Rho-independent stimulation of axon outgrowth and activation of the ERK and Akt signaling pathways by C3 transferase in sensory neurons.
Rho-independent stimulation of axon outgrowth and activation of the ERK and Akt signaling pathways by C3 transferase in sensory neurons.
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DOI：
10.3389/fncel.2012.00043
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发表时间：
2012
影响因子：
5.3
通讯作者：
Klimaschewski L
中科院分区：
文献类型：
--
作者：
Auer M;Schweigreiter R;Hausott B;Thongrong S;Höltje M;Just I;Bandtlow C;Klimaschewski L
周围神经损伤触发脊髓运动和周围感觉神经元中RhoA的激活。RhoA激活许多效应蛋白，包括Rho相关激酶ROCK，其靶向细胞骨架并导致抑制神经突生长。通过药理学手段阻断Rho/ROCK通路可改善实验性损伤后的轴突再生。C3 bot转移酶是肉毒梭菌产生的一种外切酶，通过ADP-核糖基化使RhoA失活。它已成功地应用于实验性中枢神经系统损伤，以促进轴突再生。到目前为止，尚未彻底研究C3 bot是否也对外周轴突再生产生积极影响。在本研究中，重组膜渗透性C3 bot产生了一个小的，但显着的，轴突生长的外周感觉神经元从成年大鼠背根神经节（DRG）的影响。然而，C3的神经元过表达并没有增强轴突的生长。此外，转染编码显性负性RhoA或RhoA特异性shRNA的质粒未能增加轴突生长。此外，我们表明，C3 bot突变体，C3 E174 Q，缺乏RhoA抑制活性，仍然刺激轴突生长。当分析可能的信号转导机制时，我们发现细胞外信号调节激酶（ERK）和Akt被C3 bot激活，ERK被C3 E174 Q突变体诱导。C3 bot对激酶活性的上调明显快于RhoA的失活，表明C3 bot的作用途径不依赖于RhoA。在胚胎海马神经元中也检测到C3 bot对ERK信号的诱导作用。总而言之，尽管RhoA在髓鞘衍生抑制剂抑制轴突生长方面发挥着核心作用，但它不会干扰体外允许基质上感觉神经元的轴突生长。C3 bot阻断神经元RhoA活性，但其对轴突伸长和分支的积极作用似乎是由Rho独立机制介导的，该机制涉及促进轴突生长的ERK和Akt激酶的激活。
Peripheral nerve injury triggers the activation of RhoA in spinal motor and peripheral sensory neurons. RhoA activates a number of effector proteins including the Rho-associated kinase, ROCK, which targets the cytoskeleton and leads to inhibition of neurite outgrowth. Blockade of the Rho/ROCK pathway by pharmacological means improves axon regeneration after experimental injury. C3bot transferase, an exoenzyme produced by Clostridium botulinum, inactivates RhoA by ADP-ribosylation. It has been successfully applied in experimental CNS lesions to facilitate axon regeneration. Up to now it was not investigated thoroughly whether C3bot exerts positive effects on peripheral axon regeneration as well. In the present study, recombinant membrane permeable C3bot produced a small, but significant, axon outgrowth effect on peripheral sensory neurons dissociated from adult dorsal root ganglia (DRG) of the rat. Neuronal overexpression of C3, however, did not enhance axonal growth. Moreover, transfection of plasmids encoding dominant negative RhoA or RhoA specific shRNAs failed to increase axonal growth. Furthermore, we show that the C3bot mutant, C3E174Q, which lacks RhoA inhibitory activity, still stimulates axonal growth. When analyzing possible signaling mechanisms we found that extracellular signal-regulated kinase (ERK) and Akt are activated by C3bot and ERK is induced by the C3E174Q mutant. Upregulation of kinase activities by C3bot occurs significantly faster than inactivation of RhoA indicating a RhoA-independent pathway of action by C3bot. The induction of ERK signaling by C3bot was detected in embryonic hippocampal neurons, too. Taken together, although RhoA plays a central role for inhibition of axon outgrowth by myelin-derived inhibitors, it does not interfere with axonal growth of sensory neurons on a permissive substrate in vitro. C3bot blocks neuronal RhoA activity, but its positive effects on axon elongation and branching appear to be mediated by Rho independent mechanisms involving activation of axon growth promoting ERK and Akt kinases.
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