My name is Haben Tesfamariam, and I recently graduated from the University of Texas at Austin with a B.S. in Cell and Molecular Biology. As an undergraduate research assistant working in Dr. Andrea Gore’s Lab at the University of Texas’ College of Pharmacy, I worked on a project studying the effect timing and duration of estrogen treatment have on energy balance and eating behavior in a menopausal female rat model. During menopause, dramatic decreases of ovarian estrogen cause a multitude of behavioral changes, including a change in eating behavior, mood swings, depression, and anxiety. It’s believed that the complexity of behavioral changes is due, at least in part, to the loss of estrogen. While estrogen treatments may alleviate some of these symptoms, we believe there are age-related changes in the brain of the rats that affect their sensitivity to estrogen treatment. The goal of my study was to observe how the timing and duration of estrogen treatment affect the expression of estrogen receptors (ERs) in the hypothalamus, the part of the brain that is important in feeding behavior and energy balance. I proposed that the neuronal function of estrogen receptors in the hypothalamus changed during menopause and were affected by timing and duration of hormone treatment.
For my study, we primarily focused on two specific areas (nuclei) of the hypothalamus: the medial pre-optic area (mPOA) and the arcuate nucleus (ARC). Previous studies have reported the importance of these two nuclei in controlling feeding (Mauvais-Jarvis et al., 2013). I was awarded an undergraduate research fellowship to study the level of estrogen receptor expression and the localization of those receptors within the mPOA and ARC. I used immunohistochemistry (IHC) to create a comprehensive brain atlas to compare estrogen receptor levels in rats that were treated with estrogen to untreated controls. At the end of my analysis, I observed a correlation between estrogen treatment and age-related differences in ER-α receptor expression within the nuclei of the rats. Furthermore, both estrogen treatment and ER-α receptor expression also corresponded to behavioral differences I observed within the two populations of rats. My findings established how the timing and duration of estrogen treatment affects brain development, which is important for the development of new hormone therapy treatments for women experiencing severe menopausal symptoms.
I became interested in working with SENS Research Foundation after observing the effects aging had on the rats during my research project and learning about SRF’s approach to understanding age-related diseases. I decided to apply to the SRF Summer Scholars Program because I wanted to learn how scientists approach understanding the aging process and how they use their knowledge to combat degenerative diseases.
Determining the Role of Transcription Factor Gcn4 in the Increased Lifespan of Multiple Yeast Mutants
This summer I will be working with Dr. Mark McCormick in the laboratory of Dr. Brian Kennedy at the Buck Institute for Research on Aging. We are working in the budding yeast Saccharomyces cerevisiae and the nematode worm Caenorhabditis elegans. These model organisms live for only a few weeks, making it possible to quickly study changes in their lifespan. Because they have long been used to study many other biological processes, there are many existing tools available to us when working with these organisms, such as genome-wide deletion collections. Finally, it has been shown repeatedly in many diverse biological processes that fundamental mechanisms first uncovered in simple model organisms are often conserved in higher organisms, such as humans. In the case of aging, changes in yeast genes in the TOR (target of rapamycin) signaling pathway, including the yeast gene TOR1 itself, were shown to extend lifespan (Kaeberlein et al., 2005), and subsequent work has shown that treatment with the TOR targeting drug rapamycin extends lifespan when fed to middle-aged mice (Harrison et al., 2009), leading us to hypothesize that this drug target or others we uncover may allow us to extend human lifespan as well.
Figure 1. Gcn4 is necessary for life extension in long-lived ribosomal protein deletion strains.
The life extension observed in strains rpl31aΔ and rpl20bΔ is dependent on Gcn4 (A, B). Gcn4 translation is increased in strains rpl31aΔ and rpl20bΔ (C).
Previous work from the Kennedy lab has shown that deletion of genes that encode components of the ribosome can extend the lifespan of S. cerevisiae (Steffen et al., 2008; Steffen et al., 2012). Other work has shown that knocking down the same genes in the nematode C. elegans and the fruit fly D. melanogaster can also extend lifespan (Hansen et al., 2007). The nutrient-responsive transcription factor Gcn4 is necessary for this effect in yeast (Figure 1A and 1B), and Gcn4 protein levels are increased in these long-lived ribosomal protein deletion strains (Figure 1C) (Steffen et al., 2008).
Figure 2. pVW31 dual-luciferase Gcn4 translational reporter construct.
This plasmid contains the PGK1 promoter fused to Renilla luciferase and the GCY1 terminator, followed by the promoter, 5’ untranslated region, and ORF of GCN4 fused to firefly luciferase and the CYC1 terminator. By measuring relative firefly / Renilla luciferase luminescence, we can precisely quantify translational upregulation of Gcn4.
The Kennedy lab has recently uncovered additional translation-related genes whose deletion increases lifespan. This summer I will be testing whether any of these novel long-lived deletion strains also exhibit translational up-regulation of Gcn4. By using a dual-luciferase reporter of translational regulation of Gcn4 (Figure 2), I will be able to quantify changes in Gcn4 by measuring the luminescence from a firefly protein expressed by the reporter, called luciferase. I will also determine whether any of these same genes affect the lifespan of C. elegans when knocked down. Finally, if we observe Gcn4 up-regulation in some of these strains, we will delete the GCN4 gene to determine if Gcn4 function is necessary for the observed increase in lifespan If GCN4 is required for the observed increase in lifespan, the absence of GCN4 function would revert the lifespan of the deletion strain back to normal.
Future Plans:
In the future, I plan on attending medical school with the goal of becoming a specialized surgeon. My hope is that my research at the University of Texas at Austin and the Buck Institute will give me a new insight on the aging process and ways to develop new cures for degenerative diseases. I’m hoping to develop new treatment techniques that focus on preventative care given our understanding of the aging process and human development.
References:
Hansen, M., Taubert, S., Crawford, D., Libina, N., Lee, S.J., and Kenyon, C. (2007). Lifespan extension by conditions that inhibit translation in Caenorhabditis elegans. Aging Cell 6, 95-110.
Harrison, D.E., Strong, R., Sharp, Z.D., Nelson, J.F., Astle, C.M., Flurkey, K., Nadon, N.L., Wilkinson, J.E., Frenkel, K., Carter, C.S., et al. (2009). Rapamycin fed late in life extends lifespan in genetically heterogeneous mice. Nature 460, 392-395.
Kaeberlein, M., Powers, R.W., 3rd, Steffen, K.K., Westman, E.A., Hu, D., Dang, N., Kerr, E.O., Kirkland, K.T., Fields, S., and Kennedy, B.K. (2005). Regulation of yeast replicative life span by TOR and Sch9 in response to nutrients. Science 310, 1193-1196.
Mauvais-Jarvis, F., Clegg, D.J., and Hevener, A.L. (2013). The role of estrogens in control of energy balance and glucose homeostasis. Endocrine reviews 34, 309-338.
Steffen, K.K., MacKay, V.L., Kerr, E.O., Tsuchiya, M., Hu, D., Fox, L.A., Dang, N., Johnston, E.D., Oakes, J.A., Tchao, B.N., et al. (2008). Yeast life span extension by depletion of 60s ribosomal subunits is mediated by Gcn4. Cell 133, 292-302.
Steffen, K.K., McCormick, M.A., Pham, K.M., MacKay, V.L., Delaney, J.R., Murakami, C.J., Kaeberlein, M., and Kennedy, B.K. (2012). Ribosome deficiency protects against ER stress in Saccharomyces cerevisiae. Genetics 191, 107-118.
