My name is Isha Bagga, and I am a rising junior at University of California, Los Angeles studying physiological sciences. When I found the SENS Research Foundation, I thought it would be exciting to join because everyone was working on many new concepts in aging. I find this very intriguing because that is one physical process that everyone goes through regardless of any environmental or biological effects that surround them. There are many factors that contribute to problems in aging, and I find the vast scope of these diseases very important because I know many people who have been affected by these ailments. People are now living longer, so the study of aging is even more important. I am also interested in discovering how some diseases are caused by old age and how some of those same diseases can occur in younger people as well. I was drawn to the SRF Summer Scholars Program because there are so many options under the umbrella of age-related research that are all cutting-edge and very relevant to today’s growing topics in science.
Last summer, I worked on a research project in Professor David Cheresh’s Lab in the Department of Pathology at the University of California, San Diego, under the supervision of Dr. Jay Desgrosellier. The goal of the project was to understand under what conditions breast cancer cells can survive in order to figure out better ways to treat them in a clinical setting. The Cheresh lab studies different pathways in breast cancer cells involving Integrin αvβ3 and the PUMA gene. Blockage of integrin signaling has been shown to inhibit tumor growth, with inhibition of β3 being most effective.1 PUMA is a pro-apoptotic mediator, which means it promotes a cascade of events leading to a breakdown of the cell. It is also the most upregulated gene in breast cancer cells not expressing β3. It is induced by p53, a tumor suppressor protein which is mutated in cancer cells.2
I contributed to one project focused on studying the levels of PUMA in relation to Integrin αvβ3 expression in order to see how apoptosis regulates breast cancer cells, since PUMA mediates cell death. Using qPCR, I analyzed a panel of breast cancer cell lines for relative levels of PUMA mRNA. I found that cell lines without β3 tended to show increased levels of PUMA mRNA. However, when β3 was expressed ectopically in cell lines lacking endogenous β3, PUMA mRNA levels were decreased.3 I used Western blots to confirm the differences in PUMA protein levels between control cells expressing β3 and those without β3. Using a few different cell lines to cross-check the results, I found that the PUMA mRNA concentrations did correlate with the protein concentrations. Our findings showed that Integrin αvβ3 is sufficient to suppress both PUMA mRNA and protein expression, which may enhance breast cancer cell survival and lead to more aggressive cancer since the cancerous cells are not being degraded through apoptosis.
The Induction of Autophagy by Spermidine and its Effect on Lifespan in Aging Mice
This summer, I am working in Dr. Brian Kennedy’s lab at the Buck Institute for Research on Aging under the supervision of Dr. Chen-Yu Liao. I will be testing the ability of the natural polyamine spermidine to extend healthspan by examining its role in the induction of autophagy in an aging mouse model. Autophagy is an evolutionarily conserved self-degradation process that allows cells and tissues to cope with several adverse conditions. Autophagy is necessary for a stable system because it has been shown that when the process is dysregulated, age-related diseases and heart, liver, and muscle disorders can occur.4 This process is of utmost importance to an aging cell because it ensures turnover of degraded proteins and organelles, which prevents cellular defects and ultimately enhances the health of the organism. The Kennedy Lab has reported that the drug rapamycin can improve defective autophagic-mediated degradation and enhance heart function in a mouse model with defective hearts (Lmna-/-, or Lamin A/C gene knockout), including heart diseases that are associated with aging.5 It does this by inhibiting the mTOR (mechanistic target of rapamycin) pathway, which is beneficial because reduced mTORC1 (mTOR Complex 1) activity is linked to elevated autophagy and other cellular stress responses.6
My project will involve injecting old wild-type mice (more than 24 months) with spermidine. Spermidine decreases acetylation of histone H3, leading to increased transcription of autophagy-related genes, such as Atg5. Previous studies have shown that the addition of spermidine to budding yeast (Saccharomyces cerevisiae) and fruit flies (Drosophila) extends lifespan through the induction of autophagy.
Figure 1. Application of spermidine extends the lifespan of yeast and Drosophila.
(a) Lifespan plot of BY4741 wild-type budding yeast cells after separation into old (fraction V) and young (fraction II) cells. Spermidine’s ability to increase lifespan is greater in the old yeast cells compared to the younger fraction. (b) Lifespan curve determined by the age-specific number of dead individuals of female Drosophila with and without supplementation of food with varied spermidine concentrations. The highest dosage of spermidine (1mM ) had the greatest effect in extending the lifespan in both species.7
Spermidine is a nontoxic, natural compound that is involved in growth, development, protein/nucleic acid synthesis and cell signaling. Along with other polyamines, it commonly is found in Mediterranean and Asian diets.8 It was thought to induce autophagy through mTOR-independent pathways; however, preliminary data has shown that when spermidine was administered to a Lmna-/- mouse model, mTORC1 signaling was altered.
Autophagic flux is regulated by principal protein levels such as LC3II/LC3I, SQSTM1/p62, and Beclin-1. The mTORC1 pathway can be regulated by suppression and overexpression of downstream mTORC1 targets, S6K1 and 4EBP1, respectively.5 We want to quantify autophagic- and mTORC1-related protein concentrations in different tissue types to understand how they are regulated. I will use Western blots and immunostaining techniques to visualize protein levels of LC3II/LC3I, SQSTM1/p62, and Beclin-1 to analyze autophagy activity and levels of S6K1 and 4EBP1 to understand mTORC1 activity. This project will lead to a better understanding of spermidine and may highlight new therapeutic approaches to treat heart disorders and enhance healthspan through autophagic regulation and the mTOR pathway.
Future Plans:
After graduation, I am planning on taking a gap year in which I plan to conduct research and volunteer in a hospital to gain some clinical experience. I eventually want to go to medical school and work as a physician focused on age-related diseases. Last year, I began working in Dr. Stanley Thomas Carmichael’s lab at UCLA, which is focused on stroke treatment. This upcoming fall, I plan to start a new project in the Carmichael lab that is focused on understanding brain circuits more thoroughly as well as targeting specific proteins that could be used in drugs to enhance recovery from stroke and increase the outgrowth of neurons.
References:
1. Liu, Zhaofei, Fan Wang, and Xiaoyuan Chen. “Integrin αvβ3-Targeted Cancer Therapy.” Drug Development Research 69.6 (2008): 329–339. PMC. Web. 28 June 2016.
2. Bieging, Kathryn T., Stephano Spano Mello, and Laura D. Attardi. “Unravelling Mechanisms of P53-mediated Tumour Suppression.” Nature Reviews Cancer 14.5 (2014): 359-70. Web.
3. Desgrosellier, Jay, Qi Sun, and Jacqueline Lesperance. “PUMA Expression Targets AvB3/Slug “stem-like” Tumor Cells to Prevent Breast Cancer Progression Independent of Subtype.” Proceedings of the AACR. 3323. Web.
4. Madeo, Frank, Andreas Zimmermann, Maria Chiara Maiuri, and Guido Kroemer. “Essential Role for Autophagy in Life Span Extension.” Journal of Clinical Investigation.125.1 (2015): 85-93. Web.
5. Ramos, Fresnida J. et al. “Rapamycin Reverses Elevated mTORC1 Signaling in Lamin A/C–Deficient Mice, Rescues Cardiac and Skeletal Muscle Function, and Extends Survival.” Science Translational Medicine 4.144 (2012): 1.
6. Lamming DW, Ye L, Sabatani DM, Baur JA. “Rapalogs and mTOR inhibitors as anti-aging therapeutics.” Journal of Clinical Investigation. 3.123 (2013): 980-989.
7. Eisenberg, Tobias. “Induction of Autophagy by Spermidine Promotes Longevity.” Nature Cell Biology 11.11 (2009): 1306.
8. Minois, N., Carmona-Gutierrez, D., 2011. “Polyamines in aging and disease.” Aging.
