Department of Anatomy, School of Medicine, Keio University, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan.
J Biol Chem. 2010 Feb 19;285(8):5878-87. doi: 10.1074/jbc.M109.033761. Epub 2009 Dec 18.
Neuronal migration is essential for proper cortical layer formation and brain function, because migration defects result in neurological disorders such as mental retardation and epilepsy. Neuronal migration is divided into several contiguous steps: early phase (multipolar mode), locomotion mode, and terminal translocation mode. The locomotion mode covers most of the migration route and thereby is the main contributor to cortical layer formation. However, analysis of the molecular mechanisms regulating this mode is difficult due to the secondary effects of defects at the early phase of migration. In this study, we established an ex vivo chemical inhibitor screening, allowing us to directly analyze the locomotion mode of migration. Roscovitine and PP2, inhibitors for Cdk5 and Src family kinases, respectively, suppressed the locomotion mode of migration. In line with this, a small percentage of Cdk5- or Src family kinase (Fyn)-knockdown cells exhibited locomoting morphology but retarded migration, although the majority of cells were stalled at the early phase of migration. We also showed that rottlerin, widely used as a specific inhibitor for protein kinase Cdelta (PKCdelta), suppressed the locomotion mode. Unexpectedly, however, the dominant-negative form as well as RNA interference for PKCdelta hardly affected the locomotion, whereas they may disturb terminal translocation. In addition, we found JNK to be a potential downstream target of rottlerin. Taken together, our novel chemical inhibitor screening provides evidence that Cdk5 and Src family kinases regulate the locomotion mode of neuronal migration. It also uncovered roles for Fyn and PKCdelta in the early and final phases of migration, respectively.
神经元迁移对于皮质层形成和大脑功能至关重要，因为迁移缺陷会导致神经发育障碍，如智力迟钝和癫痫。神经元迁移分为几个连续的步骤：早期阶段（多极模式）、运动模式和末端转位模式。运动模式覆盖了大部分迁移路径，因此是皮质层形成的主要贡献者。然而，由于迁移早期阶段的缺陷会产生次生效应，因此分析调节这种模式的分子机制具有一定的难度。在这项研究中，我们建立了一种体外化学抑制剂筛选方法，使我们能够直接分析迁移的运动模式。Cdk5 的抑制剂罗司维亭（Roscovitine）和Src 家族激酶的抑制剂 PP2 分别抑制了迁移的运动模式。与此一致的是，一小部分 Cdk5 或 Src 家族激酶（Fyn）敲低细胞表现出运动形态但迁移迟缓，尽管大多数细胞停滞在迁移的早期阶段。我们还表明，广泛用作蛋白激酶 C 德尔塔（PKCdelta）特异性抑制剂的罗特林（Rottlerin）抑制了运动模式。然而，出乎意料的是，PKCdelta 的显性负形式和 RNA 干扰几乎没有影响运动模式，而可能会干扰末端转位。此外，我们发现 JNK 可能是罗特林的潜在下游靶标。综上所述，我们的新型化学抑制剂筛选为 Cdk5 和 Src 家族激酶调节神经元迁移的运动模式提供了证据。它还揭示了 Fyn 和 PKCdelta 在迁移的早期和最终阶段分别发挥作用。
