Gatekeepers of Mitochondrial NAD+

项目摘要 这一建议代表了一个机会，直接在体内研究如何中间代谢物烟酰胺 腺嘌呤二核苷酸（NAD+）由于其有限的局部表达， 空房的NAD+对细胞代谢和延长老年健康至关重要。稳定下降- 国家NAD+水平与人类病理学如神经变性，心血管疾病， 代谢综合征和癌症。然而，NAD+浓度被细胞类型高度区室化， 亚细胞定位和蛋白质结合或游离部分。因此，缺乏直接监测免费的方法， 具有空间和时间信息的细胞中的NAD+阻碍了我们对相关库的学习， 阈值浓度和NAD+可能经历的导致疾病发作的时间。这就排除了我们 在NAD+水平失调之前确定干预治疗或方法的能力。 为了解决这个问题，我们免费开发了一种基因编码的荧光生物传感器 NAD+。将传感器靶向亚细胞区室揭示了细胞内的区室化。 NAD+在不同细胞类型中差异协调。令人兴奋的是，数据还表明， 一种推定的哺乳动物线粒体NAD+转运蛋白。我们相信传感器可以解决关键问题， 假设线粒体NAD+的失调是迟发性衰老的主要潜在原因， 病理学这项提议将使用传感器来识别线粒体维持其功能所需的蛋白质。 NAD+浓度升高以及这个池如何在老年神经元中改变。这具有重要 对理解细胞生物能量学和线粒体健康的影响，以及识别新的 对抗神经退化的方法了解当地NAD+水平如何波动，并可能限制 NAD+依赖性酶的功能对于评估NAD+调节是否代表一种可行的 治疗方法或干预与年龄有关的疾病。

Project summary This proposal represents an opportunity to directly study in vivo how the intermediary metabolite nicotinamide adenine dinucleotide (NAD+) contributes to the onset of age-related pathologies, due to its limited local availability. NAD+ is critical for cellular metabolism and for prolonging health in old age. Decreased steady- state NAD+ levels have been linked to human pathologies such as neurodegeneration, cardiovascular disease, metabolic syndrome, and cancer. However, NAD+ concentrations are highly compartmentalized by cell type, subcellular localization, and protein-bound or free fractions. Thus, a lack of methods to directly monitor free NAD+ in cells with spatial and temporal information has hindered our learning about the relevant pools, threshold concentrations, and timing that NAD+ may undergo leading to disease onset. This precludes our ability to identify treatments or approaches to intervene before NAD+ levels are misregulated. To address this roadblock, we have developed a genetically-encodable fluorescent biosensor for free NAD+. Targeting of the sensor to subcellular compartments has revealed compartmentalization of intracellular NAD+ that is differentially coordinated in different cell types. Excitingly, the data also pointed to the existence of a putative mammalian mitochondrial NAD+ transporter. We believe the sensor can address key problems in aging with the hypothesis that misregulation of mitochondrial NAD+ is a major underlying cause of late-onset pathologies. This proposal will use the sensor to identify the proteins required for mitochondria to sustain their elevated concentrations of NAD+ and how this pool may be altered in older neurons. This has important implications for understanding cellular bioenergetics and mitochondrial health, as well as identifying new options to combat neurodegeneration. Understanding how local NAD+ levels fluctuate and may limit the function of NAD+-dependent enzymes is important to evaluate whether NAD+ regulation represents a viable treatment approach or intervention for age-related diseases.