Mentor: Dr. Habibeh Khoshbouei
College of Medicine
"Many individuals suffer from diseases such as schizophrenia, depression, etc. The complexity of the human nervous system brings a great challenge for scientists who are trying to understand the mechanisms and causes of neurodegenerative diseases. This huge impact on the society and the mysterious nature of the brain, have made me interested in research that focuses on the effects of pharmacological agents on our nervous system and neurodegenerative diseases. I took the SFL Seminar course my freshman year and I had to interview a faculty member. After reading some of Dr. Habibeh Khoshbouei’s past publications and researching about her area of work, I was really interested to know more about her ongoing project, and I asked to volunteer in her lab. Gradually, I started working on my own independent project under her supervision. Working in the lab, attending and presenting in lab meetings every week, learning to be responsible for and committed to my project and also working with the other bright lab members have been such a great experience for me. As a student majoring in biology, working in a molecular biology lab has been very exciting for me. I can observe and apply what I learn in class every day and relate them to my project. Sometimes, I share my ideas with other lab members and my mentor and we have very open, interesting discussions. They teach me how to think critically and have an analytical mind. Apart from all the advantages, doing research for almost 15 hours a week can sometimes be stressful because I also have to focus on my schoolwork as well. I have learned the important skill of time management and being able to manage time is a critical skill for every undergraduate student at UF. Also, sometimes an interesting discussion with a lab member, a successful presentation or observation of a live cell under the microscope can turn a stressful school day to a great satisfactory day; therefore, I think that participating in this research program would open another door of great opportunities for me to learn from the great experience. As I am planning to continue research after graduation, I believe that this research program will strengthen my education and enhance my abilities as an undergraduate and therefore prepare me for my future career. I will continue my research under the supervision of Dr. Khoshbouei. We have constructed a research plan that will take at least a full year and we plan to present the data in scientific meetings. "
- Dopaminergic System
- University Scholars Program, 2012-2013
- UF HHMI Science For Life
- Best undergraduate poster award at STEM Symposium in Emory University
- Best undergraduate poster award at UF Brain Awareness Symposium
- Summer Undergraduate Research Internship (SURE)
- UF Neuroscience Club
- American Society for Pharmacology and Experimental Therapeutics (ASPET)
- North Central Florida Chapter of Society of Neuroscience
- UF Shands Hospital
- Microbiology Teaching Assistant
- Brain Awaremess Week
Hobbies and Interests
Methamphetamine and Amphetamine Differentially Influence Biophysical Properties of Dopamine Transporter
"Psychostimulants such as amphetamine and methamphetamine target dopamine transporter (DAT). The neuronal plasma membrane dopamine transporter (DAT) is essential for the maintenance of DA homeostasis in the brain. Dysregulation of DAT function is implicated in neurodegenerative diseases, drug addiction, and neuropsychiatric disorders. Previous findings at Khoshbouei lab show that amphetamine and methamphetamine differentially regulate the activity of dopamine transporter. The long-term goal of this study is to determine the molecular mechanism of amphetamine and methamphetamine regulation of dopamine transporter.
Membrane proteins are distributed in the plasma membrane; however, this distribution is not random. Membrane proteins, predicted to be segregated into distinct plasma membrane domains with specific lipid compositions. These microdomains include so-called “lipid rafts” or “membrane rafts” are defined as small (10−200 nM) dynamic sterol- and sphingolipid-enriched structures. These structures are associated with intracellular protein networks and plasma membrane microdomains that ensure appropriate spatial and temporal regulation of membrane proteins. Recent reports suggest that membrane microdomain distribution of membrane proteins regulates the function of these proteins. The dopamine transporter is distributed in the raft and non-raft membrane microdomains. The microdomains distribution of DAT influences the interaction of the transporter with the regulatory proteins, and thus DAT function. This ensures proper spatial and temporal regulation of transporter function. Although amphetamine and methamphetamine differentially alter the activity of DAT, at present, it is unknown whether amphetamine vs. methamphetamine interaction with DAT, differentially stabilizes the transporter in a specific membrane microdomains. In this study, I will use a fluorescent microscopic approach to 1) visualize the presence of lipid rafts in the Human Embryonic Kidney cells (HEK) and cultured midbrain dopamine neurons, 2) determine the membrane distribution of DAT in lipid rafts, and non-raft membrane microdomain in cells expressing YFP-DAT and 3) study the influence of amphetamine and methamphetamine on these raft-transporter complexes by time-lapse imaging of labeled dopamine transporter in the above model systems. I will test the hypothesis that DAT association with specific microdomains in the plasma membrane is differentially altered by exposure to psycostimulants.
1) Labeling of lipid raft in the cell membrane: For these experiment, I select to label a protein that is not a target for amphetamine or methamphetamine, and is shown to associate with membrane rafts. The B subunit of cholera toxin (CTxB) binds to lipid raft-enriched GM1 ganglioside and has been widely exploited to visualize lipid rafts. Therefore, to assess localization of the DAT in the raft associated membrane microdomain, before and after amphetamine and methamphetamine treatment, I will use membrane raft marker cholera toxin subunit-B (CTxB) CTXB labeling. CTXB pentavalently binds to GM1 sphingolipids, resulting in co-clustering of raft-associated proteins. These experiments will be performed inboth non-neuronal mammalian expression system, HEK-YFP-DAT cells, and neuronal MN9D cell expressing YFP-DAT. For these experiments I will use Alexa-647-conjugated CTxB (Alexa647-CTxB).
2) Labeling of non-raft microdomain in the cell membrane: For these experiment, I select a protein that is not a target for amphetamine or methamphetamine, and is not associated with membrane rafts. Na/K Atpase is a transmembrane protein that usually serves as non-raft marker and localizes in non-raft membrane microdomain. It is predicated that Na/K Atpase positive labels are not co-localized with CTXB labeled structures (GM1 positive). Therefore, the Alexa 405-Na/ K Atpase will serve as the non-raft marker.
I will calculated the co-localization of Alexa647-CXTB and DAT as well as co-localization of Na/K ATPase and DAT in the presence of vehicle and after 2 and 10 minutes of amphetamine and methamphetamine treatment. I predict to measure even distribution of YFP-DAT in the plasma membrane in untreated cells. Staining of the cells with Alexa-647-conjugated CTXB (Alexa647-CTXB), or Na/K atpase will result a punctuate localization at the surface membrane. I predict to measure a partial co-localization of YFP-DAT/Alexa647-CTXB and also a partial co-localization Na/K atpase/DAT. Only by performing the experiments we can determine whether amphetamine vs. methamphetamine influence membrane microdomain distribution of DAT.
To prevent GM1 and Na/K ATPase labeled internalization, all labeling will be performed on paraformaldehyde fixed cells.
Although amphetamine and methamphetamine have shown to induce internalization of DAT, current literature predicts the extent of DAT internalization following 2, and 10 min of amphetamine or methamphetamine exposure is negligible. Surface biotinylation of DAT following 2 and 10 min of vehicle, amphetamine or methamphetamine exposure will determine whether the hypothesized differential regulation of membrane microdmain distribution of DAT is due to differential DAT trafficking induced by these psychostimulants, or it is a direct consequence of drug exposure.
Successful completion of these studies will determine whether amphetamine and methamphetamine differentially alter membrane microdomain distribution of dopamine transporter.