This quarter has really flown by. I cannot believe it is already May!

Since my last blog so much has happened in my research lab! The experiments using the Cdk5 and GSKβ kinase inhibitor ___ significantly reduced neurofibrillary tangles in 3xTg mice. I have never seen the CA1 region of the hippocampus so black and empty of fluorescent tagged PHF1 stain. Our controls injecting 3xTg mice with PEG (the hydrophobic vehicle for ___) displayed more tangles than we have ever observed. These controls allow us to conclude that the compound does in fact clear the cells of the phosphorylated tau recognized by PHF1 antibody.

Overall, we have identified methylene blue and celastrol as Hsp70 and Hsp90 inhibitors, respectively, that increase histological observation of neurofibrillary tangles (NFTs) in the CA1 region of the hippocampus. In addition, we now know that in contrast, ___ will reduce the observation of NFTs by inhibiting phosphorylation of tau.

This ability to manipulate the levels of NFTs displayed histologically will enable us to correlate NFT levels with different mechanisms of protein degradation utilized by the cell and eventually identify which process has gone awry in diseased neurons. In addition, we will perform experiments to observe other biological markers of DNA damage in correlation with levels of NFTs. I will not bore you with all the details of the project proposal I have outlined for the next year and a half, but rather I will write on here how those projects go as they are completed.

On a personal note, I am so happy that Monday has passed! This morning I had my Organic Chemistry exam that is officially my LAST OCHEM MIDTERM EVER!!! 🙂

Just the final to go!

I also turned in the application to the scholarship I have been working on for about a month now. It was hard to finally submit the packet – I realized that the copy that left my hands was really the final draft. No more changes or revisions! Well, now the waiting begins.

My little sister turned 14 yesterday. We are nearly six years apart and so the age gap prevented us from fighting too much when we were younger. She came back to UCSB with me for three days last week, and I cannot believe how much she has matured just since I have been away at college. I am feeling old.

“Sun’s gettin’ shinery, to spotlight the finery, Spring, Spring, Spring.” – from my all-time favorite musical, Seven Brides for Seven Brothers. How I love Spring Quarter (and corny old-fashioned musicals)

The rest of this blog will be research focused, I am really excited on finally some conclusive understanding of the results from several experiments! 🙂

Abstract: Many neurodegenerative diseases, including Alzheimer’s Disease, Frontotemporal Dementia, and Niemann Pick Type C, are characterized by the histological finding of  intracellular tau aggregates termed neurofibrillary tangles (NFTs) in the CNS. We hypothesize that the amount of NFTs in hippocampal tissue is the result of dynamics between the clearance, production, and accumulation of aggregation prone proteins, particularly phosphorylated Tau. To test that idea and quantify the extent of tauopathy of aged 3xTg mice, we used phosphoTau (PHF1) immunofluorescence to observe NFTs and Hoechst to fluorescently label nuclei. Mice were either treated or left untreated with compounds that modulate the biochemical pathways of clearance or production of NFTs. The chaperone proteins Hsp70 and Hsp90 regulate tau degradation and clearance within the neuron. By treating mice with Hsp70 inhibitor methylene blue, or with Hsp90 inhibitor celastrol, we found that perturbations of the chaperones’ activity significantly increased the percentage of NFT+ cells found in the hippocampal areas of the mice brain. Phosphorylated Tau production is regulated by the activity of several kinases, of which Cdk5 and GSK3-β are of particular pathological interest. The kinase inhibitor ___ specifically reduces the activity of Cdk5 and GSK3-β. We therefore propose that treatment of mice with ___ will significantly reduce tauopathy within the hippocampus by inhibiting phosphorylation of tau and impacting the production of NFTs. In this work, immunofluorescent microscopy imaging of tauopathies for each treatment was achieved and new techniques of quantifying the extent of tauopathy developed.

Last year, I finished all of the methylene blue experiments. The past two quarters, I have been working on the celastrol portion of the research. Based upon the results of these two major projects I have formulated the abstract above and am now in the process of immunofluorescent microscopy of the ___ treated mice. ___ is a brand new compound synthesized by another member of the Kosik lab to mimic the activity of a specific micro-RNA that targeted the same binding site on CDK5 and BSK3-β.

Over the course of four quarters, the shape of this research project has developed, molded, and flipped around according to the results acquired. Hypotheses have changed and new questions have been developed. Reflecting upon my own growth throughout the past year and also the development of this research project that has broadened its scope beyond which I could have imagined, I am incredibly grateful for the opportunity I have been given to engage in research through the EUREKA internship. I am beginning to realize that the only way to really gain understanding and an appreciation for scientific research is to fully immerse oneself in its whirlpool of utter confusion, in the hope of experiencing its priceless ah-ha moments.

Is it strange that I have been counting the days until dead week and finals? Getting to this light-at-the-end-of-the-tunnel period of two weeks meant the end of 4 hour Organic Chemistry labs twice a week, those labs that managed to begin just as the sun began to warm the morning chill from your skin and end as its golden rays kissed the horizon goodnight. Dead week meant the end of two weekly lab reports of 20 pages, the end of midterms that should be labeled progress-exams-every-3-weeks. I cannot remember the last class I took in which the professor only gave one midterm. Getting to this point meant the end of leaving my room at 9:00am, rushing from here to there, and not returning until 8:00pm after dinner.

Don’t get me wrong, I am studying. With a final paper for my Honors Seminar, and separate finals for Physics, Organic Chemistry and the Lab, Spanish 100, and Spanish 25, I have enough to keep me busy.

But for someone who spent the entire quarter stressed and sleep deprived, I feel like I am in heaven. Finals week means cozy late-night tea, the sound of ocean waves crashing below the cliff through my open window, sleeping in until nine-thirty, studying in the comfort of my bed or desk all day. The serenity of a quiet room. These are the weeks I rediscover my love for knowledge, my desire for the ability to explain scientific phenomena, my relentless hunger to know “why” — I unearth the buried reasons why I challenged myself to graduate from college and pursue higher learning in the first place.

Plus I take a few minutes of the day to read one of many novels (The Forgotten Garden at the moment) who patiently waited —unread — on my shelf throughout the quarter as I trudged along through classes and worked long hours in my research lab.

Maybe everyone else will find this confession to be bizarre, or maybe just unexpected. I see it as a celebration of living in the moment – however dire the circumstances may be – because compared to the past, this optimist is thankful that the storm is over.

On a low note, the mounting of celastrol stainings of LC3B and Hsp70 that I mentioned in the last blog went horribly. The paint brush I was using was too large for the delicate procedure, but it was the only one available to me at the time. Only about 75% of the tissues were perfectly aligned for optimal microscope analysis. However, the actual staining with LC3B looked clear and distinct under the microscope with definite trends corresponding with the level of PHF-1 stainings (phosphorylated tau) observed in previous experiments. The fluorescent labeling of Hsp70 was not as successful, so Israel and I have been looking for alternate antibodies for the one we are using now.

Sometimes the college student misses the simplicity of a home cooked meal, the comfort of your mom’s routine how-was-your-day, bickering and giggling siblings in the next room, and the sound of your dad snoring after a long hard day at work. I miss family, and cannot wait to board the amtrak train 6:30am Saturday morning. Sophomore year is a tough one and the chance to cast off my Rosie the Riveter “We Can Do It” go-getter attitude and briefly take on my childhood nicknames “Princess” and “Lisa” from the Simpson’s cartoon will be more than worth the 8-hour round trip.

Round two of the Celastrol stainings with Hsp70 and LC3B are going very well, I will finish the procedure tomorrow by mounting the thin brain slices on glass slides to be viewed under the fluorescent microscope. Israel always begins one of these days with, “Ahh, you are Michelangelo today.” The technique of mounting requires a lot of practice and a tremendous amount of patience. One must be determined to use a soft paintbrush to guide the sample through a liquid medium, onto the slide, perfectly flat and without wrinkles. This often requires assembling the torn tissue fragments back into the recognizable shape of a coronal slice of mouse brain like a puzzle. Needless to say, these are the days I am sitting at a lab bench for hours on end, neck cramping and eyes squinting to arrange mosaics the size of a pea.