2015 SRF Summer Scholar Profile: June Hope

June Hope

SRF Summer Scholar, Class of 2015

Buck Institute for Research on Aging

My name is June Hope. I am a recent graduate of California Polytechnic State University, San Luis Obispo, or as we like to call it, Cal Poly SLO. I graduated with a B.S. in Biology with a concentration in Cell and Molecular Biology and a minor in Microbiology. Since my first genetics course at Cal Poly, I have discovered a deep interest in genetics and developed a passion for learning more. To me, genetics is evidence of our connection to the past and to all forms of life and, as such, is a constant source of inspiration and motivation for me.

While at Cal Poly, I was fortunate to meet and work with several amazing professors, including Dr. Ken Hillers, who runs the chromosome biology lab. The main focus of the lab’s research is discovering the mechanisms and cellular components involved in meiosis. Meiosis is the process in which haploid gametes are produced from diploid sex cells and is essential for sexual reproduction. Errors in meiosis lead to infertility, a problem in both animal and human health.

To identify proteins that are potentially involved in meiosis, the nematode worm Caenorhabditis elegans was used as a model organism. C. elegans are ideal organisms for genetic studies for several reasons. They are multicellular eukaryotes, so they share genes that are homologous to genes in mammals. They are hermaphroditic, so each individual inherits genes from a single parent, which makes genetic analysis simpler. And, they are transparent, allowing the visualization of their internal components, such as the gonads. Along with these characteristics, gene function in C. elegans can easily be inhibited through a process known as RNA interference (RNAi).

In order to study the proteins involved in meiosis, we initially used a proteomics approach. The mutant C. elegans strain glp-4 develops somatically like the wild-type strain, but the mutants do not develop gonads. Using MALDI-TOF mass spectrometry, I compared the proteins founds in the wild-type and the glp-4 strains. Since the glp-4 strain lacks the cells that undergo meiosis, proteins that were found in wild-type but not glp-4 were considered candidates for meiotic proteins.

After producing a list of candidate proteins, RNAi plasmids were constructed which, once inserted and expressed within E. coli, could be used to study candidate genes by inhibiting gene function. Each RNAi plasmid produced RNA complementary to target mRNA in C. elegans. RNAi was performed on C. elegans by feeding them E. coli. Once the RNA from the E. coli was ingested, RNAi enzymes within C. elegans would use the RNA to find complementary mRNA and destroy it, silencing the gene it encoded. In this way, the effect of the knockdown of each candidate protein could be accessed. If the protein is required for meiosis, then the knockdown of the protein would result in a significant decrease or loss in progeny born compared to wild-type. If a decrease in progeny was seen, the cells within the gonads were visualized using fluorescent confocal microscopy.

Within a C. elegans gonad, cells migrate from the distal end to the posterior end. Cells in the distal portion undergo mitotic division, while cells progressing to the distal end undergo meiotic division, eventually becoming oocytes that are available for fertilization as they reach the spermatheca. When cells within the gonads are visualized using a DNA-stain such as DAPI, errors in meiosis can be seen, which are represented by abnormal amounts of DNA within nuclei, and often a wide diversity in nuclear size. Errors in meiosis specifically can be differentiated from errors in general cellular division, which would affect mitosis as well, by comparing cells in the distal and posterior ends of the gonads.

Through this process, several proteins required for meiosis have been identified, and a database of meiotic proteins was created. My main contributions to the project were optimizing PCR reactions for the isolation of C. elegans DNA that were needed to construct the plasmids as well as designing and constructing Gateway plasmids, which was a new method used in the lab at the time. Several of the plasmids constructed were genes that had never been targeted by RNAi before, such as the gene termed D1054.11. Along with old and new members, I helped create the lab’s database of meiotic proteins, which will continue to be built, allowing for further research on meiosis and infertility.

Exploring Metallostasis and Aging in C. elegans

While at the Buck Institute for Research on Aging, I am working in the lab of Dr. Gordon Lithgow under the supervision of Dr. Daniel Edgar. The Lithgow lab has recently begun investigating the relationship between alterations in metals that affect cell homeostasis and how these changes affect the aging process.

Metal levels play a crucial role in cell function [1]. As we age, the amount of metal found in cells changes, with some metals, such as iron (Fe) and manganese (Mn), increasing and some, such as potassium (K), [2] decreasing (Fig. 1). As we age, the amount of insoluble proteins also increases, and it is thought that these insoluble proteins lead to the formation of harmful aggregates [3]. These aggregates have been implicated as the cause of several age-related neurodegenerative diseases such as Alzheimer’s and Parkinson’s disease [3][4].

The fact that both metal composition and protein solubility changes with age prompts four different possibilities that can be explored. The first is that changes in metal composition within cells causes proteins to become insoluble, and most of the evidence so far agrees with this possibility. However, there are other possibilities – the increase in insoluble proteins causes changes in metal composition or the changes in metals and protein solubility are both being affected by a separate mechanism. A final possibility, although the least likely according to studies so far, is that the increase in protein insolubility and changes in metal composition is a correlation, and there is no causative relationship occurring.

The Lithgow lab has recently begun investigating these possibilities by first studying the relationship between metal composition in cells and changes in protein solubility and investigating how these changes in turn affect aging and neurodegenerative diseases. A previous study showed that increases in iron led to increases in protein insolubility, and this, in turn, led to significant decreases in longevity of C. elegans, confirming that there is a correlation between metals, protein insolubility, and aging [2].

Figure 1. Comparison of the metal profile in C. elegans as they age.

Metal ICPaes analysis was used to determine the metal profiles of synchronous populations that were analyzed on day 1, day 5, day 9, and day 11. Each bar represents the mean of three biological replicates, each in triplicate. Error bars represent the standard error.[2]

During my time here at the Buck I will be working to determine if there is a causal relationship between alterations in metals and increased protein insolubility as well as investigating if specific changes in metals can increase longevity in C. elegans. Again, C. elegans make an ideal model organism because it has genes that are associated with aging and are homologous to those found in mammals [5]. Previous studies have demonstrated that changes in metal composition within C. elegans causes changes in protein solubility, which has been associated with changes in longevity and locomotion in the worms. The decreases in locomotion, which often leads to paralysis, are caused by protein aggregates found in neurons. This phenomenon is reflective of age-related neurodegenerative diseases in humans, such as Parkinson’s disease [6].

For my project, I will study the metal composition of worms with and without insoluble proteins by using Lithium and Vitamin D3, which have been shown in previous studies to inhibit protein insolubility as C. elegans age [7]. This will help me establish if there is a direct relationship between metal composition and protein solubility. I also plan to study how protein solubility and longevity are affected by increasing metals that normally decrease with age or by decreasing metals that normal increase with age. Finally, I will be studying the effect of compounds that are known to inhibit the formation of protein aggregates on both metal composition and longevity.

Future Plans:

My future goals focus around combining my interest in genetics and research with my passion for helping animals. I currently volunteer with wildlife and zoo conservation programs and hope to work in an animal diagnostics or animal genetics laboratory over the next year or so. I am also planning on teaching abroad for a year, while volunteering in animal sanctuaries, and hope to gain a Ph.D. in either evolutionary biology or molecular biology following that year. My ultimate career goal is to be able to work with endangered species and use genetic research to help protect and re-populate them.

References:

1. Farina, M., Avila, D.S., de Rocha, J.B.T., and Aschner, M. 2013. Metals, oxidative stress and neurodegeneration: A focus on iron, manganese and mercury. Neurochemistry International. 62: 575-594.

2. Klang, I.M., Schilling, B., Sorensen D.J., Sahu, A.K., Kapahi, P., Andersen, J.K., Swoboda, P., Killilea, D.W., Gibson, B.W., and Lithgow, G.J. 2014. Iron promotes protein insolubility and aging in C. elegans. Aging. 6(11): 975-991.

3. David, D.C., Ollikainen, N., Trinidad, J.C., Cary, M.P., Burlingame, A.L., and Kenyon, C. 2010. Widespread Protein Aggregation as an Inherent Part of Aging in C. elegans. PLoS Biol. 8(8): e1000450. doi:10.1371/journal.pbio.1000450

4. Taylor, J.P., Hardy, J., and Fischbeck, K.H. 2002. Toxic Proteins in Neurodegenerative Disease. Science. 296: 1991-1995.

5. Chen, C., Chen, Y., Jiang, H., Chen, C., and Pan, C. 2013. Neuronal aging: learning from C. elegans. J. Mol. Sig. 8 (14): 1-10.

6. McColl, G., Killilea, D.W., Hubbard, A.E., Vantipalli, M.C., Melov, S., and Lithgow, G.J. 2008. Pharmacogenetic Analysis of Lithium-induced Delayed Aging in Caenorhabditis elegans. J Biol Chem. 283(1): 350-357.

7. Kraemer, B.C., Zhang, B., Leverenz, J.B., Thomas, J.H., Trojanowski, and Schellenberg, G.,D. 2003. Neurodegenerative and defective neurotransmission in a Caenorhabditis elegans model of tauopathy. PNAS. 100 (17): 9980-9985.

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