Cheng C, Webber C A, Wang J, Xu Y, Martinez J A, Liu W Q, McDonald D, Guo G F, Nguyen M D, Zochodne D W
University of Calgary, 3330 Hospital Drive NW, Calgary, Alberta, Canada T2N 4N1.
Exp Neurol. 2008 Aug;212(2):358-69. doi: 10.1016/j.expneurol.2008.04.023. Epub 2008 Apr 30.
The regeneration of adult peripheral neurons after transection is slow, incomplete and encumbered by severe barriers to proper regrowth. The role of RHOA GTPase has not been examined in this context. We examined the expression, activity and functional role of RHOA GTPase and its ROK effector, inhibitors of regeneration, during peripheral axon outgrowth. We used qRT-PCR, quantitative immunohistochemistry, and assays of RHOA activation to examine expression in sensory neurons of rats with sciatic transection injuries. In vitro, we exposed dissociated adult sensory neurons, not grown on inhibitory substrates, to a RHOA-ROK inhibitor HA-1077 and measured neurite initiation and outgrowth. In vivo, we exposed early regenerating axons and Schwann cells directly to HA-1077 in a conduit connecting the proximal and distal stumps of transected sciatic nerves. Intact adult dorsal root ganglia sensory neurons expressed RHOA and ROK 1 mRNAs and protein and there were rises in RHOA after injury. Activated GTP-bound RHOA, undetectable in intact ganglia, was dramatically upregulated in both neurons and axons after injury. Adult rat sensory neurons in vitro demonstrated a dose-related increase in the initiation of neurite outgrowth, and in the proportion with long neurites when they were exposed to a ROK antagonist. Regenerative bridges that were directly exposed to the ROK inhibitor had a dose-related rise in the extent and distance of in vivo axon and partnered Schwann cell regrowth within them. RHOA activation and signaling are features of adult peripheral axon regeneration within its own milieu, independent of myelin. Inhibition of its activation may benefit peripheral axon lesions.
成年外周神经元横断后再生缓慢、不完全，且受到正常生长严重障碍的阻碍。在这种情况下，RHOA GTP酶的作用尚未得到研究。我们研究了RHOA GTP酶及其ROK效应器（再生抑制剂）在外周轴突生长过程中的表达、活性和功能作用。我们使用qRT-PCR、定量免疫组化和RHOA激活检测，来研究坐骨神经横断损伤大鼠感觉神经元中的表达情况。在体外，我们将未生长在抑制性底物上的成年解离感觉神经元暴露于RHOA-ROK抑制剂HA-1077，并测量神经突的起始和生长情况。在体内，我们将早期再生轴突和雪旺细胞直接暴露于连接横断坐骨神经近端和远端残端的导管中的HA-1077。完整的成年背根神经节感觉神经元表达RHOA和ROK 1的mRNA及蛋白，损伤后RHOA水平升高。完整神经节中无法检测到的活化GTP结合型RHOA，在损伤后的神经元和轴突中均显著上调。体外培养的成年大鼠感觉神经元在暴露于ROK拮抗剂时，神经突生长起始呈剂量相关增加，且长神经突的比例也增加。直接暴露于ROK抑制剂的再生桥中，体内轴突和伴行雪旺细胞在其中的生长范围和距离呈剂量相关增加。RHOA激活和信号传导是成年外周轴突在自身环境中再生的特征，与髓磷脂无关。抑制其激活可能有利于外周轴突损伤。
