Mutalik Sampada P, Ho Chris T, O'Shaughnessy Ellen C, Frasineanu Anca G, Shah Aneri B, Gupton Stephanie L
Department of Cell Biology and Physiology, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Neuroscience Center, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
J Neurochem. 2025 Jan;169(1):e70002. doi: 10.1111/jnc.70002.
The guidance cue netrin-1 promotes both growth cone attraction and growth cone repulsion. How netrin-1 elicits diverse axonal responses, beyond engaging the netrin receptor DCC and UNC5 family members, remains elusive. Here, we demonstrate that murine netrin-1 induces biphasic axonal responses in cortical neurons: Attraction at lower concentrations and repulsion at higher concentrations using both a microfluidic-based netrin-1 gradient and bath application of netrin-1. We find that repulsive turning in a netrin gradient is blocked by knockdown of UNC5C, whereas attractive turning is impaired by knockdown of DCC. TRIM9 is a brain-enriched E3 ubiquitin ligase previously shown to bind and cluster the attractive receptor DCC at the plasma membrane and regulate netrin-dependent attractive responses. However, whether TRIM9 also regulated repulsive responses to netrin-1 remained to be seen. In this study, we show that TRIM9 localizes and interacts with both the attractive netrin receptor DCC and the repulsive netrin receptor, UNC5C. We find that deletion of murine Trim9 alters both attractive and repulsive axon turning and changes in growth cones size in response to murine netrin-1. TRIM9 was required for netrin-1-dependent changes in the surface levels of DCC and UNC5C in the growth cone during morphogenesis. We demonstrate that DCC at the membrane regulates the growth cone area and show that TRIM9 negatively regulates FAK activity in the absence of both repulsive and attractive concentrations of netrin-1. Together, our work demonstrates that TRIM9 interacts with and regulates both DCC and UNC5C during attractive and repulsive axonal responses to netrin-1.
导向信号分子网蛋白-1既能促进生长锥吸引，也能促进生长锥排斥。除了与网蛋白受体DCC和UNC5家族成员结合外，网蛋白-1如何引发多样的轴突反应仍不清楚。在这里，我们证明，使用基于微流控的网蛋白-1梯度和网蛋白-1的浴式应用，小鼠网蛋白-1在皮质神经元中诱导双相轴突反应：较低浓度时吸引，较高浓度时排斥。我们发现，UNC5C基因敲低可阻断在网蛋白梯度中的排斥性转向，而DCC基因敲低则损害吸引性转向。TRIM9是一种在脑中富集的E3泛素连接酶，先前已证明它能在质膜上结合并聚集有吸引力的受体DCC，并调节网蛋白依赖性吸引反应。然而，TRIM9是否也调节对网蛋白-1的排斥反应仍有待观察。在本研究中，我们表明TRIM9定位于有吸引力的网蛋白受体DCC和排斥性网蛋白受体UNC5C，并与其相互作用。我们发现，删除小鼠Trim9基因会改变对小鼠网蛋白-1的吸引性和排斥性轴突转向以及生长锥大小的变化。在形态发生过程中，TRIM9是网蛋白-1依赖性生长锥中DCC和UNC5C表面水平变化所必需的。我们证明，膜上的DCC调节生长锥面积，并表明在没有排斥性和吸引性浓度网蛋白-1的情况下，TRIM9负向调节粘着斑激酶（FAK）活性。总之，我们的工作表明，在对网蛋白-1的吸引性和排斥性轴突反应过程中，TRIM9与DCC和UNC5C相互作用并对其进行调节。
