Yes-Associated Protein(YAP) 在脊髓损伤胶质疤痕形成中的作用及其机制

Glial scarring after spinal cord injury(SCI) is mainly due to proliferation of reactive astrocytes, however, the underlying molecular mechanism of regulating the reactive gliosis response to injury remains unclear. The Hippo/YAP(Yes-Associated Protein) signaling pathway plays a key role in regulating cell proliferation, in addition, our preliminary data has shown that, ① YAP was specifically, highly expressed in astrocytes of spinal cord; ②In scratched injury model, nuclear YAP was dramatically decreased, whereas, cytoplasmic p-YAP was increased in injuried astrocytes; ③Knockdown YAP by RNAi promoted the activation but inhibited the proliferation of astocytes; ④YAP was dramatically decreased in early stage, and increased in late stage of spinal cord injury. Based on these preliminary data, we propose our hypothesis: during spinal cord injury, activation of astrocytic YAP promotes the proliferation of astrocytes, and leads to the formation of glial scars. To test this hypothesis, firstly, we will further examine the roles and mechanisms of YAP in astrocyte activation and proliferation by using cellular and molecular technologies. Secondly, we will exmamine the effects of astrocytic YAP in glial scars, and neural regeneration by using inducible and conditional YAP knockout mice with spinal cord injuried model. This study will help to understand the roles and mechanisms of Hippo/YAP pathways in glia scar formation after spinal cord injury, and provide new targets and new insights for spinal cord injury therapies.

胶质疤痕形成是脊髓损伤重要的病理学特征，主要由活化的星形胶质细胞增殖引起，然而诱导胶质细胞活化和增殖机制尙不清楚。研究报道Hippo通路调节机体细胞增殖，我们预实验发现：①YAP特异性高表达于脊髓星形胶质细胞；②在划痕损伤细胞模型中，损伤区内星形胶质细胞核定位YAP显著降低， p-YAP增多；③沉默YAP后促进星形胶质细胞活化并抑制其增殖；④YAP表达量在脊髓损伤早期下调而在晚期显著上调。由此我们设想：脊髓损伤后是否通过激活YAP促进星形胶质细胞增殖，从而诱导胶质疤痕形成。本课题拟采用细胞生物学等技术进一步阐明YAP在星形胶质细胞激活和增殖中的作用及其分子机制。构建可诱导性、条件性YAP基因敲除小鼠，观察在脊髓损伤动物模型各个时期，YAP对胶质疤痕形成及对脊髓损伤再生修复的作用。本研究将首次深入阐述YAP信号通路在脊髓损伤胶质疤痕形成中的作用及分子机制，为今后的临床治疗提供新靶点和新思路。

中枢神经损伤与再生修复一直是神经科学研究的热点和难点，如在临床上，由交通事故、坠落伤、战争等导致的脊髓损伤（Spinal Cord Injury, SCI）是较为常见的中枢神经损伤疾病。脊髓损伤在全球发病率高，致残率高，对社会经济损失和患者家庭造成巨大负担。因此，神经系统再生修复的分子基础研究不仅有着重要的科学意义，而且具有重大的社会效益。目前脊髓损伤中胶质瘢痕的研究已然成为热点，脊髓损伤后，星形胶质细胞细胞肥大、增殖并且迁移至损伤区形成致密的胶质瘢痕，其不仅限制炎症细胞的浸润同样也会影响神经再生。然而，目前对于胶质瘢痕的分子机制研究甚少。YAP（yes-associated protein）是一种受Hippo通路负调控的重要共转录因子，在发育过程中参与肝脏等多个器官大小的调控。先前文章报道YAP在中枢神经系统中主要表达于星形胶质细胞，但是YAP是否参与脊髓损伤后胶质瘢痕的形成尚不清楚。本课题主要利用YAP条件性敲除小鼠、碱性成纤维细胞生长因子（bFGF）体外刺激模型、bFGF及XMU-MP-1（选择性MST1/2抑制剂）在体注射模型，研究YAP对脊髓损伤后胶质瘢痕形成和神经再生修复中的作用并探讨其相关分子机制。我们实验中发现C57BL/6小鼠在脊髓损伤后以Hippo通路依赖的方式促进星形胶质细胞的YAP表达上调并被激活。特异性敲除星形胶质细胞中YAP后显著抑制星形胶质细胞增殖和胶质瘢痕形成，抑制轴突再生和运动功能的恢复。机制上，脊髓损伤后碱性纤维细胞生长因子（bFGF）分泌增多。增多的bFGF通过RhoA通路诱导YAP激活从而促进胶质瘢痕形成。进一步YAP通过负调控CRM1介导的p27Kip1核分布促进bFGF诱导的星形胶质细胞增殖。最后bFGF和XMU-MP-1（Hippo通路激酶MST1/2抑制剂）注射脊髓损伤小鼠，通过激活YAP信号通路促进胶质瘢痕形成和运动功能恢复。这些结果表明bFGF-RhoA-YAP-p27Kip1通路在胶质瘢痕形成中的作用，可以为脊髓损伤患者提供治疗策略和理论基础。