Hi everyone! My name is Savan Patel, a rising junior, and this summer I am working at Brown University under Dr. John Marshall in the Biomedical Engineering building. The lab that I am working in focuses on various projects, but the one that I was assigned to along with a graduate student and masters student involves Angelman Syndrome. Specifically, Angelman syndrome is a disease state that is identified in some children at an early age. Biologically, it results from the deletion of the UBE3A gene from your maternal chromosome. Many of the associated symptoms are similar to that of autism which is also heavily linked to the signalling pathway that I am currently researching.
The BDNF-TrkB signalling pathway has a growth factor of brain derived neurotrophic factor and a rececptor called Tyrosine receptor kinase B. These two when the receptor is activated leads to a series of other pathways including AKT/mTOR which is a cell cycle proliferation and death pathway. The overall pathway is responsible for cell survival, differentiation, and synaptic plasticity, all of which are important to addressing Angelman Syndrome.
June 28th - June 30th:
For the first few days, I spent time accustoming to lab procedures and learning about the lab itself. This included doing some background research on the research area and also learning about the basis for the project itself. One aspect of the project is to do live animal testing to evaluate the effectiveness of the drug that the lab developed, CN 2097. The graduate student and masters student conducted various tests on live mice shocking them with an electrical impulse. The process is called fear conditioning:
1) Shock the mice on the first day three times.
2) On the next day, conduct a recall experiment to see if the mice freeze when placed in the "unsafe environment"
3) Every four days conduct a recall experiment (without actually shocking them) to see their reaction.
The main data that you would look for is how often the mice freeze, which you can detect and analyze using a monitoring program. Control mice without the drug will freeze and eventually undergo extinction which means they start to see that the environment is no longer unsafe because they were only shocked the first time. So, initially, the mice will freeze on the second day because they are expecting to be shocked, but after a period of time they start to realize that the environment is safe. Control mice with the drug should ideally behave normally, because we don’t necessarily want the compound interacting with parts of the neurological system that are not affected by AS (Angelman Syndrome). Angelman Syndrome mice during recall, do not freeze when put in the “unsafe” environment. This suggests that the syndrome is leading them to forget the conditions of the environment. AS mice, when treated with the CN compound, remember that they were placed in an unsafe environment and freeze, staying in the corner of the cage. Their freezing graphs have a drop off after 12 days (can’t remember exact value) but this is either because of extinction or because they forgot that they are in an unsafe environment.
July 1 - July 3
Over the weekend, I came into the lab to observe SDS PAGE runs and Western blot processes so that during the week I could do the procedures myself.
An SDS PAGE run involves taking lysed cell contents (specifically proteins and DNA) and applying an electrical current that will separate the components of the sample according to molecular weight. The Western Blot (a common lab practice in biomedical labs) involves transferring the samples from the SDS gel to a membrane which can then be analyzed using chemiluminescence.
I also got to see the first 4-day recall of the control mice that they were testing at the time.
July 4 - July 7
I ran the samples that my grad student had been culturing and transfecting into for PSD-95. The specific experiment was involving the overexpression, knock-down, and control of PSD-95 in neuronal cells. So, for the lab we use in vitro SH cells which are human-derived cells that are very similar to nerve cells. PSD-95 is a post-synaptic density protein that is highly abundant in nerve cells, which makes it hard to work with. Currently, the hypothesis is that PSD-95 participates and facilitates a positive feedback loop to the BDNF-TrkB signalling pathway. Thus, we are conducting various experiments to test this hypothesis and determine how the drug will also interact with this pathway.
The samples were in a lab-made gel:
Well 1: PSD-95 Knockdown in Media
Well 2: PSD-95 Knockdown after Starvation only
Well 3: PSD-95 Knockdown in Serum (FetalClone)
Well 4: Control in Media
Well 5: Control in Serum
Well 6: PSD-95 Overexpression in Media
Well 7: PSD-95 Overexpression after Starvation only
Well 8: PSD-95 Overexpression in Serum
Well 9: Protein Standard
Well 10: Same as Well 1
Well 11: Same as Well 2
Well 12: Same as Well 3
Well 13: Same as Well 5
Well 14: Same as Well 6
Well 15: Same as Well 7
I ran this gel and transferred it to a membrane, cut out two parts of the membrane (around the 95 kDa mark and on either side of the protein standard in Well 9. Then, for the first 8 wells I applied Millipore (mouse-derived) primary antibody and to the right side I applied Cell Signalling (rabbit-derived) primary. Then, anti-mouse as secondary for the left and anti-rabbit for the right. After that, I ran the development in the chemiluminescence chamber and analyzed results.
Unfortunately the results are saved on a computer at the lab and I will try to upload pictures sometime this week, but otherwise, the last two days of the week (Thursday and Friday), I reran the experiment again to analyze results (research is all about running your experiments multiple times).
July 3-8
This week mainly involved splitting cells into 6-well dishes as preparation for experiments in the following weeks. I split at 0.5 mL of a 4.5 mL stock solution of ~80% confluent cells.
July 17-21
For the first part of the week, I helped a masters student with one of their inhibitor experiments. In the past few weeks, our lab has been focused on trying to figure out what enzymes are involved in the phosphorylation of PSD-95. Under starving conditions, cells experience stress which seems to result in primary antibody being unable to bind at the epitope of the protein. This means that if we want to determine the role of PSD-95 in the BDNF-TrkB signalling pathway, we can't just assume that the protein is acting functionally the same. We have to figure out a way to keep PSD-95 in its dephosphorylated state during starving. Thus, we have been trying a series of inhibitors and oxidizing agents/ antioxidants to determine what is specifically going on with PSD-95.
For this experiment, we analyzed CK2 inhibitors, CSA (PP2A inhibitor), and KN93 (CAMKII Inhibitor). These reagents were added before starvation in order to allow the cells to absorb the chemicals and influence potential kinases and phosphatases. Unfortunately though, after running the Western blot, we were unable to see PSD-95.
Background info on how Western Blots work: Protein samples are first treated with a primary antibody that attaches to the epitope (binding site) of the protein. Then, a secondary antibody is applied which binds to the primary antibody. When the sample is placed in a chemiluminescence chamber, it will produce a band of saturation that can then be analyzed both qualitatively and quantitatively (with a program like Image J).
For the second part of the week, I was given a small experiment. A Chinese manufacturer had tried to develop CN 2097 at their facility and sent some of that sample over to us. My experiment was to determine the effect of the Chinese CN on AKT and ERK signalling in comparison with the American CN and control. Fortunately, the Chinese CN seemed to increase AKT and ERK signalling in comparison to the control. We also did a variant NGF stimulation to diversify the data.
Wells 1-3: American CN (0 min, 15 min, 30 min NGF)
Wells 4-6: Chinese CN (0 min, 15 min, 30 min NGF)
Wells 7-9: Control (0 min, 15 min, 30 min NGF)
Wells 10-12: Chinese CN (0 min, 15 min, 30 min NGF)
July 24-26:
On Monday I was given another experiment with a different combination of chemicals (NAC, Hydrogen peroxide, Thapsigargin (released Calcium ion stores), and Orthovanadate). I hope to determine the effect of H2O2 with Thapsigargin vs. Orthovanadate (two drugs).
1) Media control
2) Starve + NAC (30') @ 10mM
3) Media + NAC (30') @ 10mM
4) Media + NAC (30') @ 10mM + Thapsigargin (50') @ 1uM
5) Media + H2O2(30') @ 10mM + Thapsigargin (50') @ 1uM
5) Media + H2O2(30') @ 10mM + Orthovanadate (50') @ 10uM
On Wednesday, I put the samples in primary, so tomorrow I will be developing it and posting my results.