Andrew Romney and Dr. Sterling Sudweeks, Physiology & Developmental Biology
For the past two years, I have worked in Dr. Sterling Sudweeks’ research lab experimenting with neurotransmitter receptors that are implicated in the development of Alzheimer’s Disease. When functioning normally, these receptors act as ion channels which open upon binding nicotine or acetylcholine and are thus appropriately named nicotinic acetylcholine receptors (nAcRs). nAcRs’s are found at the neuromuscular junction as well as in nervous tissue and are comprised of 5 subunits. Dr. Sudweeks’ previous research suggests that the alpha 3 and beta 2 subunits are the most common subunits in receptors in the hippocampus, which is the primary area of the brain damaged in patients with Alzheimer’s Disease. Alzheimer’s patients have increased levels of the protein beta amyloid in the brain, which has been shown in hinder the function of local nAcRs. My research has focused on the alpha 3 and beta 2 receptor subunits and their interaction with beta amyloid in an effort to better understand how nAcR function is altered in Alzheimer’s patients.
It is difficult to experiment on these receptors in natural tissue because there are generally several other types of receptors nearby, so to obtain more accurate results we create a more ideal experimental environment by injecting mRNA coding for the desired receptor subunits (alpha 3 and beta 2) into Xenopus oocytes. After about 48 hours the mRNA is expressed and the nAcRs are inserted on the cell membrane. This setup is useful because there are no other kinds of neurotransmitter receptors on the membrane and because the cell is large enough to be seen with the naked eye. If the mRNA is properly expressed, ion channels open in the presence of acetylcholine, causing a measurable change in the membrane potential. Using voltage-clamp electrophysiology, the resting membrane potential is measured before application of acetylcholine, and we record on a computer the change in membrane potential as acetylcholine is added. We analyze this data by considering such aspects as the time it took for the channels to open, the length of time they remained open, and the extent of desensitization upon sustained acetylcholine application.
Other researchers have shown that the kinetics of nAcRs comprised of different subunits are not identical to each other. Likewise, receptors made of the same subunits but in different ratios also have dissimilar kinetics. Our research lab hypothesized that the effects of beta amyloid on nAcRs comprised of two alpha 3 and three beta 2 subunits would differ from the effects on receptors with three alpha 3 and two beta 2 subunits. Unfortunately, we have not yet been able to obtain the data needed to support this hypothesis. There were been several unforeseen obstacles that delayed our research. We began with 10 Xenopus frogs to rotate through and collect oocytes from which allowed us to gather eggs every week for our experiments, but over the course of 5 months 4 of the frogs died, leaving us unable to experiment nearly every other week. One of our pieces of equipment broke in March which delayed us another week while we waited for the replacement to be shipped. The main problem, however, was that we were unable to consistently produce oocytes with functioning nAcRs. Because beta amyloid is so expensive, we didn’t want to purchase it until we were confident that we wouldn’t be wasting it. After brainstorming things we could do to help assure we would regularly construct eggs with functioning receptors, we made a few changes to our protocol. We decided to use gloves with RNA-ase Away more often and avoid breathing on the RNA, we purchased a new microinjector to make cleaner RNA injections into the eggs as well as new plasmid DNA to make new uncontaminated mRNA from, and we began making fresh batches of acetylcholine solution every week. Following these fine-tunings, we finally ordered some beta amyloid in April, but I never had to chance to experiment with it because I moved shortly after graduation. With continued effort, I expect our lab will obtain the desired results by the end of this year.
Despite all the difficulties we faced, Dr. Sudweeks submitted an abstract of our research to Alzheimer’s Association and we were invited to present a poster of our research at the International Conference of Alzheimer’s Disease in Honolulu on July 12. Six of us will attend the conference with Dr. Sudweeks to represent our lab and share our ideas with researchers from across the world.