Summer 2018: Researching ALS across Different Model Organisms

By Sophia Nelson

One of the world’s leading neurodegenerative diseases, Amyotrophic Lateral Sclerosis (ALS), causes its destruction of the nervous system in largely unknown ways. Recent research has shown that a hexanucleotide repeat, GGGGCC in intron one of c9orf72, is the most frequent genetic cause of ALS, with the number of repeats in affected patients ranging anywhere from hundreds to thousands. While these repeat sequences are found in the non-coding region of the gene, they still appear to contribute to toxicity through repeat associated non-ATG translation, which can begin at any point without the presence of the start codon, ATG. This unconventional translation allows repeats to be translated into five different dipeptide repeat proteins (DPRs): GA, GP, GR, AP, and PR. One of these proteins, GA, forms paranuclear amyloidogenic aggregates which have been found in the brains of human ALS patients. However, the direct role of GA—or any DPR— in disease pathology and toxicity is not yet known, particularly because the toxicity of GA varies heavily based on the model system in use.

As a Velay Scholar this past summer, I got the chance to work with professors Robert Fairman and Roshan Jain to investigate the protein GA through a comparative study characterizing the aggregation and toxicity of GA in three model systems: worms (C. elegans), zebrafish (D. rerio), and fruit flies (D. melanogaster). The bulk of my work was in worms and flies, as my fish have not yet grown large enough for testing! I expressed GA in the neurons— ALS is known to attack motor neurons, so neuronal expression is important to study— in each of the two model systems and then performed behavioral and confocal imaging analysis for comparison. In the imaging studies, I was looking for multiple things: firstly, the large, paranuclear puncta that are hallmark of GA aggregation; and second, the localization pattern within each organism. Behaviorally, I was attempting to understand whether or not GA expression within neurons was toxic by comparing organism performance in simple behavioral assays both with and without GA. For worms, this behavior was thrashing; for flies, it was the ability of larvae to crawl. I learned so much throughout the summer! I dissected fruit flies (both larvae and adults) and removed and imaged their brains, which are barely the size of a poppyseed. I learned behavioral testing across all three organisms, as well as PCR genotyping, staining and imaging techniques, confocal microscopy, and the beginning stages of biochemical assays as well, such as lysate preparation.

The dissection of fruit fly larvae and brains showed that GA was heavily concentrated within their developing brains, particularly in the progenitor of the neural column and the neuronal ganglia that develop and associate with the eyes. The confocal images of my fly brains were likely the coolest result of my whole summer! In worms, GA puncta is found throughout the body; heavily concentrated in the brain and along both the ventral and dorsal cord. Behavioral assays indicated that the presence of GA had a negative effect on C. elegans thrashing. Larval crawling data was collected and differences between controls and GA positive larvae were found, but this data is still being followed up on to conclusively determine any effects. Biochemical results, obtained through SDD-AGE, will be gathered this spring. Overall, this study indicates that the form of GA aggregation is consistent across species despite slight differences in localization, and the presence of GA appears to have a negative effect on behavior, indicating it may have a role in disease toxicity and should be tested further.

Performing these experiments was an incredible experience! My mentors were both amazing and taught me so much. It was my first time working in a research lab, and as a biology major with minors in neuroscience minor and health studies, it has only confirmed my interests further. After graduation, I now plan on going to medical school and graduate school for an M.D.-Ph.D so that I can continue to perform research while also gain a clinical perspective and directly help patients. I am so excited to be able to apply the knowledge I gained this summer (and will continue to gain in my last year at Haverford) in my future career and work with a topic that has the potential to have a direct impact on many people’s lives.

Dissected and removed fruit fly larval brain neuronally expressing the protein GA. The middle section is the progenitor of the neural column.

How I got in Whalen’s Lab

As another internship season approaches, many friends are asking me how I got my summer 2017 biology research position without having taken any biology at Haverford. [1]

The process was not straightforward. I set out considering a major at Bryn Mawr, then halfway switched to Swarthmore, and ended up determined, “If biology, then Haverford.” Last summer was to get a taste of biology at Haverford.

Once the decision was made, I immediately reached out to Professor Whalen, whom I had chatted with at academic tea, a casual gathering of all departmental representatives at the beginning of every semester, to answer students’ questions. I had also browsed professors’ Haverford webpages, where their CVs and research directions are listed. Although my tentative major was biology, I could not understand the content of any project our biology professors listed. Still, marine science, drug resistance, and a photo of Professor Whalen smiling from a solid blue ocean-sky background attracted me above all.

I sent out my first email on Dec. 22, 2016, and went to play in New Haven. When I came back, a reply had lain in my mailbox since the day I left — what a quick response! To day, Professor Whalen’s efficiency is still surprising, motivating, and scaring me from time to time. Back then, I immediately arranged to meet with my first-ever mentor.

She showed me all her undertakings and some summer opportunities, and asked me to show up at lab meetings the coming semester. Since I could only work on-campus as an international student, we decided to begin with the “bacterial response to a chemical” project ongoing at her lab. That was it! I became part of Whalen’s lab. When the summer scholarship application season came, she instructed me to apply (Kovaric Fellowship [2]). When I failed, she applied funding from Provost for me, so I could get paid. Everything was settled as early as Mar.18, 2017, after which I just sat back and pictured the richness of the coming summer.

 

Takeaways for new applicants:

  1. Start collecting information early; browse professor/institution’s webpages, and from there find out more
  2. After narrowing down your choices, reach out (sometimes it takes longer to receive reply; don’t feel discouraged or overly anxious)
  3. Academic tea is a great space to ask any lay (or expert) questions about subject/ courses/ major/ internship/ …; professors are there for you
  4. Don’t be afraid if professor’s research seems hard to understand!

 

[1] only sophomores and above could take biology courses at Haverford

[2] funding opportunities please see: www.haverford.edu/integrated-natural-sciences-center/programs-funding/student-research-funding

Soybeans, Blood, and Robots: The Epigenetics of Cancer

This summer I’m working in the lab of Dr. Laura Rozek, a molecular epidemiologist who specializes in environmental carcinogenesis. This lab is part of a team of researchers at the University of Michigan receiving a SPORE grant (Specialized Program of Research Excellence) from the NIH’s National Cancer Institute to investigate head and neck cancer. Most of the time when I tell people “head and neck cancer,” they hear “brain cancer,” which is not the case. More specifically, the cancers we are studying are called head and neck squamous cell carcinomas (HNSCC), which are tumors that form in the mouth and throat (oral cavity, oropharynx, and larynx). The most common contributing factors the the development of this disease is tobacco use and certain strains of HPV (human papilloma virus). While this kind of cancer is a small fraction of cancers in the US, it is much more prevalent in other regions of the globe, such as Southeast Asia.

The researchers here at Michigan are interested in understanding how epigenetic changes to certain genes arise and turn normal cells into cancerous cells, and how we might be able to reverse the process. For those of you who don’t know or have forgotten what epigenetics is, it’s the study of the chemical changes to DNA that activate or deactivate different parts of the genome at specific times. The specific kind of change studied in the Rozek lab is methylation, which involves methyl groups being attached to different nucleotides. Too much methylation, called hypermethylation, in the promoter region of a gene can turn it off. In cancers, genes for proteins called “tumor suppressors” are often turned off in this way. Research has also shown that cigarette smoking is directly related to the hypermethylation of the important tumor suppressor p16.

The project I’m working on is a phase II clinical trial to investigate the cancer preventative (“chemopreventative”) properties of compounds found in soybeans and other legumes called soy isoflavones. The study recruited both smokers and nonsmokers who had developed the cancer and gave them capsules containing soy isoflavones, mainly the one called genistein, which they ingested daily. The cancer patients who smoked were helped to quit. Tumor tissue and blood samples were taken from the patients before and after the soy treatment to analyze the changes in methylation levels.

Why soy? There are a couple reasons why soy is seen as a promising agent for nontoxic preventative therapy to undo damaging methylation levels. Past projects at Michigan have looked at diet in connection with HNSCC survival rates. They found a correlation to better rates of survival with higher consumption of fruits and vegetables, in particular soy and other leguminous foods high in soy isoflavones. Other research has found that genistein can inhibit the growth of other cancers (breast and prostrate) in vivo and in vitro through regulation of signaling pathways.

My role has been focused on the blood DNA component of the soy study. First, I extracted the DNA from the blood samples using a robot called the QIAsymphony from Qiagen. It basically uses a pipette-equipped arm controlled by a very smart computer to distribute reagents. It looks like this:

yes this is actually a robot

DNA extraction robot. I wanted to add a video of it running, but videos were too big to upload.

After obtaining the pure DNA, I bisulfite converted (BSC) the samples along with controls. This makes the methylation levels readable to the pyrosequencing machine. After this step, I have to copy the DNA using PCR (polymerase chain reaction) for each of seven genes that I am investigating. I have done two of these plates so far and run them through the Pyrosequencer, giving me data for two out of seven total genes. One of these genes is particularly tricky so I will not be sequencing it myself but sending it to a different department at UMich.

the computer for this thing is like from 1999 but the camera inside it is like $$$$$$$$$$$$$

The PyroMark MD pyrosequencer and the special plate that goes in it. The black tick marks indicate wells with controls.

So now I am equipped and trained on all the techniques needed to finish collecting data. I’ve been delayed by the PCR process because we were having difficulty making primer mixes that were not contaminated. My lab manager suspects that the primers are just old so we’ve ordered fresh new batches. In the meantime, I’ve been helping out in another phase of the HNSCC research by receiving and entering clinical information about tumor blocks and slides from patients who are enrolled in Michigan studies.

I don't even have to wear gloves

Tumor suspended in a block of wax to be stored at room temperature. This technique is called FFPE.

I thought they looked pretty

Stacked slides containing stained slices of tumor.

 

 

 

 

 

 

 

 

 

 

My next steps involve PCR of the next five genes and measuring their methylation levels. I’m on track to finish this with plenty of time left in July, so I may be helping out on other projects or shadowing the bio statisticians who will analyze my data.

-Claire Côté, ’17