“A molecular mechanism for Alzheimer’s disease: the effects of WT and mutant Presenilin D257A on TrpC5 channel function” Blog #2

Summer research at Pace in the biology labs with Dr. Buraei has been an incredible learning experience. Right away we began by preparing competent cells by growing E.Coli overnight, then utilizing ZymoBroth and buffers to obtain the highest efficiency cells as possible.
During our first trial using the Zymo Mix & Go kit, we followed each procedure exactly as directed. The largest concern was making sure that each step was carried out at the correct temperature. As a majority of the procedure required conditions which were 0-4 degrees Celsius, we made sure to have a container of ice on hand at all times, and precooled the equipment needed. This is crucial since a cells competence can decrease immensely when it reaches temperatures higher than 4 degrees Celsius. Additionally, once prepared, the cells must be stored at -80 degrees Celsius, so in order to prepare we labeled the micro centrifuge tubes ahead of time as well as cooled them using ice.
Although our first trail was successful, after further research we learned the competency can be greatly increased by using a few troubleshooting techniques. For our research, we want to use cells with the highest transformation efficiency as possible, meaning the cell has the best chance of accepting the foreign genetic sequence including our desired mutation. Two of the main challenges in the second trail were growing the E.Coli culture in the ZymoBroth at room temperature, as opposed to 37 degrees Celsius, and being even more precise with keeping everything at a low temperature once the bacteria grew to our desired level. Since bacteria will optimally grow at 37 degrees Celsius, lowering the temperature will result in a much longer doubling time, taking a greater amount of time to reach our desired optical density. However, once grown, we decided that in order to further reduce the temperature of the culture to rise we could keep all tubes and pipettes which we would use in a freezer, in addition to labeling the micro centrifuge tubes and placing them in the -80 degree Celsius prior to use. Unfortunately, we found that we had allowed the overnight culture to grow in the ZymoBroth for an extended amount of time and it had passed the optimal optical density. However, since the cells were still usable for a different step in our research, we were able to quickly finish that trial and begin again.
In our last trial making the cells we followed the same procedure as the second trial, this time adjusted the calculated doubling time and monitoring the culture every two hours. This adjustment, along with preparing the tubes and pipettes before hand resulted in very successful competent cells which will be crucial in our next steps in research!
Over the next few months I am thrilled to continue this research with Dr. Buraei and to perform electrophysiological studies to hopefully draw a sound conclusion!

“A molecular mechanism for Alzheimer’s disease: the effects of WT and mutant Presenilin D257A on TrpC5 channel function.” Blog #1

My name is Paris Hanson, I am a rising junior at Pace, a biology major, and this summer I am so excited to be working with Dr. Zafir Buraei on our research “A molecular mechanism for Alzheimer’s disease: the effects of WT and mutant Presenilin D257A on TrpC5 channel function.”
Mutations found in Presenilin genes (PSEN1 & PSEN2) can be accounted for majority of the early onset FAD mutations. Presenilin FAD mutations have been shown to alter calcium signaling. Recently, studies show that Presenilin itself can be a ‘leak’ channel on the ER that depletes Ca2+ ions from the smooth ER. This discovery led us to investigate the possible crosstalk between Presenilin and intracellular calcium signaling, as it relates to TrpC5 chanel activation. TrpC5 is a multi-pass membrane protein and is thought to form a receptor-activated non-selective calcium permeant cation channel.
Previous research conducted by resent graduate Sukhjinder Kaur and Dr. Buraei investigated the effects of Presenilin on TrpC5 Channel function. A result of the experiment found that that co-expression of WT Presenilin resulted in a significant decrease in agonist evoked TrpC5 channel currents. A plausible explanation is that Presenilin expression renders the ER somewhat Ca2+-poor, and subsequent Ca2+ efflux through IP3 receptors -required for TrpC5 activation is significantly diminished. They also tested the effect of FAD mutant Presenilin M146V on TrpC5 channel activity, finding that that Pres-WT decreases TRPC5 currents, whereas, Pres-M146V does not inhibit TrpC5 as effectively. This may have a significant impact in the pathogenesis of Alzheimer’s disease, especially when Presenilin1 is mutated. Presenilin FAD mutation D257A, a mutation that is known to abolish γ-secretase activity, does not seem to impact the function of presenilin as an ER leak channel.
Thus, our purpose is to determine how different presenilin mutants effect TrpC5 activity. We predict that, similarly to presenilin, presenilin FAD mutation D257A would act like the WT channel and decrease TrpC5 channel activation. Another mutation found in Presenilin D385A was shown to completely abolish calcium leak channel activity on the ER. Testing D385A co-expressed with TRPC5 in oocytes may reveal an effect similar to (xyz, the mutation we detest) and shine a new light on the etiology of Alzheimer’s disease.
Overall, our goal is to use mutant Pres-M146V and wild-type presenilin to confirm the previous studies as well as to synthesize Presenilin FAD mutation D257A in order to perform electrophysiological studies and analyze our results.