Author Archives: Ian Levitan
Dr. Anne West’s seminar was particularly intriguing because of its focus on the “basic sciences”. Oftentimes, I hear undergrads who say they want to do research in cancer or immunology “because it’s cool,” and yet many of the major scientific discoveries came from so-called basic sciences that may appear to be separated from clinical implications. In fact, as Dr. Anne West pointed out, a large portion of the discovery of CRISPR came from studies of bacteria in the food industry. I was also fascinated by Dr. West’s desire to write a book of anecdotes designed to bridge the gap between the abstract world of academia and life. Her passion for her work and the reasons for her continued motivation were truly inspiring.
Limitations to dopamine D2 ligands for antipsychotic efficacy.
The D2 receptor has often been examined as the target for antipsychotic drugs due to its distribution in critical areas of the brain involved in movement and reward. Beta-arrestin biased drugs such as UNC9994 function as antagonists through G-protein signaling and partial agonists through beta-arrestin signaling, which has been shown to decrease psychotic properties while minimizing unwanted side effects. Of the five dopamine receptors, D2 closely mirrors D3 and D4. Therefore, it has been proposed that these novel therapeutics may be having an effect on the dopamine D3 and D4 receptors. Using BRET assays with HEK293 cell lines, aripiprazole and its congener UNC9994 have been tested on the dopamine D2-D4 receptors, and their effects were recorded as dose-response curves. By comparing the effect of the drugs to the effect of the natural ligand dopamine, it has been discovered that these agents do have an effect at the D3 and D4 receptors. Due to the role of non-D2 dopamine receptors in contributing to many of the symptoms of schizophrenia, novel therapeutic agents remain to be developed that could have the receptor specificity needed to attenuate psychotic phenotypes while minimizing unwanted side effects.
In the Caron lab, my project has largely been testing new schizophrenia drugs on the dopamine receptors. Because my interests in the sciences have largely revolved around pharmacology and cell biology, I have usually approached the concept of neuropsychiatric disorders as a dysregulation of neurotransmitter systems that can be fixed by the administration of a molecular substance (i.e. drug). This would involve knowledge of the receptor and the subsequent effects it has on the cell. This approach to these disorders focuses on abating symptoms once they are present in a patient (or forced in an animal model), and does not truly examine the causes of such disorders (but merely the effects). Thus, I was quite fascinated to hear about some of my colleague’s labs which focus on proposed causes for certain disorders, like the role of gut microbiota in contributing to depression.
One chalk talk that really grabbed my attention was the one given by Annika Sharma. In the talk, she stressed that experiments transferring fecal matter from a depressed mouse (mice eat poop) into a healthy mouse was able to induce depression in the healthy mouse, suggesting a role for the gut microbiota in facilitating connections with the brain (i.e. the gut-brain access). It is also known that Major Depressive Disorder (MDD) patients have altered microbial compositions and many metabolites which play a role in depression are byproducts of gut microbiota. I was also rather shocked by the way in which her lab generates what it calls the social defeat paradigm. Essentially, to create a depressed mouse, it is left in the company of older, more aggressive mice that beat it up. The lab then extracts fecal matter from the depressed mouse and is able to run any series of tests that they want to determine, for instance, the presence or absence of certain metabolites. Overall, I was just interested in learning the many different ways in which researchers have approached certain disorders.
For me, each day of the week in the Caron lab is dedicated to a different procedure with the overall goal of generating data for my project. Thanks to my dedicated mentor who goes to lab on Sunday, I am able to run three rounds of BRET in a week.
On Mondays, I transfect the cells that my mentor split the day before. I also split cells for transfections on Tuesdays. On Tuesdays, I split the transfected cells to a 96 well plate for BRET and transfect the cells split on Monday. On Wednesday, I am able to finally use the BRET machine and see if my transfections and plating reveal any interesting results. Like is expected for good science, I try to achieve many rounds of similar results with BRET assay to assure that the data is not due to error on my part. Thus, I run BRET on Thursdays and Fridays as well.
While the BRET assay is my main objective in lab, it is not the only thing I do. Each time I transfect, I have to use some DNA from the stocks we keep in the fridge. After many rounds of BRET, the DNA starts to run low. Therefore, I occasionally engage in cloning projects in order to make more DNA. Sometimes, I am lucky that other people in the lab have glycerol stocks of the DNA I need. If so, I am able to utilize a Qiagen MidiPrep in order to extract more DNA. However, if no glycerol stocks are available, I have to transform the DNA in bacteria and let the bacteria grow up copies for me.
In addition to all of these small mini cloning projects, I did partake in a fairly lengthy cloning project that lasted an entire week. I attached a fluorescent probe to the D3 receptor.
So, this is how I have been keeping busy in lab. Sometimes, if I finish everything I have to in a day (or if there ends up being too few cells to split), my mentor will keep me busy with random projects that usually involve new techniques. One thing is for sure, there is never a boring day in the Caron lab.
Research Question: To what extent do the D3 and D4 receptors play a role in mediating a response to a schizophrenia drug? What implications would this have for the need for more highly specific drugs in the future?
Metabotropic (7-TM GPCRs) receptors are a major family of membrane receptors (the largest, most diverse group in eukaryotes comprising the largest class in the human genome). GPCRs are also the target for ~50% of drugs on the market, and yet only a small portion of GPCRs have been tested, leaving the possibility for great potential in the field of receptor pharmacology. All 5 dopamine receptors are metabotropic, with the D2/D3/D4 receptors exhibiting close homology and the D1/D5 receptors exhibiting a close homology. While the D2 receptor is often implicated in contributing to reward in the human midbrain, the question of the effects of drugs on the D3 and D4 receptor suddenly becomes relevant. Many of the dopaminergic pathways originate in the midbrain (like the substantia nigra) and one of the hallmarks of schizophrenia is dysregulated doapminergic pathways (like excess dopamine in the midbrain). Traditional antipsychotics are antagonists of the D2 receptor which can alleviate the symptoms associated with the midbrain, but not with the forebrain where the dopamine is already depleted.
Using BRET assay, it is my hope that I will be successful in seeing how a new schizophrenia drug may act on the Dopamine 3 and 4 receptors which previously have been essentially disregarded. Due to recent publications commenting on the widespread distribution of the D3 and D4 receptors in the cortex, it is now important to not only observe the effects of biased signaling but also of signaling associated with receptor specificity.
In the early 60s, Dr. Caron became interested in the sciences while attending Laval University in Quebec. In this decade, steroid metabolism was at the forefront of biomedical research. This field within biochemistry prompted Dr. Caron to attend the University of Miami where he received his Ph.D. Following his time at U Miami, Dr. Caron sought a postdoctoral fellowship and ended up with Dr. Bob Lefkowitz at Duke. It was during this time that Dr. Caron’s research really began to evolve.
The main focus of Dr. Lefkowitz’s lab has always primarily been GPCRs, the largest and most diverse group of membrane receptors in eukaryotes. GPCRs are distributed widely in the human body, with notable examples including rhodopsin (in the eye), taste receptors such as sweet and bitter, adrenergic receptors (i.e. beta-adrenergic receptors involved in the flight-or-fight response), and other transmitter/hormone receptors (including for dopamine and endogenous opioids). Dr. Caron spent most of his time with Dr. Lefkowitz attempting to purify these receptors and found success in 1986 with the beta-adrenergic receptor. At this time, the homology between different GPCR receptor subtypes was discovered to be extremely close. In other words, the mechanism of action of the beta-adrenergic receptor closely mirrors that of a dopamine receptor, for example.
Nowadays, the focus of the Caron lab is GPCR cell signaling with emphasis placed on neuropsychiatric disorders like schizophrenia and Parkinsons. These disorders are largely a result of neurotransmitter imbalances (amongst other things) such as dopamine with schizophrenia. Thus, by studying receptors such as the dopamine receptors and their subsequent effects (i.e. with known or newly developed drugs), it is our hope to further our understanding of GPCRs to combat such disorders. In fact, 40-50% of drugs on the market specifically target GPCRs. However, many other GPCR subtypes remain to be tested within the realm of this exciting field.
Starting research in the Caron lab has been an exciting, yet terrifying experience. As the recipient of prestigious awards such as the Lieber Prize for Outstanding Achievement in Schizophrenia Research, Dr. Caron has set the standards high for his lab.
Upon first arriving, I was thrown in at the deep end and expectations were high. I witnessed many procedures and took notes as I watched, hoping that I would be able to replicate the techniques on my own when needed. Such is the case with transfecting and splitting cells, which is how I spent most of my time in lab this past week. While my mentor, Tom Pack, no longer has to overbook the hood for me (though he still does just in case), I find that I still have much to learn in the next 7 weeks.
As the summer progresses, I hope to not only increase my technical proficiency in lab, but also gain a greater understanding of GPCR signaling and the lab equipment that exists for studying it. While my current focus is on arrestin signaling following dopamine receptor activation by ligand, I believe I will be branching out to new topics and hope to gain as much exposure to the workings of lab as possible in 8 weeks time. I also hope to have my research experience guide my undergraduate experience for the remainder of my time at Duke.