecule that plays a role in neuronal cell adhesion, migration, myelination, neuronal differentiation, and neurite outgrowth (Zhou, 2012). Thus, our goal for the study was to show and determine through various methods that BACE I inhibition prevents the cleavage of L1/CHL1, resulting in the reduction of neurodegeneration following induced injury.
This led us to the beginning of summer and developing our conditions and inhibitors for our study. I still remember when I met my post doc and summed up being in science as “everything usually goes wrong, or doesn't work 90% of the time.” I got to fully experience this summer. With other researchers echoing this sentiment we went forward with our project and experienced some of these difficulties. We initially had some trouble finding the right inhibitor and the right conditions/ controls that would not kill our cells. We initially were using a larger concentration of DMSO which was the standard of our lab, but were able to find the right concentration. We also troubleshooted the right time of incubation for our cells to be in the prime time to perform the scratch essay, the experiment that simulates injury and collect protein from the scratch. We moved forward and worked out the kinks of our experiments and collected data and performed experiments every week. The experiments included culturing cells every week, changing media, collecting protein samples, performing scratch assays and western blots every week.
A few weeks in, our lab had a contamination problem and dealt with a bit of contamination that set us back in the beginning portion of our research project. At the same time, my mentors would always say “you’re getting the full science experience, you get contamination when you need data the most” With a good attitude and hard work we kept at it and got the project to go smoothly for the remainder of the summer up to the end of the project where we performed western blotting to analyze the protein collected. At the end of the project we got data indicating that our hypothesis was correct and we saw an effect after inhibiting BACE 1. We were able to see an increase inneurite outgrowth as we hypothesized.
Overall, my experience in research this summer was amazing. I was able to a part of a team of world class researchers and work together on a common scientific goal. I got the opportunity to learn and perform new techniques like Scratch Assays, cell culture, Western blotting, cell staining and live imaging. It is amazing that I was able to get the opportunity to perform and learn these techniques at this stage of my Penn career. This research experience solidified my passion for science and my interest in pursuing a career in science. We are proud of our project and thankfully there is still a lot left to explore and different mechanisms to look at in the future! I’m really thankful for the donors, the CURF Staff, my PI and post doc, and the CURF Jumpstart Junior grant for helping make it possible and giving me an amazing research experience!