The Kaeberlein laboratory utilizes the budding yeast Saccharomyces cerevisiae to study aging, a progression characterized by the gradual deterioration of cellular components and bodily functions. Aging is a progression every organism eventually undergoes, and by better understanding the molecular mechanisms behind it, we can determine how to delay its progression and increase our healthspan, the portion of our lifespan in which we are healthy and free from serious disease. S. cerevisiae functions as an effective model organism because it contains many conserved biological processes, making lifespan simple and fast to measure. The replicative lifespan assay (RLS) is used to quantify the lifespan of the yeast. Individual yeast cells undergo asymmetric mitotic division producing a daughter cell that is distinctively smaller than the original mother cell. Yeast are incubated on growth promoting plates, and the yeast produce daughter cells that are microdissected, counted, and moved away. The cycle repeats until the original mother cell can no longer produce a daughter. The total number of daughter cells produced by individual mother cells is then used to understand the effect of the gene on the lifespan of the yeast. This technique aids in developing models that can be used to understand the conserved biological pathways that influence longevity. Aging is an underlying risk factor for many non-communicable diseases such as Alzheimer’s Disease, Cancer, and heart disease. Our laboratory has performed thousands of gene knockouts on S. cerevisiae and has identified several genes that modulate replicative lifespan. This research is focused on identifying the genes that play a role in reducing the rate of aging to delay the onset of age associated diseases. By understanding how specific genes play a role in aging, we can apply this research to other organisms, with the ultimate intent of increasing the length of a healthier human life.

The incidence of many human diseases, such as cancer, Alzheimer's and heart disease, increases with age. Studying the aging process could provide insight into new treatments and therapies for these aging-related diseases. Our research uses the yeast species, Saccharomyces cerevisiae, as a model organism to explore the effects of specific genetic and environmental factors on aging. Aging in yeast can be measured in terms of replicative lifespan, which is the number of progeny (daughter) cells produced by a parental (mother) cell. In order to measure the replicative lifespan, we utilize a microdissection microscope outfitted with a fiber optic needle that is used to separate individual mother cells from their daughter cells in order to quantify the number of daughter cells produced. The total number of daughters produced by a mother cell is the replicative lifespan of that cell. To determine which genes play an instrumental role in yeast replicative lifespan, our lab has studied the lifespan of 4,698 yeast mutants containing single gene deletions. Many mutants were identified that increase the lifespan of S. cerevisiae. In this study, we have re-evaluated our data to identify potential false-negative results from our initial work, in order to identify additional genes that may influence lifespan. Identifying the false-negative results will be beneficial in finding genes that may play a role in the aging process and had been previously overlooked. This will allow future research to continue in developing treatments that target the prevention of age-associated disease in humans, potentially allowing people to live healthier lives and decreasing the prevalence of cardiovascular disease and cancers. 

Mechanistic Target of Rapamycin (mTOR) is a highly conserved serine/threonine kinase originally discovered in yeast as a central regulator of cellular growth and proliferation in response to adequate nutrients and energy levels. mTOR is activated in a complex with an essential co-activator called Raptor. Our lab recently discovered that mTOR holds an essential role in early murine B-cell development; B-cell specific disruption of Raptor inhibited mTOR activity and resulted in a complete block in B cell development at the precursor-B (pre-B) cell stage. We hypothesize that B-cell specific disruption of Raptor will inhibit B-cell transformation induced by the Myc oncogene, and/or will inhibit the survival of B-cell lymphoma cells, by inhibiting cell growth induced by mTOR. We conditionally disrupted Raptor using gene targeting strategies in a mouse model of Burkitts Lymphoma whereby the c-Myc oncogene is expressed under control of the immunoglobulin heavy chain enhancer. We found that Raptor deficient mice took significantly longer to develop B-cell tumors when bred to c-Myc transgenic mice; our Kaplan-Meier Survival curve revealed that disruption of Raptor improved survival rates significantly, such that Raptor knockouts rarely showed growth of tumors. To better understand cellular mechanisms by which Raptor may inhibit B-cell lymphoma formation, we utilized flow cytometry to measure cell proliferation and survival of B cell lymphoma cells in the presence or absence of Raptor. Our results revealed a distinct reduction in both cellular proliferation and survival in the absence of Raptor, which suggests Myc driven B-cell proliferation and survival is highly dependent on Raptor and mTOR activity. In the next part of our studies, we will inducibly disrupt Raptor in already established primary Myc-induced B-cell lymphomas to determine whether Raptor and mTOR activity are essential for lymphoma cell survival. Through these results we will be able to determine whether inhibition of Raptor could be utilized as a strategy to inhibit cancer cell proliferation and/or survival.

Alzheimer's disease (AD) is a neurodegenerative illness responsible for 60-80% of dementia cases in the United States. Studies have shown that the formation of two primary pathologies, amyloid plaques and tau tangles, are present prior to neuronal death that results in this loss of memory and worsened motor function. Our research focuses on understanding the role of tau in disease progression. Tau stabilizes microtubules in the cell, which function to maintain cell structure and assist in intracellular transport. To successfully regulate microtubules, tau is modulated by site-specific phosphorylation. Evidence points specifically to the hyperphosphorylation of tau by certain kinases in playing a key role in neurodegenerative dieases such as AD. One kinase that has been identified as a tau-phosphorylating agent is tau-tubulin kinase 2 (TTBK2). To understand the role of TTBK2 kinase activity in the context of tau, we created double transgenic C. elegans lines expressing both human tau and TTBK2. We assessed behavioral abnormalities in our subsequent populations using locomotor assays of the homozygous crosses. From the data that was generated, we observed a significant difference in the movement of the double transgenic tau/TTBK2 strains as compared to their controls. The crosses moved substantially less than the other strains, and exhibited a more uncoordinated phenotype. These results support the hypothesis that the abnormal phosphorylation of tau by TTBK2 results in worsened motor control and general health of affected individuals. Additional testing of protein levels within the double transgenic lines will enable us to determine the mechanisms underlying this effect, and could eventually lead to information that might inform treatments counteracting the activity of kinases such as TTBK2 involved in hyperphosphorylation of tau proteins. Furthermore, this research has the potential to aid in finding a cure for a currently incurable disease and providing a hopeful future for Alzheimer's-affected individuals.

Working with the Northwest Avalanche Center (NWAC), the Trailhead Outreach Project researched levels of avalanche awareness, education, and preparedness of different demographics and user groups who enter avalanche terrain in the winter backcountry. Washington state itself is responsible for 54% of hiker avalanche fatalities nationwide in the past ten years. This statistic, combined with the increasing popularity of mountain recreation, made it necessary to understand who uses the backcountry and how they can be reached with the education required to make informed decisions when traveling near or in avalanche terrain. It was believed that backcountry skiers are generally well versed in avalanche risk and protocol while snowshoers and hikers are not. To obtain this information, NWAC set up a tent staffed with volunteers at four popular backcountry trailheads in the Cascade mountains on 12 separate weekend days (three at each location) over the winter of 2017-2018. Users were asked to take a survey that consisted of 14 quick questions to identify their mode of travel and levels of education, awareness, and preparedness. General demographic questions were also included. Preliminary results indicated that hikers and snowshoers do in fact have lower levels of avalanche awareness, education, and preparedness than skiers, and that people are heading into the backcountry with low levels of avalanche education at astounding rates. The results from the survey help the Northwest Avalanche Center direct their education and awareness programs toward the user groups shown to be less informed and potentially more vulnerable to an avalanche related accident.

Alzheimer’s disease (AD) is a progressive degenerative disorder that affects over 5.5 million Americans, resulting in memory loss and cognitive decline over time. AD is characterized by the accumulation of neurofibrillary tangles composed of pathogenic proteins, such as alpha-synuclein. Recent findings suggest that extracellular vesicles (EVs), which primarily function in intercellular communication, may play a critical role in the propagation of these pathogenic proteins in the brain. However, the cellular pathways that load AD-associated toxic proteins into EVs remain unknown. This research project aims to investigate the cellular pathways that influence the biogenesis, secretion, and uptake of EVs carrying AD-associated toxic proteinsusing the powerful genetic model system Saccharomyces cerevisiae. This simple brewer’s yeast has already been proven to be a useful model for understanding AD-related toxicity. When alpha synuclein is overexpressed in S. cerevisiae, it localizes to cell membranes and results in cytoplasmic aggregation that is observed in humans. We purified EVs from yeast expressing human AD-associated toxic proteins (Tau, alpha-synuclein) to establish whether they contain the human transgenes using Western blot analysis. We developed an immunohistochemistry protocol to rapidly quantify the levels of proteins secreted into the extracellular enviroment. We cultured yeast, optimized Western blotting conditions, purified extracellular vesicles, and used imaging techniques in our project.