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

职业：通过内源细胞荧光整合线粒体结构和功能的定量生物标志物

• 批准号: 1846853
• 负责人: Kyle Quinn
• 金额: $ 50万
• 依托单位: University of Arkansas
• 依托单位国家: 美国
• 项目类别: Continuing Grant
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-06-01 至 2024-05-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1846853&HistoricalAwards=false
• 关键词: CAREER; Integrating; quantitative; biomarkers; mitochondrial

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.

线粒体是细胞内动态的能量产生结构，在细胞代谢中发挥着核心作用。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、内应力增加以及细胞内线粒体组织改变与衰老生物学有关。 因此，需要建立线粒体中这些与年龄相关的变化的定量、非侵入性读数。该项目将基于两种代谢分子（称为辅因子）的天然荧光开发新的图像分析工具和统计模型，这对于能量产生过程至关重要。 独特的光学特征将通过基于对单个细胞内两个辅助因子的强度、基于时间的响应和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评估细胞。这些模型还将用于了解衰老和肥胖如何影响细胞代谢。这项研究将被纳入教育和招聘工作中，以促进科学和工程的发展。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，让来自弱势群体的高中生接触多学科生物医学研究。首席研究员的长期职业目标是推动代谢辅因子 NADH 和 FAD 成像的能力，为发育、修复、衰老和疾病期间观察到的代谢动态变化提供真正的新见解。 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天然荧光的代谢功能障碍的定量生物标志物，即无标记成像，并利用这些生物标志物促进对衰老生物学的理解。 该项目将建立在 PI 先前在 NADH 和 FAD 自发荧光成像方面的经验的基础上，并将建立一种新的分析技术，用于快速、单细胞评估细胞内线粒体分形维数 (FD)。 该研究计划根据三个目标进行组织。 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。 研究旨在检验这样的假设：多光子指标的不同组合可用于单独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仅基于内源细胞荧光来预测氧化还原状态、ETC（电子传递链）活性和氧化应激来实现。 第二个目标是表征线粒体生物标志物对实际年龄和常见抗衰老治疗的敏感性。 研究旨在检验以下假设：在老年患者的细胞中会检测到 ETC 活性降低和氧化应激增加，并且抗衰老治疗的敏感性将取决于其作用机制。 这将通过评估健康女性捐赠的年轻人和老年人（31 岁和 88 岁）角质形成细胞（皮肤细胞）之间线粒体指标的差异并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。 研究旨在检验以下假设：显着的代谢变化与 HFD（高脂饮食）小鼠和雌性小鼠在大约 18 个月大时进入围绝经期最为密切相关。 这将通过在小鼠的整个生命周期（大约 2.5 年）中每两个月量化角质形成细胞线粒体结构和功能的年龄依赖性变化（性别比例为 50:50）来实现。尽管该项目的工作重点是角质形成细胞的细胞衰老，但研究结果可能适用于已报道代谢变化的其他细胞类型和应用，包括癌症、心血管疾病和 该奖项反映了 NSF 的法定使命，并通过使用基金会的智力价值和更广泛的影响审查标准进行评估，被认为值得支持。

项目成果

Label-free metabolic biomarkers for assessing valve interstitial cell calcific progression

用于评估瓣膜间质细胞钙化进展的无标记代谢生物标志物

• DOI: 10.1038/s41598-020-66960-4
• 发表时间: 2020
• 期刊: Scientific Reports
• 影响因子: 4.6
• 作者: Tandon, Ishita;Kolenc, Olivia I.;Cross, Delaney;Vargas, Isaac;Johns, Shelby;Quinn, Kyle P.;Balachandran, Kartik
• 通讯作者: Balachandran, Kartik

Label-Free Optical Metabolic Imaging in Cells and Tissues.

• DOI: 10.1146/annurev-bioeng-071516-044730
• 发表时间: 2023-06-08
• 期刊: Annual review of biomedical engineering
• 影响因子: 9.7