Tang Fangjun, Kalil Katherine
University of Wisconsin-Madison, Madison, Wisconsin 53706, USA.
J Neurosci. 2005 Jul 13;25(28):6702-15. doi: 10.1523/JNEUROSCI.0871-05.2005.
A single axon can innervate multiple targets by collateral branching. Axon branching is thus essential for establishing CNS connectivity. However, surprisingly little is known about the mechanisms by which branching is regulated. Axons often stop elongating before branches develop and anatomical and molecular data suggest that axon branching occurs independent of axon outgrowth. We found that netrin-1 dramatically increases cortical axon branching. Here, we sought to identify intracellular signaling components involved in netrin-1-induced axon branching. Using live cell imaging of dissociated developing cortical neurons, we show that netrin-1 rapidly increases the frequency of repetitive calcium transients. These transients are often restricted to small regions of the axon. Simultaneous imaging of calcium activity and development of axon branches revealed that Ca2+ transients coincide spatially and temporally with protrusion of branches from the axon. Remarkably, fully formed branches with motile growth cones could develop de novo within 20 min. Netrin-1-induced Ca2+ transients involve release from intracellular stores and Ca2+ signaling is essential for netrin-1-induced axon branching. Using techniques to overexpress or suppress kinase activity, we find that calcium/calmodulin-dependent protein kinase II (CaMKII) and mitogen-activated protein kinase (MAPK) are major downstream targets of the netrin-1 calcium signaling pathway and are required for axon branching. CaMKII, but not MAPK, is also involved in axon outgrowth. The role of CaMKII and MAPKs in axon branching is consistent with the sensitivity of these kinases to changes in the frequency Ca2+ transients. Together, these novel findings define calcium signaling mechanisms required for development of new axon branches promoted by a guidance cue.
单个轴突可通过侧支分支支配多个靶点。因此，轴突分支对于建立中枢神经系统的连接至关重要。然而，令人惊讶的是，对于轴突分支的调控机制却知之甚少。轴突通常在分支形成之前就停止伸长，解剖学和分子数据表明轴突分支的发生独立于轴突生长。我们发现，netrin-1可显著增加皮质轴突分支。在此，我们试图确定参与netrin-1诱导轴突分支的细胞内信号成分。利用解离的发育中皮质神经元的活细胞成像技术，我们发现netrin-1能迅速增加重复性钙瞬变的频率。这些瞬变通常局限于轴突的小区域。同时对钙活性和轴突分支发育进行成像显示，Ca2+瞬变在空间和时间上与轴突分支的突出相吻合。值得注意的是，具有活动生长锥的完全形成的分支可在20分钟内从头发育形成。netrin-1诱导的Ca2+瞬变涉及细胞内钙库的释放，并且Ca2+信号对于netrin-1诱导的轴突分支至关重要。利用过表达或抑制激酶活性的技术，我们发现钙/钙调蛋白依赖性蛋白激酶II（CaMKII）和丝裂原活化蛋白激酶（MAPK）是netrin-1钙信号通路的主要下游靶点，并且是轴突分支所必需的。CaMKII而非MAPK也参与轴突生长。CaMKII和MAPK在轴突分支中的作用与这些激酶对Ca2+瞬变频率变化的敏感性一致。总之，这些新发现确定了由一种导向信号促进新轴突分支发育所需的钙信号机制。
