Imbalances in Copper or Zinc Trigger Further Trace Metal Dyshomeostasis in Amyloid-Beta Producing Caenorhabditis elegans
Abstract:While the email sent by Mr. Wong on 3/4/22 indicates that we should not submit an abstract, the portal will not allow me to submit without this field filled out. Therefore, here is my abstract...
Alzheimer's Disease (AD), a progressive neurodegenerative disease characterized by the buildup of amyloid-beta (Aβ) plaques, is believed to be a disease of trace metal dyshomeostasis. Amyloid-beta is known to bind with high affinity to trace metals copper and zinc. This binding is believed to cause a conformational change in Aβ, transforming Aβ into a configuration more amenable to forming aggregations. Currently, the impact of Aβ-trace metal binding on trace metal homeostasis and the role of trace metals copper and zinc as deleterious or beneficial in AD remain elusive. Given that Alzheimer's Disease is the sixth leading cause of adult death in the U.S., elucidating the molecular interactions that characterize Alzheimer's Disease pathogenesis will allow for better treatment options. To that end, the model organism C. elegans is used in this study. C. elegans, a transparent nematode whose connectome has been fully established, is an amenable model to study AD phenomena using a multi-layered, interconnected approach. Aβ-producing and non-Aβ-producing C. elegans were individually supplemented with copper and zinc. On day 6 and day 9 after synchronization, the percent of worms paralyzed, concentration of copper, and concentration of zinc were measured in both groups of worms. This study demonstrates that dyshomeostasis of trace metals copper or zinc triggers further trace metal dyshomeostasis in Aβ-producing worms, while dyshomeostasis of copper or zinc triggers a return to equilibrium in non-Aβ-producing worms. This supports the characterization of Alzheimer's Disease as a disease of trace metal dyshomeostasis. This might indicate that the inability to return to trace metal homeostasis in Aβ-producing C. elegans is part of the cause of higher amyloid-beta aggregations.
Citations related to my procedure are below
Additional citations related to my research paper are included in the pdf of the research paper attached below
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Additional Project Information
Demonstrate whether dyshomeostasis of a given trace metal triggers the dyshomeostasis of other trace metals and how the addition of a given trace metal to a C. elegans model affects the development of amyloid-beta (Aβ) aggregations. This will elucidate the correlation between trace metals, copper and zinc, and amyloid-beta in wild type C. elegans compared to an Alzheimer’s Disease Model.
I hypothesized that if amyloid-beta aggregations increase in an Alzheimer’s Disease model, then these aggregations are induced by trace metal imbalances because dyshomeostasis of one trace metal triggers further dyshomeostasis of other trace metals.
Nematode Strains and Maintenance
- Aβ aggregation-producing (strain CL2006) and wild type (strain N2) C. elegans were propagated at 20°C on Nematode Growth Media (NGM) plates seeded with the bacterial strain OP50
- Perform synchronization to ensure all C. elegans are synchronized to the same age using the following method: Adult hermaphrodite worms were transferred to fresh plates and allowed to lay eggs for 2–4 h. After removal of the adult parental worms, the synchronized progeny were allowed to reach adulthood
Supplementation with Copper and Zinc
*Begin supplementation, once the worms reach adulthood, stage L4 to ensure the treatments do not affect development
- Once the worms reach adulthood (day 3), a group of synchronized CL2006 worms and a group of synchronized N2 worms were placed on the copper-supplemented plates. CuCl2 stock solution was diluted into a live E. coli OP50 suspension, reaching a final concentration of 150 μM, and was placed on the surface of the NGM plates.
- Also on day 3, a group of synchronized CL2006 worms and a group of synchronized N2 worms were placed on the zinc-supplemented plates. ZnSO4 stock solution was diluted into a live E. coli OP50 suspension, reaching a final concentration of 500 μM, and was placed on the surface of the NGM plates.
- On days 6 and 9, thirty worms from each of the four groups: (1) copper-supplemented CL2006, (2) zinc-supplemented CL2006, (3) copper-supplemented N2, (4) zinc-supplemented N2, were lysed in preparation for copper and zinc colorimetric assays:
- Spin worms in microfuge at 4,000 rpm for 1 min to a pellet
- Remove supernatant and wash in 1.5ml of cold L15 buffer
- Spin to pellet. Remove supe.
- Pipette ~25µl dauer pellet onto glass slide
- Add 50 mm glass coverslip
- Hold the coverslip and slide into place while applying pressure to the coverslip using a pipette tip. Perform under dissecting microscope to visualize head disruption. Repeat applying pressure until most of the worms are exploded
- Remove coverslip from slide over a glass petri dish
- Wash coverslip and slide with 1ml of cold L15. Repeat.
- Pipette L15/cell mixture vigorously 25 times with pipette to ensure C. elegans are lysed.
Copper and Zinc Levels Quantification
- On days 6 and 9, the amount of copper in 30 lysed C. elegans from each of the four groups was quantified. To do so, a copper colorimetric assay (Elabscience) was applied to the lysed C. elegans solution.
- Also on days 6 and 9, the amount of zinc in 30 lysed C. elegans from each of the four groups was quantified. A zinc colorimetric assay (Elabscience) was applied to the lysed C. elegans solution.
- Once the standard wells were created for both assays, the percent transmittance of the standards and test groups was measured using a colorimeter. The percent transmittance was converted to ion content (μmol/L) as specified by the Elabscience assays.
- On days 6 and 9 after synchronization, 20 worms from the copper-supplemented and zinc-supplemented CL2006 and N2 groups were tested for paralysis, indicating the level of Aβ.
- The worms were tested for paralysis by tapping their noses with a platinum wire pick. Worms that moved their noses but failed to move their bodies were scored as “paralyzed”
- Plot results and compare the levels of Cu, Zn, and Aβ over time and between control and Aβ producing C. elegans (CL2006).
- Statistical analysis involving two groups was conducted using a t-test. Statistical analysis involving more than two groups was conducted using a one-way analysis of variance (ANOVA) followed by a post-hoc analysis using Tukey test. The differences were considered to be significant at p < 0.05.
The following potentially hazardous materials were used: 1)150 μM CuCl2 (NFPA Rating: (estimated) Health: 2; Flammability: 0; Instability: 1), and 2)500 μM ZnSO4 (NFPA Rating: (estimated) Health: 2; Flammability: 0; Instability: 0). The above materials can cause eye, skin, gastrointestinal, and respiratory tract irritation if they are inhaled, ingested, or come in contact with skin or eyes. Due to the potential safety risks associated, several safety precautions will be taken. To avoid inhalation, the experiment will be conducted in a well ventilated area with a hood. To avoid skin or eye irritation the researcher will use gloves, a lab coat, and goggles and conduct the experiment in a facility equipped with an eyewash facility and safety shower for emergencies. Any excess materials will be disposed of at a chemical waste disposal center at my high school. This experiment can safely be run with the appropriate safety precautions under a mentor supervision, considering the NFPA ratings are low.
For more information please refer to the following msds sheets:
Questions and Answers
1. What was the major objective of your project and what was your plan to achieve it?
a. Was that goal the result of any specific situation, experience, or problem you encountered?
b. Were you trying to solve a problem, answer a question, or test a hypothesis?
I first became interested in conducting neuroscience related research when an extended family member, with whom I am close, suffered a mental health challenge. This was a very difficult time for my family as we tried to get the appropriate treatment and adjust to the new reality.
Thus, I was determined to conduct neuroscience research when I was accepted to my high school’s 3 year research program, which accepts the top 5% of students, as a sophomore in September 2019. After reading dozens of publications to better understand current research in the neuroscience field, I came across a super interesting publication that found high trace metal levels in amyloid-beta plaques: the primary biomarker in Alzheimer's Disease.
This really piqued my interest because I had recently made a facebook advocacy page about the importance of water safety after hearing about the Flint, Michigan water crisis in which toxic trace metals were leaked into drinking water. I had become very passionate about making sure that the water we were drinking was safe and free of toxic trace metals. So, the publication greatly piqued my interest because it made a connection between high trace metals and the development of the neurodegenerative disease Alzheimer's.
I spent several months reading all of the publications I could find about the role of trace metal imbalances in the development of Alzheimer's Disease (AD). It became abundantly clear that the role of trace metals copper and zinc as deleterious or beneficial in AD remains elusive. In addition, the molecular mechanisms that characterize trace metal - amyloid-beta binding also remain elusive. After pinpointing these unknowns that persist in the field, I independently developed a hypothesis and procedure about the role of trace metal dyshomeostasis in Alzheimer's Disease pathology.
Specifically, I sought out to determine whether the dyshomeostasis of a given trace metal further triggers the dyshomeostasis of other trace metals in Alzheimer's Disease and how this affects amyloid-beta levels, which had not been previously researched. I chose to research the effect of high copper levels (simulated through supplementation) on zinc levels and the effects of high zinc (simulated through supplementation) on copper levels in Alzheimer’s Disease because previous publications had found that copper and zinc avidly bind to amyloid-beta. Therefore, I hypothesized that if amyloid-beta aggregations increase in an Alzheimer’s Disease model, then these aggregations are induced by trace metal imbalances because dyshomeostasis of one trace metal triggers further dyshomeostasis of other trace metals
2. What were the major tasks you had to perform in order to complete your project?
a. For teams, describe what each member worked on.
After developing my research procedure, I initiated conversations with faculty from surrounding universities to make sure my procedure was as comprehensive as possible. Dr. Rachel Kaletsky (Princeton University, molecular biology) provided me with the lysis procedure for dauer C. elegans, as I mention in the acknowledgements section of my manuscript.
Since I would be conducting experimentation in my high school’s rudimentary science lab, I needed to independently purchase several pieces of equipment. Overall, I needed $2,250 worth of materials! Therefore, I spent four months (September- December 2020) fundraising on the research crowdfunding website experiment.com: https://experiment.com/tracemetalsalzheimers. After raising the full sum, I spent every free moment between classes and during lunch speaking with material manufacturers to ensure the materials would fit in my school’s rudimentary lab and arrive promptly despite the pandemic induced shipping delays. Finally, I began experimentation while persevering through Covid-19 school closure challenges and covid-induced supply chain delays (which I detail further in questions 4 and 7).
I conducted experimentation and recorded data independently from March to June 2021. Overall, gathering data required being adaptive and a great deal of dedication. I spent at least 2 hours in the lab 4 times per week to conduct experimentation, with some days becoming 5 hour long lab days. Given that I am also a track and field athlete and captain of the team, balancing both long days in the lab and track practice was challenging but ultimately successful. Overall, I was so passionate about conducting my research that I was happy to spend long hours in the lab.
After finishing experimentation, I spent summer 2021 statistically analyzing the raw data, formulating conclusions, and reading dozens more previously-published papers to fully pinpoint the unique contributions of my research. Overall, my study newly demonstrates that dyshomeostasis of trace metals copper or zinc triggers further trace metal dyshomeostasis in Aβ-producing worms, while dyshomeostasis of copper or zinc triggers a return to equilibrium in non-Aβ-producing worms. This might indicate that the inability to return to trace metal homeostasis in Aβ-producing C. elegans is part of the cause of higher amyloid-beta aggregations.
I submitted my full research paper to Frontiers in Neuroscience: the most cited, international neuroscience journal. After several rounds of blind peer-review, my paper was accepted and published in October 2021 (https://www.frontiersin.org/articles/10.3389/fnins.2021.755475/full).
3. What is new or novel about your project?
a. Is there some aspect of your project's objective, or how you achieved it that you haven't done before?
b. Is your project's objective, or the way you implemented it, different from anything you have seen?
c. If you believe your work to be unique in some way, what research have you done to confirm that it is?
As the 6th leading cause of U.S. deaths, Alzheimer's Disease is a life threatening illness that affects over 44 million people worldwide. It is very hard seeing loved ones' memories slowly slipping away. My research brings us one step closer to finding a cure.
Previous research on the role of trace metals in Alzheimer’s Disease has focused on: a) the dysregulation of trace metals (such as Cu and Zn) in the brains of Alzheimer’s Disease patients, b) the binding affinities and sites between trace metals and Aβ plaques, and c) trace metal induced Aβ-aggregation in Alzheimer’s Disease models. The molecular mechanisms that characterize the binding between trace metals and amyloid-beta are not completely understood. In addition, the effect of the imbalance of a given trace metal on the homeostasis of other trace metals in an Alzheimer’s Disease model is also elusive. The novelty of my research is that it demonstrates the key differences in trace metal - amyloid-beta coordination between an Alzheimer’s Disease model and a non-Alzheimer’s Disease model. My study demonstrates that dyshomeostasis of trace metals copper or zinc triggers further trace metal dyshomeostasis in Aβ-producing worms, while dyshomeostasis of copper or zinc triggers a return to equilibrium in non-Aβ-producing worms. This might indicate that the inability to return to trace metal homeostasis in Aβ-producing C. elegans is part of the cause of higher amyloid-beta aggregations. Therefore, the molecular interaction between trace metals and amyloid-beta can be targeted in Alzheimer’s Disease therapies.
The model organism C. elegans, used in my research, is an excellent in vivo model for the study of Alzheimer's Disease and roughly 38% of worm genes have a human ortholog, such as APP and tau which are largely involved in Alzheimer’s Disease. Given that many biochemical pathways are conserved in C. elegans, my study reveals key insights about the molecular mechanisms between trace metals and amyloid-beta present in Alzheimer’s Disease.
As the large baby boomer generation continues to age, better understanding the molecular mechanisms characterizing Alzheimer’s Disease, particularly as a disease of trace metal dyshomeostasis, is key in helping develop a cure that targets the correct molecular pathways.
4. What was the most challenging part of completing your project?
a. What problems did you encounter, and how did you overcome them?
b. What did you learn from overcoming these problems?
During experimentation, there were several challenges that I overcame. For instance, I had originally planned on using Congo Red dye to strain amyloid-beta aggregations under the microscope and qualitatively analyze the extent of stained amyloid-beta aggregations. Upon trying this method, however, the C. elegans were not becoming effectively stained. Therefore, after consulting the literature, I adjusted my method to solely use paralysis as a quantitative measure for amyloid-beta aggregations.
Additionally, balancing my responsibilities as track and field captain with long lab days was challenging. As a National level qualifying track athlete, I am expected to be at practice every day and at multiple hour-long meets which are often on weekdays. Given that I needed to stay after school to finish lab experimentation, it was often a challenge to finish the trials in time before practice started. Overall, by maintaining focus and preparing any materials ahead of time whenever possible, I was able to successfully balance both experimentation in the lab and track athlete responsibilities.
Also, I had several pandemic induced challenges that I creatively overcame, which I detail in question 7.
Overall, my research journey has been an invaluable experience. To overcome procedural and material-constraint-related challenges, I have further developed my problem solving skills and perseverance. I have also practiced reading scientific literature to find methods proposed by other scientists in the field. By initiating conversations about my procedure with several university professors, I learned the power of networking and collaborating with other scientists. To fundraise a large sum of money to support my experimentation, I practiced using persistence and outreach methods. Finally, I further developed my analytical skills while analyzing data and formulating conclusions.
5. If you were going to do this project again, are there any things you would you do differently the next time?
I used a colorimeter to determine the zinc ion content and copper ion content of various experimental groups of C. elegans. A spectrophotometer might have yielded even more precise values. However, my high school lab already had a working, accurate colorimeter and the cost of a spectrophotometer is quite high, which would have made fundraising a much longer and more difficult process. Additionally, in this study, the levels of copper and zinc in Aβ-producing and non-Aβ-producing C. elegans were measured at several time points in response to copper and zinc supplementation. Future work includes measuring the levels of copper and zinc in Aβ-producing and non-Aβ-producing C. elegans without any supplementation to determine the copper and zinc homeostatic ranges.
6. Did working on this project give you any ideas for other projects?
Amyloid-beta and trace metals copper and zinc interact with high affinity and this binding is believed to cause a conformational change in the shape of amyloid-beta, transforming it into a configuration more amenable to forming aggregations. My study indicates that an imbalance in a given trace metal causes a cascading effect resulting in further trace metal imbalances and more amyloid-beta aggregations. Therefore, I am interested in exploring the mediating factors that allow the interaction between trace metals and amyloid-beta in an Alzheimer’s Disease model.
HSP-16.2 is an oxidative stress response chaperone protein. As a chaperone protein, HSP-16.2 facilitates normal folding of proteins and handles misfolded and aggregated proteins. Thus, I would be interested in exploring the levels of protein HSP-16.2 as amyloid-beta aggregations increase in response to trace metal imbalances.
7. How did COVID-19 affect the completion of your project?
Throughout my 2.5 year long research journey, dealing with the Covid-19 pandemic has been challenging. Firstly, the scientific crowdfunding website experiment.com was severely behind schedule at approving projects due to delays imposed by Covid-19. It took over 2 months of sending emails to inquire about the delays and find ways to speed up the process to simply receive approval for my project to be listed on the site. Later, when ordering materials, shipping and manufacturing were greatly delayed due to the pandemic. I had several phone calls with manufacturers to find products that would arrive in the shortest possible time and that would fit in my school’s rudimentary science lab. In addition, my school had decreased class time to minimize the time in school during the pandemic. I simply would not have been able to finish my experimentation with such limited in-person class time. Therefore, I stayed after school to conduct experimentation, spending 2 hours in the lab between the end of the school day and the start of my track practice. Overall, I have used perseverance and problem solving skills to successfully conduct my experimentation, despite the pandemic-induced challenges.