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.

Summer 2018: Mitigating ACEs at Vanderbilt Medical Center

“It’s easier to build strong children than to repair broken men.” – Frederick Douglass

Adverse childhood experiences (ACEs) come in many shapes and forms, including neglect, abuse, and household dysfunction. But how influential are they in a child’s health outcomes? Research has repeatedly shown that ACEs can significantly affect brain health enough to contribute to cognitive impairment, risky behavior in adult life, and long term risks of disease and mental illness. Therefore, we move onto the next question: How do we mitigate ACEs? That’s when I come in.

ACEs can range from being parenting related, to environmental.

This summer, I’m working alongside Dr. Seth Scholer, a pediatrician at Vanderbilt University Medical Center Children’s Hospital. Dr. Scholer has spent over a decade conducting research regarding ACEs, and how to successfully assess and alleviate them through pediatric primary care. With funding from the state of Tennessee, my research this summer has mostly focused on a randomized control trial (RCT) in which we hope to demonstrate that a brief parenting intervention can reduce unhealthy parenting tactics, thus nurture brain health in the clinic’s patients.

The utilization of an ACEs Screening Tool can improve health outcomes of children by identifying and addressing ACEs early in life.

My personal research project this summer is definitely simpler than an RCT, but has its own challenges. All previous research utilizing ACEs screening tools have taken place in pediatric clinics associated with research institutions such as Vanderbilt. However, the next step from here is employing a screening tool state-wide, which requires additional research that addresses how to implement the screening tool in private medical practices.

Therefore, I have been implementing an ACEs Algorithm and screening tool at a private pediatric primary care clinic for my summer research project. The screening tool is a quick survey that measures a child’s household/environmental stressors, and the degree to which their parent(s) use healthy discipline strategies. The ACEs Algorithm helps health-care providers interpret their patients’ scores, and points out when children are at low-high risk of ACEs. This is the first research study of its kind, and it requires working hands-on with the doctors and nurses at the private clinic to maximize the efficiency and effectiveness of the screening tool. Overall, this project has been a great opportunity to work along the front lines of ACEs research.

Health care providers use this ACEs Algorithm to interpret a child’s parenting-related ACEs and environmental ACEs (or other childhood stressors), after their caregiver completes a short ACEs Screening Survey. I worked with Dr. Scholer on the development of this algorithm throughout the summer, and this image is our final result.

As a Psychology major with minors in Neuroscience and Health Studies, this research experience perfectly fits the little niche formed from the intersection of my three fields of study. A typical day for me involves lots of patient/provider interaction and data management, with some manuscript and literature review writing stuck in between. This has helped me build concrete clinical research skills that are hard to learn in a classroom. Furthermore, I’m ecstatic about my ability to work within a research topic that is having a direct impact on people’s lives.