- Title of Project:
Regulating Neural Exosome Production in Alzheimer’s Patients
- Statement of Purpose:
Through this research we hope to determine which genes and chemicals regulate exosome production in neurons. By regulating exosome production, we hope to limit the spread of the Tau tangles in the hippocampus and the neocortex that are prevalent in patients with Alzheimer’s Disease (AD). The goal is that with limited Tau spread, the tangles that interfere with brain function and memory can be prevented.
- Background:
I have taken Honors Chemistry and Biology, AP Chemistry, Capstone Chemistry (which covers first semester Organic Chemistry) and am in the process of taking an online AP Biology course. I have planned on studying the sciences since I was ten, and this research is the perfect opportunity to learn more about the subject and typical laboratory procedures. More recently I have decided that I want to major in Biochemistry, and this is the perfect internship in order to prepare me for that future.
- Significance:
According to the Alzheimer’s Association’s “2012 Alzheimer’s Disease Facts and Figures,”
Alzheimer’s Disease is the sixth-leading cause of death in the US. One in eight people age 65 and older has Alzheimer’s, and the total number of people of all ages with Alzheimer’s is estimated to be 5.4 million, ad that number is expected to rise with the aging of the baby-boom generation.
Alzheimer’s patients suffer from impaired brain function and memory due to the spread of Tau tangles in the hippocampus and neocortex. By containing the Tau to the entorhinal cortex, we would prevent the buildup of tangles in the hippocampus, the memory center of the brain. While it wouldn’t cure AD per se, it would certainly limit the damage it could do a patient’s brain and life. Similarly, because the Tau is spread through trans-synaptic gaps, all of the areas monosynaptically connected to the entorhinal cortex would be likewise free of Tau tangles.
We hypothesize that regulating exosome production will help limit Tau tangles. Exosomes were discovered relatively recently, and were thought to be just little garbage cans of the cells. But in 1996, they were discovered to also function as a type of intercellular communication. They could be packaged with anything from proteins to digested pathogens to bits of RNA, all of which can get transferred and integrated with other cells. They are involved in maintaining the immune system, although they can cause both beneficial and harmful effects. They also have the same relationship with cancer, sometimes hindering and sometimes helping its progression. They often have receptors on their membrane that correspond to different types of cells that they communicate with. Occasionally, they will carry in them the necessary component to trigger apoptosis (cell death) in the receiving cell.
- Description:
I will be doing laboratory research at TGen for most of the time. Time not spent at TGen will be spent learning protocols and reading the articles that are sent to everyone in the lab via email. The hours are very irregular, and some weeks will consist only of making sure the cells are growing properly. Other weeks, however, considerable time will be spent in the lab on an almost full-time basis.
- Methodology:
First, we will take six different kinds of cells (Cath.a, Neuro-2a, PC-12, SH-SY5Y, H4, and HELA) and grow them in a cell culture until they are at 90% confluency (a measure of cell density on the bottom of the dish) at which point we will split them, one to return to the freezer, one to use as a sample. We will then force the exosomes to package a protein called PLAP to use as a marker. After 48 hours, we will centrifuge the samples and collect the supernatant containing the exosomes. Then we will lyse the cells, color the PLAP proteins and keep two of the cell lines based on exosome count. The two chosen cells will then be run through a drug screen and a gene screen to determine which chemicals and genes control exosome production.
- Problems:
There is a potential problem in that I probably won’t be able to finish this project before the end of the school year, however, I hope to continue the research in the summer.
The samples could get contaminated during one of the many processes that we have to put them through.
- Bibliography:
Liu, L., Drouet, V., Wu, J. W., Witter, M. P., Small, S. A., Clelland, C., & Duff, K. (2012) Trans-synaptic spread of Tau pathology In Vivo. PLoS ONE 7(2): e31302. Retrieved from: http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0031302
Phelan, M., (2006) Appendix 3F, Techniques for Mammalian Cell Culture. Current Protocols in Molecular Biology
Downing, M. (February 2, 2012) Basic Disinfection Practices for Tissue Culture Laboratories. Retrieved from: http://www.brighttalk.com/webcast/6639/40843
Slish, D. (1999) Bacterial Transformation Retrieved from: http://faculty.plattsburgh.edu/donald.slish/transformation.html
Théry, C., Clayton, A., Amigorena, S., Raposo, G. (2006) Unit 3.22, Isolation and Characterization of Exosomes from Culture Supernatants and Biological Fluids. Current Protocols in Cell Biology
Théry, C. (July 2011). Exosome Explosion. The Scientist, 25, 7.