Nicotinamide adenine dinucleotides and their precursor NMN have no direct effect on microtubule dynamics in purified brain tubulin

Microtubules are dynamic cytoskeletal polymers that provide mechanical support for cellular structures, and play important roles in cell division, migration, and intracellular transport. Their intrinsic dynamic instability, primarily controlled by polymerization-dependent GTP hydrolysis, allows for rapid rearrangements of microtubule arrays in response to signaling cues. In neurons, increases in intracellular levels of nicotinamide adenine dinucleotide (NAD+) can protect against microtubule loss and axonal degeneration elicited by axonal transection. The protective effects of NAD+ on microtubule loss have been shown to be indirect in some systems, for example through the sirtuin-3 pathway. However, it is still possible that NAD+ and related metabolites have direct effects on microtubule dynamics to promote assembly or inhibit disassembly. To address this question, we reconstituted microtubule dynamics in an in vitro assay with purified bovine brain tubulin and examined the effects of NAD+, NADH, and NMN. We found that the compounds had only small effects on the dynamics at the plus and minus ends of the microtubules. Furthermore, these effects were not statistically significant. Consequently, our data support earlier findings that NADs and their precursors influence microtubule growth through indirect mechanisms.

微管（Microtubules）是一类动态细胞骨架聚合物，可为细胞结构提供机械支撑，并在细胞分裂、细胞迁移及胞内运输过程中发挥关键作用。其固有的动态不稳定性主要由依赖聚合作用的GTP水解调控，可使微管阵列响应信号刺激而快速发生重排。在神经元中，胞内烟酰胺腺嘌呤二核苷酸（NAD+）水平升高，能够抵御轴突横断诱导的微管丢失与轴突变性。在部分研究体系中，NAD+对微管丢失的保护作用被证实为间接效应，例如通过沉默信息调节因子3（Sirtuin-3）通路介导。然而，NAD+及其相关代谢物仍可能直接作用于微管动态过程，以促进微管组装或抑制其解聚。为解答这一科学问题，我们利用纯化的牛脑微管蛋白构建体外实验体系以重构微管动态过程，并考察了NAD+、烟酰胺腺嘌呤二核苷酸还原型（NADH）以及烟酰胺单核苷酸（NMN）的相关影响。研究结果显示，上述化合物对微管正负两端的动态过程仅存在微弱影响，且该影响均未达到统计学显著性水平。因此，本研究数据支持此前的结论：烟酰胺腺嘌呤二核苷酸类物质及其前体通过间接机制调控微管生长。