Cancer Research Program, Research Institute of the McGill University Health Center (RI-MUHC), Montréal, Québec, Canada.
Department of Anatomy and Cell Biology, McGill University, Montréal, Québec, Canada.
J Neurochem. 2020 Feb;152(3):315-332. doi: 10.1111/jnc.14837. Epub 2019 Aug 22.
During development, neurons extend axons toward their appropriate synaptic targets to establish functional neuronal connections. The growth cone, a highly motile structure at the tip of the axon, is capable of recognizing extracellular guidance cues and translating them into directed axon outgrowth through modulation of the actin cytoskeleton. Netrin-1 mediates its attractive function through the receptor deleted in colorectal cancer (DCC) to promote axon outgrowth and guidance. The calcium-activated protease calpain is involved in the cleavage of cytoskeletal proteins, which plays an important role during adhesion turnover and cell migration. However, its function during neuronal development is less understood. Here we demonstrate that netrin-1 activated calpain in embryonic rat cortical neurons in an extracellular-regulated kinase 1/2-dependent manner. In addition, we found that netrin-1 stimulation led to an increase in calpain-1 localization in the axon, whereas its endogenous inhibitor calpastatin was decreased in the growth cones of cortical neurons by indirect immunofluorescence. Interestingly, calpain-1 was able to cleave DCC in vitro. Furthermore, netrin-1 induced the cleavage of the cytoskeletal proteins spectrin and focal adhesion kinase concomitantly with the intracellular domain of DCC in a calpain-dependent manner in embryonic rat cortical neurons. Cortical neurons over-expressing calpastatin or calpain-depleted neurons displayed increased basal axon length and were unresponsive to netrin-1 stimulation. Altogether, we propose a novel model whereby netrin-1/DCC-mediated axon outgrowth is modulated by calpain-mediated proteolysis of DCC and cytoskeletal targets in embryonic cortical neurons. Open Science: This manuscript was awarded with the Open Materials Badge For more information see: https://cos.io/our-services/open-science-badges/.
在发育过程中，神经元会向其适当的突触靶标延伸轴突，以建立功能性的神经元连接。轴突末端的生长锥是一种高度活跃的结构，能够识别细胞外的导向线索，并通过调节肌动蛋白细胞骨架将其转化为定向的轴突生长。轴突导向因子 netrin-1 通过受体Deleted in colorectal cancer（DCC）发挥其吸引作用，促进轴突生长和导向。钙激活蛋白酶 calpain 参与细胞骨架蛋白的切割，在细胞黏附转化和细胞迁移过程中发挥重要作用。然而，其在神经元发育过程中的功能知之甚少。本研究证明，netrin-1 通过细胞外调节激酶 1/2 依赖性方式激活胚胎大鼠皮质神经元中的 calpain。此外，我们发现 netrin-1 刺激导致 calpain-1 在轴突中的定位增加，而间接免疫荧光显示其内源性抑制剂 calpastatin 在皮质神经元的生长锥中减少。有趣的是，calpain-1 能够在体外切割 DCC。此外，netrin-1 诱导的骨架蛋白 spectrin 和粘着斑激酶的切割与 DCC 的细胞内结构域一起，以 calpain 依赖的方式，在胚胎大鼠皮质神经元中发生。过表达 calpastatin 或耗尽 calpain 的皮质神经元显示出基础轴突长度增加，并且对 netrin-1 刺激无反应。总之，我们提出了一个新的模型，即 netrin-1/DCC 介导的轴突生长通过 calpain 介导的胚胎皮质神经元中 DCC 和细胞骨架靶标的蛋白水解来调节。开放科学：本文获得了开放材料徽章。更多信息请参见：https://cos.io/our-services/open-science-badges/。
