Winter quarter is deep underway, and as a result sickness has permeated through the student body of UCSB. Unfortunately, my cloak of vitamin C, airborne, and continuous hand washing did not prove to have the power of invisibility, and sickness found me. All through my horror week of 4 midterms (OChem, Physics, Spanish 100, and Spanish 25) my voice had abandoned me, my throat ached from coughing, my body shivered with a 101 degree fever, and my only consolations were boxes of nyquil and sudafed. After two weeks without symptom relief, I finally convinced myself to take a trip to the urgent care doctor who gave me antibiotics that have finally kicked in, and kicked out whatever sinus infection had decided to keep my ears plugged for so long.

Today is Monday, and this is the first week I have felt human again. So I dropped into my research lab to check on the Celastrol brain stainings that I had somehow finished (in an incoherent state, no doubt) over the course of the past two weeks. Now, keep in mind that these stainings are supposed to begin with a primary antibody 48-52 hour incubation, continue with a secondary antibody 24-48 hour incubation, and conclude with an afternoon of tissue washing and the process of mounting the brain slices on slides. This process generally occurs, Friday, Monday, and then Wednesday mounting. So less than one week.

My Celastrol stainings have been sitting in incubation buffer (contains a soap to perforate the tissues and allow antibodies to enter individual cells) and antibodies for TWO weeks, not less than one. You might think: no big deal, the brains were sitting in soap, so they will just be extra clean! But, no. Soap + brain tissue = thousands of tiny holes in all the membranes and extremely delicate slices. I tried to mount them on the slides and most of them fell apart simply in the transportation process. It was an epic fail, and although some of the tissues remained intact, the experiment will have to be repeated. So that’s what I will be doing this Friday after the next Ochem midterm.

On the bright side, the tissues that survived the mounting process will probably appear beautifully stained. Due to the abundance of tiny holes in the membranes, the antibody should have had free access to the proteins inside the cells. I guess we will see.

Well, back to studying for my Spanish Linguistics midterm tomorrow!

I see I am getting behind on updating this blog!

Sleep, eat, eat, sleep, cuddle, watch soccer, sleep, eat, laugh, bake cookies, shop, sleep, read novels, sleep, repeat — that about sums up my winter break in a simple string of words.

Study, sleep, study, go to class, research, study, eat, study, research, applications, RA meetings, study, sleep, repeat — note the scarcity of the word sleep — these represent my first two weeks of winter quarter.

Now you, my reader, are caught up on my life between blogs! It has been pretty busy, but after this friday when scholarships and applications for next year are submitted, my load will lighten up 🙂

My research project is moving forward, and the data from last quarter is so motivating. Although the results of the Celastrol treatments suggest the opposite effect upon tauopathies of what Israel and I were expecting from our hypothesis, there is a definite trend. In research I have learned that this characteristic of results – consistency and a distinct trend as one variable is changed – is the most valuable result. A trend can be studied – it can be questioned and tested. Random inconsistent results are the only ones to be feared.

I am now in the process of digitally quantifying the images from the Celastrol experiments from fall quarter. However, based upon a rough estimate of the fluorescent signal of tauopathies by eye, it appears that treatment with Celastrol had an identical increasing effect upon tauopathies as Methylene Blue. (Remember that the presence of Hsp90 in the cell has been shown to regulate the levels of Hsp70 through a negative feedback mechanism). Therefore, inhibition of Hsp70 and Hsp90 in separate models had an identical effect – an increase in tauopathies. These results suggest the hypothesis that if the cell undergoes stress relating to any alteration of Hsp70/Hsp90 activity, then tauopathies will increase within that cell, regardless of whether Hsp70 or Hsp90 activity is the protein targeted by the drug.

Now, these conclusions are dependent upon the assumption that Methylene blue inhibits Hsp70 (spiking Hsp90 levels) in neurons, and Celastrol inhibits Hsp90 (spiking Hsp70 levels) in neurons. To ensure that this is true, I have just finished designing an antibody staining of Hsp70 in these same Celastrol tissues. By choosing samples with high, low, and no signs of tauopathies from treated and untreated mice, and then staining them with antibodies targeting Hsp70, I can answer two questions: 1) Do tissues with high levels of tauopathies also display high levels of Hsp70 indicating a correlation between the two? and 2) Are Hsp70 levels dependent upon (augmented by) high doses of celastrol? In other words, is the celastrol increasing Hsp70 levels?

Simultaneously, I have chosen samples identical to the ones chosen for the Hsp70 staining to determine the levels of the protein LC3B, a protein associated with the level of autophagy (degradation of tau in these neurons by lysosomes). Last year, we performed a similar staining with the Methylene Blue tissue samples, and found that an increased dosage of MB actually increased the levels of LC3B, regardless of the fact that tauopathies increased. It will be interesting to see whether Celastrol has the same effect. If the results illustrate an increase in autophagy, then the consistency of both the MB and Celastrol experiments will provide a strong case for the hypothesis that increasing lysosomal activity will actually increase the level of tauopathies within neurons in the hippocampus of the brain.

Finally, because this post is steadily lengthening, we have proposed that Methylene Blue and Celastrol may have the ability to induce tauopathies. This new hypothesis will be tested by designing an experiment to determine the effect of Methylene Blue and Celastrol upon tauopathy levels in wild type mice.

Dr. Kosik has given my mentor and I permission to begin writing a research paper to be published about this project. For this reason, I am looking forward to the exciting possibility of staying here at UCSB this summer and having the time to catalyze that process. But there is always a catch, I need funding to pay for living expenses here. Hence the applications mentioned above. Back to writing my proposal for the Crowe Family Summer Research Scholarship. Wish me luck!

What a hectic quarter this one has been! Between attending meetings, planning events, and working shifts as an RA, dedicating an average of 10 hours a week to my research internship, and keeping up with 19 units of fast-paced science classes, it is a wonder that I still have time to breath and sleep a few hours at the end of the day!

Although my grades are not suffering from all this busyness, I can feel myself burning out. Like the Energizer bunny looking confident in pink and shades, moving forward, always moving forward in chase of some invisible carrot, and banging its drum as if its life depended on it, I convince myself that I am superwoman and do not give myself a break. I bang my drum, studying late into the night, baking fresh batches of cookies for my residents because they taste better, staying in my lab overtime because I want to reach the results of an experiment, tying ribbons with sparkly tags around lotions to make holiday gifts for each girl in my house, redoing Physics and OChem practice problems, and signing up for even more classes next quarter. There is always the carrot, the motivation to work harder, strive higher, do more, be better, to give my all in everything I do, and to truly make a difference.

To accomplish those things, or to reach the carrot so to speak, I need to be realistic. I am only a Sophomore Undergraduate. In the future, there are many more years of school. Burning out is not an option. So, time management and prioritizing come into play. Unlike the Energizer bunny that “keeps going, and going, and going…”, I am not a fictitious animation with an infinite source of energy. I am a 19-year old human being who needs (1) Sleep, (2) Exercise, and (3) Guilt-free relaxation. I am afraid I took on more than I could chew happily this quarter, because to be honest anyone looking at my record would think this quarter went smoothly like all the rest. Happily is the key word in the former sentence. I have been able to manage all of these responsibilities simultaneously and do well, but I simply do not have the time to fully enjoy and engage in each one.

After reflecting upon all of these things I have come to this conclusion: Just because I can, does not necessarily mean that I should.

So, next quarter, I am taking the second parts of Physics and Organic Chemistry. However, instead of additional science classes, I will be working towards my double major and taking two Spanish courses. I am hoping that the these classes will add some variety into my course load and give certain areas of my brain a break from a quarter of Newton’s laws-cAMP-bromination-delocalized electrons-science-thinking-overload.

On a more positive note, the Celatrol experiment is going very well, I have sliced, stained, and fixed the brain slices and will finish mounting them on glass slides on Friday. We should be viewing them under the fluorescent microscope next week. I cannot wait to see the results!

Hello! My name is Madison Cornwell, and I am an undergraduate researcher and CNSI EUREKA Intern currently working in the lab of Dr. Kenneth Kosik through the Neuroscience Research Institute at UCSB.

During my freshman year, I determined to break through the obstacles and fierce competition restricting many students from becoming an integral member of a research team. After dabbling in a Marine Biology Lab in my first quarter, the EUREKA internship empowered me to obtain a position in a cutting edge medical research lab.

It only took a few days to realize I had found my passion.

Alzheimer’s (AD) is a neurodegenerative disease characterized by both intracellular neurofibrillary tangles (NFTs) and extracellular aggregates of beta-amyloid found within specific regions of the brain including the hippocampus, which is responsible for memory. In an Alzheimer’s patient, hyperphosphorylated tau detaches from microtubules in the neuronal axon and accumulates within the cell, forming NFTs that cause neuron malfunction and eventual cell death. Developing a drug that would target these aggregates of tau could improve the lives of millions of Alzheimer’s patients worldwide.

During the winter and spring quarter of my freshman year, my research focused upon one candidate of interest; the phenothiazine methylene blue (MB) that had been shown to disintegrate tau tangles in vitro. To test the viability of this interaction in an in vivo model, Israel and I studied the effects of MB upon NFTs using a transgenic AD mouse model. Through intraperitoneal injection, we treated mice with different concentrations of MB daily for one week. We then collected the brain tissues and used fluorescent immunohistochemistry to detect NFTs in the hippocampus. Intriguingly, when viewed under the microscope, we observed that the MB treatments seemed to actually increase the fluorescent signal of hyperphosphorylated tau. Quantification of the intensity of fluorescence in histological samples of treated and untreated mice reinforced this visual observation. Through this experiment, our hypothesis, stating that Methylene blue decreases levels of hyperphosphorylated tau in vivo as it does in vitro, was proven incorrect. Concluding in early June, this project left me simultaneously curious, confused, and eager to investigate as I boarded the Amtrak train for a summer with my family.

Returning in the fall, Israel and I discussed published research exploring the different roles of Methylene Blue. We discovered that it had been identified as an inhibitor of Hsp70 ATPase activity. Hsp70 is part of multichaperone complex consisting of Hsp70 and Hsp90 connected by the protein Hop. Within this complex, Hsp70 is the chaperone protein responsible for the disposal and degradation of damaged or defective proteins through interaction with CHIP (Carboxyl-terminus of Hsp70 Interacting Protein) an E3 ubiquitin ligase. Our data illustrated an increase in hyperphosphorylated tau with the addition of Methylene Blue. If Methylene blue inhibits the activity of Hsp70 in vivo, and this inhibition of Hsp70 increases the level of hyperphosphorylated tau within the neurons of the hippocampus, then this interaction would explain our results from the spring.

Following this conversation, we decided to test our adjusted hypothesis by designing a new experiment to observe the effect of an Hsp90 inhibitor upon the level of hyperphosphorylated tau within the hippocampus of the same transgenic AD model mice. We chose the drug Celastrol.

Today marked the last of 15 daily injections of 3xTg mice with different concentrations of Celastrol. After so many days of treatment, I am feeling pretty confident with the process of IP injections. We are wondering whether we extended the treatment too long, however. By the last few days, the area of injection seemed to thicken with scar tissue, making treatment more difficult. Tomorrow Israel and I will perform heart perfusions with PBS to clear the brain of blood, and then extract and the brains to be soaked in paraformaldehyde. This forms methylene bridges or cross-links between the peptides (chains of amino acids forming proteins) of the tissue so that they are firm and ready for vibratome slicing on Thursday.

I am the Resident Assistant of the Women in Science and Technology Residence Hall at UCSB. While heading to dinner today, I realized I spent the entirety of my one-on-one session with the Assistant Resident Director (who was a Business and Economics major) explaining my research project and answering all his questions. That is when you know you are doing what you love.