Regulation of mitochondrial dehdyrogenases and neuronal NADH levels via interaction of NIPSNAP1 and APP intracellular domain

摘要 线粒体功能障碍与衰老以及一些与年龄相关的神经退行性变有关 包括阿尔茨海默病(AD)在内的各种疾病。早发性阿尔茨海默病与淀粉样蛋白突变有关 前体蛋白(APP)和早老素1和2(PS1和PS2)，导致APP和PS2的异常切割 释放有毒的淀粉样β蛋白(A肽。越来越多的证据表明，APP细胞内结构域 (AICD)也可能参与AD的发病。为了深入了解人的正常和病理作用 AICD，我们之前使用了一种生化亲和蛋白质组策略，发现AICD直接与 一种新的线粒体蛋白NipSnap1(4-硝基苯基磷酸酶结构域和非神经性SNAP-25样蛋白 蛋白质同源1)。尽管NipSnap1在进化上是保守的，但人们对其功能知之甚少。我们的 长期目标是研究NipSnap1的分子和细胞功能，并确定其在 神经退行性变。为此，我们培育了一只靶向破坏NipSnap1基因的小鼠。 NipSnap1表达的中断深刻影响中间代谢并显著增加 大脑中的细胞凋亡和神经变性。蛋白质结构建模与虚拟配体筛选 提示NipSnap1可能与NADH和NADPH结合。使用体外生化分析，我们发现对于 这是NipSnap1首次直接与NADH和NADPH结合。此外，我们发现更低的 NipSnap1缺陷型脑组织中NAD+/NADH比值的变化NAD+和NADH之间的平衡对于 ATP的产生，线粒体潜力的维持和细胞内还原剂的再生 中和活性氧自由基。根据这些初步结果，我们假设NipSnap1发挥作用 通过调节脱氢酶活性和NAD(P)H水平在神经元存活中发挥重要作用。在这 项目中，我们将使用生化方法和源自WT和NipSnap1的原代神经元培养 缺陷小鼠以确定：1)NipSnap1是否与多种脱氢酶相互作用并调节 线粒体；2)AICD与NipSnap1的相互作用影响脱氢酶活性和神经元NAD+/NADH 级别。我们的工作将提供对NipSnap1分子功能的见解，并可能提供新的机制 使AICD产生神经毒性。

Abstract Mitochondrial dysfunction is associated with ageing as well as a number of age-related neurodegenerative diseases including Alzheimer’s Disease (AD). Early onset AD has been linked to mutations in amyloid precursor protein (APP) and presenilins 1 and 2 (PS1 and PS2), which result in abnormal cleavage of APP and release of toxic amyloid beta (Apeptides. Accumulating evidence suggests that APP intracellular domain (AICD) may also contribute to pathogenesis of AD. To gain insights into the normal and pathological roles of AICD, we previously used a biochemical affinity proteomic strategy and found that AICD directly interacts with a novel mitochondrial protein, Nipsnap1 (4-nitrophenyl phosphatase domain and non-neuronal SNAP-25 like protein homolog1). Although Nipsnap1 is evolutionarily conserved, very little is known about its function. Our long-term goal is to investigate the molecular and cellular function of Nipsnap1 and to determine its role in neurodegeneration. Toward this end, we generated a mouse with a targeted disruption of the Nipsnap1 gene. Disruption of Nipsnap1 expression profoundly affects intermediate metabolism and significantly increased apoptosis and neurodegeneration in the brain. Protein structure modeling and virtual ligand screening suggested that Nipsnap1 may bind to NADH and NADPH. Using in vitro biochemical assays, we found for the first time that Nipsnap1 directly binds to both NADH and NADPH. Moreover, we found significantly lower NAD+/NADH ratios in Nipsnap1 deficient brain. The balance between NAD+ and NADH is critical for production of ATP, maintenance of mitochondrial potential and regeneration of reducing agents within cells to counteract reactive oxygen radicals. Based on these preliminary results, we hypothesize that Nipsnap1 plays an important role in neuronal survival by modulating dehydrogenase activities and NAD(P)H levels. In this project, we will use biochemical approaches and primary neuronal cultures derived from WT and Nipsnap1 deficient mice to determine if: 1) Nipsnap1 interacts with and regulates multiple dehydrogenases in the mitochondria; 2) AICD interaction with Nipsnap1 affects dehydrogenase activity and neuronal NAD+/NADH levels. Our work will provide insights into the molecular function of Nipsnap1 and possibly a new mechanism by which AICD produces neurotoxicity.