Discipline: Biological Sciences
Brennetta Crenshaw - Alabama State University
Co-Author(s): Sparkle D.Williams2, B. Sims3, Q.L. Matthews1 1Microbiology Program, Department of Biological Sciences, College of Science, Technology, Engineering and Mathematics, Alabama State University, 2Departments of Pediatrics, Neurobiology and Cell, Developmental and Integrative Biology, Division of Neonatology, University of Alabama at Birmingham
Exosomes are small extracellular vesicles that are formed during the maturation of endosomes. The biogenesis of exosome changes based on external factors, such as alcohol exposure. As a small molecule, alcohol can easily cross membrane barriers and reach different parts of the body very quickly. Alcohol interacts with brain receptors, interfering with communication between nerve cells, and suppressing the excitatory nerve pathway. We hypothesize that exosomes protect against alcohol mediated apotosis in microglia cells. Therefore, the purpose of this project was to investigate the effects of alcohol exposure on the biogenesis and composition of exosomes derived from microglia brain cells, BV2. The BV2 cell lines was cultured in exosome-free medium and was either not treated (control) or treated with 50mM or 100mM of alcohol for 24, 48, or 72 hours. The cell morphology was examined through light microscopy. The groups of exosomes were isolated using a series of high-speed ultracentrifugation and quantitated using the Lowry dilution method. NanoSight analysis was used to identify and quantify the size of exosomes. Sodium dodecyl sulfate polyacrylamide gel electrophoresis was used to separate the exosomal proteins based on size and Coomassie blue staining was performed to identify the presence of exosomal proteins each treatment group. Enzyme-linked immunosorbent assay was performed on the exosomes to confirm the presence of exosomal proteins on BV2 cells.
Cell viability decreased after alcohol administration. Cells showed apoptotic behavior after 48-hour and 72-hour treatments after dosing with both concentrations of alcohol. Cells appeared to be shrunken compared to the control untreated cells. The average of three experiments demonstrated through fold change that alcohol treatment at 48 hours at the high dose caused a significant increase of tetraspanin CD63 expression in exosomes. In addition, alcohol treatment after 48 hours caused significant increases of heat shock protein 90 beta compared to the control. At 72 hour treatment with alcohol, we observed a significant increase of cytoskeletal protein actin expression in exosomes when compared to control. Our studies revealed that exosome biogenesis and composition was affected by alcohol treatment. However, more experiments will be conducted to gain additional knowledge on the impact of alcohol on exosome biogenesis in brain resident cells.
Funder Acknowledgement(s): This work was supported by the American Association of Immunologists Careers in Immunology Fellowship Program (to QLM), Gorgas Memorial Foundation Research Grant Award (QLM). National Science Foundation (NSF) grant HRD 1401075 (to GTJ). National Institutes of Health grant #5R01AI089337-04 (to QLM), and University of Alabama at Birmingham (UAB) Center for AIDS Research (CFAR), an NIH-funded program (P30 AI027767).
Faculty Advisor: Qiana Matthews, firstname.lastname@example.org
Role: I did majority of the work (cell culture, exosome purification and quantitation, ELISA, etc) on this project.