Gatekeepers of Mitochondrial NAD+

项目总结 这一提议提供了一个在体内直接研究中间代谢物烟酰胺如何 腺嘌呤二核苷酸(NAD+)由于其局部局限性，参与了年龄相关病理的发生 可用性。NAD+对细胞新陈代谢和延年益寿至关重要。稳定下降- 状态NAD+水平与人类病理有关，如神经退化、心血管疾病、 代谢综合征和癌症。然而，NAD+浓度是按细胞类型高度划分的， 亚细胞定位，以及蛋白质结合或游离部分。因此，缺乏直接监控免费的方法 具有时空信息的细胞中的NAD+阻碍了我们对相关池的学习， 阈值浓度，以及NAD+可能经历的导致疾病发作的时间。这排除了我们的 有能力在NAD+水平被错误调控之前确定干预的治疗方法或方法。 为了解决这个障碍，我们免费开发了一种可遗传编码的荧光生物传感器。 NAD+。将传感器定位到亚细胞隔间揭示了细胞内的隔间 NAD+在不同的细胞类型中有不同的协调作用。令人兴奋的是，数据还指出了 一个可能的哺乳动物线粒体NAD+转运蛋白。我们相信该传感器可以解决 假设线粒体NAD+的错误调节是迟发性的主要潜在原因的衰老 病理学。这项提议将使用传感器来识别线粒体维持其生存所需的蛋白质。 NAD+浓度升高以及这一池可能如何在老年神经元中改变。这一点很重要 对理解细胞生物能量学和线粒体健康以及识别新的 对抗神经退行性变的选择。了解当地NAD+水平如何波动，可能会限制 NAD+依赖的酶的功能对于评估NAD+调节是否代表一种活的 对与年龄有关的疾病的治疗方法或干预。

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.