Gatekeepers of Mitochondrial NAD+

项目摘要 我们正在研究新的体内方法来研究中间代谢物烟酰胺腺嘌呤二核苷酸 (NAD+). NAD+对细胞代谢和健康至关重要，其稳态水平的降低一直是一个问题。 与人类病理学如神经变性、心血管疾病、代谢综合征和 癌NAD+浓度是高度区室化的细胞类型，亚细胞定位，其蛋白质， 结合或自由分数。缺乏可以在空间和时间上监测细胞中游离NAD+的方法， 信息阻碍了我们对相关池，阈值浓度和波动的了解， NAD+可能导致疾病的发生。这使得我们无法确定治疗方法或方法， 在NAD+水平失调之前进行干预。特别是，没有基因编码的方法来 体内线粒体NAD+测量。游离线粒体NAD+是细胞呼吸所必需的， 由于遗传、年龄或感染导致的水平降低可促进还原性代谢。因此，为了弥补这一差距， 我们将提高当前NAD+传感器的一个变体的亮度（目标1），该变体能够免费监测 线粒体NAD+。我们还将生成一系列用于测试新方法的构造 用于改善荧光读出的动态范围（目标2）。总之，这些目标的数据将确定 一种可行的方法，以稳健地获得游离线粒体NAD+测量体内。

Project summary We are working on new in vivo methods to study the intermediary metabolite nicotinamide adenine dinucleotide (NAD+). NAD+ is critical for cellular metabolism and health, and its decreased steady-state levels have been linked to human pathologies such as neurodegeneration, cardiovascular disease, metabolic syndrome, and cancer. NAD+ concentrations are highly compartmentalized by cell type, subcellular localization, and its protein- bound or free fractions. A lack of methods that can monitor free NAD+ in cells with spatial and temporal information has hindered our learning about the relevant pools, threshold concentrations, and fluctuations that NAD+ may undergo leading to disease onset. This precludes our ability to identify treatments or approaches to intervene before NAD+ levels are misregulated. In particular, there are no genetically-encoded methods for in vivo mitochondrial NAD+ measurements. Free mitochondrial NAD+ is required for cellular respiration, and diminished levels due to genetics, age, or infection can promote reductive metabolism. Thus, to address this gap we will improve the brightness (Aim 1) of a variant of the current NAD+ sensor, which is able to monitor free mitochondrial NAD+ in cells. We will also generate a series of constructs that will be used to test a new approach for improving the dynamic range of fluorescent readout (Aim 2). Together, data from these the aims will identify a feasible method to robustly obtain free mitochondrial NAD+ measurements in vivo.