CAREER: Integrating quantitative biomarkers of mitochondrial structure and function through endogenous cellular fluorescence

University Of Arkansas, Fayetteville AR

Investigators

Abstract

Mitochondria are dynamic energy-producing structures within cells that play a central role in cellular metabolism. With advanced age, mitochondrial dysfunction can arise and contribute to many common degenerative diseases. Reduced mitochondrial efficiency, increased internal stresses, and altered mitochondrial organization within cells are associated with the biology of aging. Therefore, there is a need to establish quantitative, non-invasive readouts of these age-related changes in mitochondria. This project will develop new image analysis tools and statistical models based on the natural fluorescence of two metabolic molecules, called cofactors, that are critical for the energy producing processes. Distinct optical signatures will be identified through statistical models based on measurements of the intensity, time-based response, and spatial organization of the two cofactors within individual cells. These models of mitochondrial function will be validated and then applied to assess cells during anti-aging treatments. The models will also be used to understand how aging and obesity affect cellular metabolism. This research will be incorporated into educational and recruitment efforts to promote science and engineering. In particular, the project will allow for the expansion and development of new content for a biomedical engineering camp that exposes high school students from underrepresented groups to multidisciplinary biomedical research. The principal investigator's long-term career goal is to push the capabilities of imaging metabolic cofactors NADH and FAD to offer truly new insights into the dynamic changes in metabolism observed during development, repair, aging and disease. Toward this goal, this project will develop advanced multiphoton imaging and analysis techniques to provide quantitative biomarkers of metabolic dysfunction based on the natural fluorescence of NADH and FAD, i.e., label-free imaging, and to use these biomarkers to advance understanding of the biology of aging. The project will build on the PI's previous experience in NADH and FAD autofluorescence imaging and will establish a new analysis technique for rapid, single cell assessments of mitochondrial fractal dimension (FD) within cells. The Research Plan is organized under three objectives. The FIRST OBJECTIVE is to establish a set of optical biomarkers and statistical models to predict changes in mitochondrial structure and function. Studies are designed to test the hypothesis that different combinations of multiphoton metrics can be used to separately predict different metabolic responses. This will be accomplished by validating the FD-based method to rapidly quantify mitochondrial organization in individual cells and developing a linear mixed-effects model to predict redox state, ETC (electron transport chain) activity, and oxidative stress based only on endogenous cellular fluorescence. The SECOND OBJECTIVE is to characterize the sensitivity of mitochondrial biomarkers to chronological age and common anti-aging treatments. Studies are designed to test the hypothesis that decreased ETC activity and increased oxidative stress will be detected in cells from older patients and that sensitivity to anti-aging treatments will be dependent on their mechanism of action. This will be accomplished by evaluating differences in mitochondrial metrics between young and old (31 and 88 year old) keratinocytes (skin cells) donated by healthy females and assessing the sensitivity of optical biomarkers to anti-aging treatments such as rapamycin. The THIRD OBJECTIVE is to monitor the aging process over the entire lifespan of mice through non-invasive skin imaging and to evaluate the effect of normal and high-calorie diets. Studies are designed to test the hypothesis that significant metabolic changes will be most strongly associated with HFD (High Fat Diet) mice and female mice upon the onset of peri-menopause at approximately 18 months of age. This will be accomplished by quantifying the age-dependent changes in mitochondrial structure and function of keratinocytes in individual mice (50:50 sex split) every 2 months over their entire lifespan (approximately 2.5 years.) Though the project's efforts are focused on cellular aging in keratinocytes, findings could be applicable to other cell types and applications in which metabolic changes have been reported, including cancer, cardiovascular disease and wound healing. This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.