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

线粒体是细胞内的动态能量产生结构，在细胞新陈代谢中起着核心作用。随着年龄的增长，线粒体功能障碍可能会出现，并导致许多常见的退行性疾病。线粒体效率降低，内部压力增加，细胞内线粒体组织改变与衰老的生物学相关。因此，有必要建立这些与年龄相关的线粒体变化的定量、非侵入性读数。该项目将开发新的图像分析工具和统计模型，基于两种被称为辅因子的代谢分子的自然荧光，这两种分子对能量产生过程至关重要。不同的光学特征将通过基于强度、基于时间的响应和单个细胞内两个辅助因素的空间组织的测量的统计模型来识别。这些线粒体功能模型将得到验证，然后应用于抗衰老治疗期间的细胞评估。这些模型还将用于了解衰老和肥胖如何影响细胞新陈代谢。这项研究将被纳入教育和招聘工作，以促进科学和工程。特别是，该项目将允许扩展和开发生物医学工程营的新内容，使高中生从代表性不足的群体接触到多学科的生物医学研究。主要研究人员的长期职业目标是推动成像代谢辅助因子NADH和FAD的能力，以提供对发育、修复、衰老和疾病期间观察到的代谢动态变化的真正新的见解。为了实现这一目标，该项目将开发先进的多光子成像和分析技术，以提供基于NADH和FAD的自然荧光的代谢功能障碍的定量生物标记物，即无标记成像，并利用这些生物标记物来促进对衰老生物学的理解。该项目将建立在PI以前在NADH和FAD自发荧光成像方面的经验基础上，并将建立一种新的分析技术，用于快速、单细胞地评估细胞内的线粒体分维(FD)。研究计划是按照三个目标组织的。第一个目标是建立一套光学生物标志物和统计模型来预测线粒体结构和功能的变化。研究的目的是检验这样一个假设，即多光子度量的不同组合可以用来分别预测不同的代谢反应。这将通过验证基于FD的方法来快速量化单个细胞中的线粒体组织并开发线性混合效应模型来预测氧化还原状态、ETC(电子传输链)活性和仅基于内源细胞荧光的氧化应激来实现。第二个目标是表征线粒体生物标记物对时序年龄和常用抗衰老治疗的敏感性。研究旨在验证这样的假设，即在老年患者的细胞中将检测到ETC活性降低和氧化应激增加，并且对抗衰老治疗的敏感性将取决于它们的作用机制。这将通过评估健康女性捐赠的年轻和老年(31岁和88岁)角质形成细胞(皮肤细胞)之间的线粒体指标差异，以及评估光学生物标记物对雷帕霉素等抗衰老治疗的敏感性来实现。第三个目标是通过非侵入性皮肤成像监测小鼠整个生命周期的衰老过程，并评估正常和高热量饮食的效果。研究旨在验证这一假设，即在大约18个月大的围绝经期开始时，显着的代谢变化与HFD(高脂肪饮食)小鼠和雌性小鼠最密切相关。这将通过量化单个小鼠(性别比例为50：50)的角质形成细胞线粒体结构和功能随年龄的变化来实现，在它们的整个生命周期(约2.5年)中每2个月一次。虽然该项目的工作重点是角质形成细胞的细胞老化，但研究结果可能适用于已报告代谢变化的其他细胞类型和应用，包括癌症、心血管疾病和伤口愈合。该奖项反映了NSF的法定使命，并通过使用基金会的智力优势和更广泛的影响审查标准进行评估，被认为值得支持。

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.