靶向性沉默AMPK促轴索再生治疗急性运动轴索型神经病的实验研究

Acute motor axonal neuropathy (AMAN) is the most common subtype of Guillain-Barre syndrome with poor prognosis because of the difficulty for impaired axon to regenerate. Our preliminary results suggested that the energy monitoring protein, adenosine monophosphate-activated protein kinase (AMPK) may act as the key inhibitory factor for the axonal regeneration of AMAN through reducing the synthesis of axon-sprouting-related protein and energy consumption. In this project, the cell model of AMAN will be established to confirm the effects of AMPK on the energy metabolism and axon-sprouting of motor neuron in AMAN and explore the possible molecular mechanism via the bioluminescence resonance energy transfer technology, dominant negative mutants and luciferase report gene strategies. And then, the neurotropic rabies virus glycoprotein combined with the clustered regularly interspaced short palindromic repeats (CRISPR)/ CRISPR-associated (Cas) 9 technology mediated by adeno-associated virus will be used for the targeting silence of AMPK expression in motor neuron of the mice model of AMAN. The effects of AMPK silence on axonal regeneration of AMAN will be evaluated based on the analysis of clinical, pathological and molecular level. Finally, this project is aimed to confirm whether AMPK can serve as the new target in the therapy of AMAN and targeting to modulate the energy metabolism of motor neuron could be taken as a new strategy for AMAN.

急性运动轴索型神经病(AMAN)是吉兰-巴雷综合征(GBS)最常见的、预后差的发病类型，其根本原因是被破坏的轴索难以再生。课题组前期的研究表明，能量监测蛋白-单磷酸腺苷活化蛋白激酶(AMPK)可能是AMAN中轴索再生的关键性抑制因子，AMPK可能通过减少轴索生长相关的蛋白合成和能量消耗而抑制AMAN中的轴索再生。本项目拟首先建立AMAN细胞模型，利用生物发光共振能量转移技术、显性失活负突变体及荧光素酶报告基因策略明确AMPK对AMAN运动神经元能量代谢和轴索生长的影响及其可能的分子机制；然后利用狂犬病毒糖蛋白结合腺相关病毒介导的CRISPR/Cas9技术靶向性沉默AMAN小鼠运动神经元中AMPK的表达，从临床、病理和分子水平充分评估AMPK缺失对AMAN小鼠轴索再生的影响，最终明确AMPK能否成为治疗AMAN的新靶点以及靶向性调控运动神经元能量代谢能否成为治疗AMAN的新策略。

急性运动轴索型神经病(Acute motor axonal neuropathy, AMAN)是吉兰-巴雷综合征(Guillain-Barre syndrome, GBS)最常见的、预后差的发病类型，其根本原因在于被破坏的轴索难以再生。单磷酸腺苷活化蛋白激酶(Adenosine monophosphate-activated protein kinase, AMPK) 是一种关键性的能量代谢调节蛋白。本项目旨在前期的研究基础上，利用AMAN细胞模型和动物模型证明AMPK对轴索再生的抑制作用并探讨其具体的分子机制，明确AMPK能否成为治疗AMAN的新靶点。我们首先利用抗GD1a抗体干预脊髓前角运动神经元建立AMAN细胞模型并证明了AMPK在AMAN神经元中的显著升高，抑制AMPK可以促进AMAN神经元的轴索生长；然后我们构建了脊髓前角运动神经元靶向性的AMPK沉默腺相关病毒载体-AAV2/2Retro Plus-Hb9-eGFP-miRNAi (Prkaa1)-miRNAi (Prkaa2)-WPRE并验证了其有效性；最后我们利用6-8周龄的GD3s-/-小鼠通过坐骨神经挤压联合腹腔注射抗GD1a抗体建立了AMAN小鼠模型，分别给予AAV2/2Retro Plus-Hb9-eGFP-miRNAi（Prkaa1）-miRNAi（Prkaa2）-WPRE及空白病毒载体，在治疗2周后以电生理测定坐骨神经传导速度、以甲苯胺蓝染色观察坐骨神经中的轴索再生、以步态分析试验观察坐骨神经的运动功能；结果显示：靶向性沉默脊髓前角运动神经元中AMPK的表达可以显著促进AMAN小鼠的轴索再生、改善其临床症状。本项目的结果提示：靶向性干预脊髓前角运动神经元AMPK的表达、增强其能量代谢水平有望成为临床治疗AMAN的新策略。