Disease-associated mutations in human TUBB3 disturb netrin repulsive signaling

Missense mutations in the human TUBB3 gene cause a variety of neurological disorders associated with defects in axon guidance and neuronal migration, but the underlying molecular mechanisms are not well understood. Recent studies have shown that direct coupling of dynamic TUBB3 in microtubules with netrin receptors is required for netrin-1-mediated axon guidance, and the interaction of netrin-1 repulsive receptor UNC5C with TUBB3 is involved in netrin-1 mediated axonal repulsion. Here, we report that TUBB3 mutations perturb netrin-1/UNC5C repulsive signaling in the developing nervous system. Among twelve mutants reported in previous studies, five of them show significantly reduced interaction with UNC5C in comparison to the wild-type TUBB3. TUBB3 mutants R262C and R62Q exhibit decreased subcellular colocalization with UNC5C in the peripheral area of the growth cone of primary mouse neurons. Netrin-1 reduces the colocalization of UNC5C with wild-type TUBB3, but not TUBB3 mutants R262C or R62Q, in the growth cone. Results from the in vitro cosedimentation assay indicate that netrin-1 inhibits cosedimentation of UNC5C with polymerized microtubules in primary mouse neurons expressing the wild-type TUBB3, but not R262C or R62Q. Expression of either R262C or R62Q not only blocks netrin-1-induced growth cone collapse and axonal repulsion of primary EGL cells in vitro, but also results in axon projections defects of chicken dorsal root ganglion neurons in ovo. Our study reveals that human TUBB3 mutations specifically perturb netrin-1/UNC5C-mediated repulsion.人TUBB3基因（human TUBB3 gene）的错义突变可引发多种伴随轴突导向（axon guidance）与神经元迁移（neuronal migration）缺陷的神经系统疾病，但其潜在分子机制尚未完全明晰。已有研究表明，微管（microtubules）内动态形式的TUBB3与诱导向素受体（netrin receptors）的直接耦联，是诱导向素-1（netrin-1）介导轴突导向的必要条件；而诱导向素-1的排斥受体UNC5C（Uncoordinated-5 homolog C）与TUBB3的相互作用，参与了诱导向素-1介导的轴突排斥过程。本研究发现，TUBB3突变会扰乱发育神经系统中的诱导向素-1/UNC5C排斥信号通路。在既往研究报道的12种TUBB3突变体中，有5种与野生型TUBB3相比，与UNC5C的相互作用显著减弱。TUBB3突变体R262C与R62Q，在原代小鼠神经元生长锥的外周区域，与UNC5C的亚细胞共定位水平显著降低。诱导向素-1可降低生长锥内野生型TUBB3与UNC5C的共定位水平，但对R262C或R62Q突变体无此调控作用。体外共沉降实验结果显示，在表达野生型TUBB3的原代小鼠神经元中，诱导向素-1会抑制UNC5C与聚合态微管的共沉降，但在表达R262C或R62Q的神经元中无此效应。无论是R262C还是R62Q突变体的表达，不仅可在体外阻断诱导向素-1诱导的原代外颗粒层（EGL）细胞生长锥塌陷与轴突排斥反应，还可在鸡胚卵内引发背根神经节神经元的轴突投射缺陷。本研究揭示，人TUBB3突变会特异性扰乱诱导向素-1/UNC5C介导的轴突排斥信号通路。