Islam Md Ariful, Sharif Syeda Ridita, Lee HyunSook, Moon Il Soo
Department of Anatomy, College of Medicine Dongguk University, Gyeongju 780-714, Korea.
Dongguk Medical Institute, College of Medicine Dongguk University, Gyeongju 780-714, Korea.
Mol Cells. 2015 Oct;38(10):876-85. doi: 10.14348/molcells.2015.0120. Epub 2015 Oct 15.
N-acetyl-D-glucosamine kinase (NAGK) plays an enzyme activity-independent, non-canonical role in the dendritogenesis of hippocampal neurons in culture. In this study, we investigated its role in axonal development. We found NAGK was distributed throughout neurons until developmental stage 3 (axonal outgrowth), and that its axonal expression remarkably decreased during stage 4 (dendritic outgrowth) and became negligible in stage 5 (mature). Immunocytochemistry (ICC) showed colocalization of NAGK with tubulin in hippocampal neurons and with Golgi in somata, dendrites, and nascent axons. A proximity ligation assay (PLA) for NAGK and Golgi marker protein followed by ICC for tubulin or dynein light chain roadblock type 1 (DYNLRB1) in stage 3 neurons showed NAGK-Golgi complex colocalized with DYNLRB1 at the tips of microtubule (MT) fibers in axonal growth cones and in somatodendritic areas. PLAs for NAGK-dynein combined with tubulin or Golgi ICC showed similar signal patterns, indicating a three way interaction between NAGK, dynein, and Golgi in growing axons. In addition, overexpression of the NAGK gene and of kinase mutant NAGK genes increased axonal lengths, and knockdown of NAGK by small hairpin (sh) RNA reduced axonal lengths; suggesting a structural role for NAGK in axonal growth. Finally, transfection of 'DYNLRB1 (74-96)', a small peptide derived from DYNLRB1's C-terminal, which binds with NAGK, resulted in neurons with shorter axons in culture. The authors suggest a NAGK-dynein-Golgi tripartite interaction in growing axons is instrumental during early axonal development.
N-乙酰-D-葡萄糖胺激酶（NAGK）在培养的海马神经元树突形成中发挥非酶活性依赖性的非经典作用。在本研究中，我们研究了其在轴突发育中的作用。我们发现，直到发育阶段3（轴突长出），NAGK在整个神经元中均有分布，而在阶段4（树突长出）其轴突表达显著下降，在阶段5（成熟）时变得微不足道。免疫细胞化学（ICC）显示，NAGK在海马神经元中与微管蛋白共定位，在胞体、树突和新生轴突中与高尔基体共定位。对阶段3神经元进行NAGK与高尔基体标记蛋白的邻近连接分析（PLA），随后对微管蛋白或动力蛋白轻链路障型1（DYNLRB1）进行ICC，结果显示NAGK-高尔基体复合物在轴突生长锥和体树突区域的微管（MT）纤维末端与DYNLRB1共定位。NAGK-动力蛋白与微管蛋白或高尔基体ICC结合的PLA显示出相似的信号模式，表明在生长的轴突中，NAGK、动力蛋白和高尔基体之间存在三方相互作用。此外，NAGK基因和激酶突变体NAGK基因的过表达增加了轴突长度，而小发夹（sh）RNA敲低NAGK则缩短了轴突长度；这表明NAGK在轴突生长中具有结构作用。最后，转染源自DYNLRB1 C末端的小肽“DYNLRB1（74-96）”（其与NAGK结合），导致培养中的神经元轴突较短。作者认为，生长轴突中的NAGK-动力蛋白-高尔基体三方相互作用在早期轴突发育过程中起重要作用。
