Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, Pennsylvania 19129, and.
Albert Einstein College of Medicine, Department of Physiology and Biophysics, Bronx, New York 10461.
J Neurosci. 2019 Mar 13;39(11):2011-2024. doi: 10.1523/JNEUROSCI.1888-18.2018. Epub 2019 Jan 15.
Fidgetin is a microtubule-severing protein that pares back the labile domains of microtubules in the axon. Experimental depletion of fidgetin results in elongation of the labile domains of microtubules and faster axonal growth. To test whether knockdown assists axonal regeneration, we plated dissociated adult rat DRGs transduced using AAV5-shRNA- on a laminin substrate with spots of aggrecan, a growth-inhibitory chondroitin sulfate proteoglycan. This cell culture assay mimics the glial scar formed after CNS injury. Aggrecan is more concentrated at the edge of the spot, such that axons growing from within the spot toward the edge encounter a concentration gradient that causes growth cones to become dystrophic and axons to retract or curve back on themselves. knockdown resulted in faster-growing axons on both laminin and aggrecan and enhanced crossing of axons from laminin onto aggrecan. Strikingly, axons from within the spot grew more avidly against the inhibitory aggrecan concentration gradient to cross onto laminin, without retracting or curving back. We also tested whether depleting fidgetin improves axonal regeneration after a dorsal root crush in adult female rats. Whereas control DRG neurons failed to extend axons across the dorsal root entry zone after injury, DRG neurons in which was knocked down displayed enhanced regeneration of axons across the dorsal root entry zone into the spinal cord. Collectively, these results establish fidgetin as a novel therapeutic target to augment nerve regeneration and provide a workflow template by which microtubule-related targets can be compared in the future. Here we establish a workflow template from cell culture to animals in which microtubule-based treatments can be tested and compared with one another for their effectiveness in augmenting regeneration of injured axons relevant to spinal cord injury. The present work uses a viral transduction approach to knock down from rat neurons, which coaxes nerve regeneration by elevating microtubule mass in their axons. Unlike previous strategies using microtubule-stabilizing drugs, knockdown adds microtubule mass that is labile (rather than stable), thereby better recapitulating the growth status of a developing axon.
Fidgetin 是一种微管切割蛋白，可减少轴突中微管的不稳定域。实验性耗尽 fidgetin 会导致微管不稳定域伸长和更快的轴突生长。为了测试 knockdown 是否有助于轴突再生，我们将使用 AAV5-shRNA 转导的成年大鼠 DRG 细胞在层粘连蛋白底物上进行分离培养，该底物上有聚集蛋白的斑点，聚集蛋白是一种生长抑制性软骨素硫酸蛋白聚糖。这种细胞培养测定法模拟了中枢神经系统损伤后形成的神经胶质瘢痕。聚集蛋白在斑点的边缘更为集中，因此从斑点内部生长的轴突会遇到一个浓度梯度，导致生长锥变得营养不良，轴突回缩或自身弯曲。 knockdown 导致在层粘连蛋白和聚集蛋白上生长的轴突更快，并且增强了从层粘连蛋白到聚集蛋白的轴突交叉。引人注目的是，从斑点内部生长的轴突在抑制性聚集蛋白浓度梯度的作用下更强烈地生长，以越过层粘连蛋白，而不会回缩或弯曲。我们还测试了在成年雌性大鼠的背根挤压后耗尽 fidgetin 是否可以改善轴突再生。在损伤后，对照 DRG 神经元未能将轴突延伸穿过背根入口区，而 被敲低的 DRG 神经元显示出增强的再生，将轴突穿过背根入口区进入脊髓。总之，这些结果确立了 fidgetin 作为一种新的治疗靶标，以增强神经再生，并为未来比较微管相关靶标提供了工作流程模板。在这里，我们建立了一个从细胞培养到动物的工作流程模板，在该模板中可以测试基于微管的治疗方法，并比较它们在增强与脊髓损伤相关的损伤轴突再生方面的有效性。本工作使用病毒转导方法来敲低大鼠神经元中的 ，通过增加其轴突中的微管质量来促使神经再生。与以前使用微管稳定药物的策略不同， 敲低增加了微管质量的不稳定性（而不是稳定性），从而更好地模拟了发育中轴突的生长状态。
