Department of Orthopaedics, The First Affiliated Hospital of Jinan University, Guangzhou.
Clinical Laboratory Center, The First Affiliated Hospital of Jinan University, Guangzhou.
Eur J Histochem. 2023 Jan 12;67(1):3594. doi: 10.4081/ejh.2023.3594.
Spastin, a microtubule-severing enzyme, is known to be important for neurite outgrowth. However, the role of spastin post-translational modification, particularly its phosphorylation regulation in neuronal outgrowth, remains unclear. This study aimed to investigate the effects of eliminating spastin phosphorylation on the neurite outgrowth of rat hippocampal neurons. To accomplish this, we constructed a spastin mutant with eleven potential phosphorylation sites mutated to alanine. The phosphorylation levels of the wildtype spastin (WT) and the mutant (11A) were then detected using Phos-tag SDS-PAGE. The spastin constructs were transfected into COS7 cells for the observation of microtubule severing, and into rat hippocampal neurons for the detection of neuronal outgrowth. The results showed that compared to the spastin WT, the phosphorylation levels were significantly reduced in the spastin 11A mutant. The spastin mutant 11A impaired its ability to promote neurite length, branching, and complexity in hippocampal neurons, but did not affect its ability to sever microtubules in COS7 cells. In conclusion, the data suggest that mutations at multiple phosphorylation sites of spastin do not impair its microtubule cleavage ability in COS7 cells, but reduce its ability to promote neurite outgrowth in rat hippocampal neurons.
Spastin 是一种微管切割酶，已知对神经突生长很重要。然而，spastin 的翻译后修饰作用，特别是其磷酸化调节在神经元生长中的作用仍不清楚。本研究旨在探讨消除 spastin 磷酸化对大鼠海马神经元突起生长的影响。为此，我们构建了一个 11 个潜在磷酸化位点突变为丙氨酸的 spastin 突变体。然后使用 Phos-tag SDS-PAGE 检测野生型 spastin (WT) 和突变体 (11A) 的磷酸化水平。将 spastin 构建体转染到 COS7 细胞中观察微管切割，转染到大鼠海马神经元中检测神经元突起生长。结果表明，与 spastin WT 相比，spastin 11A 突变体的磷酸化水平显著降低。spastin 突变体 11A 削弱了其促进海马神经元突起长度、分支和复杂性的能力，但不影响其在 COS7 细胞中切断微管的能力。综上所述，数据表明 spastin 多个磷酸化位点的突变不会损害其在 COS7 细胞中切断微管的能力，但会降低其促进大鼠海马神经元突起生长的能力。
