P2Y1受体调控Neuritin的信号转导机制及对喉返神经再生作用

Vocal fold paralysis (VFP) caused by the injury to recurrent laryngeal nerve (RLN) is a common disease in otorhinolaryngology department, which severely impairs the quality of life for those patients. Our previous investigations revealed that a varying degree of spontaneous axons regeneration occurred in symptomatic VFP patients who exhibited typical denervation potential and poor regenerative potential in laryngeal electromyography examination. These histologic and electrophysiologic evidences indicated that inadequately regenerative degree of RLN axons resulted in the unsatisfactory recovery of laryngeal function in those symptomatic VFP patients, and after undergoing laryngeal reinnervation as well. Hithreto, the mechanism of regulating axonal regeneration remains unclear. Recent study focused on Neuritin, coded by candidate plasticity-related gene 15 (CPG15), which plays an important role in the central and peripheral nervous systems. It was reported that both NMDA/CaMKs and BDNF/TrkB signaling pathways can modulate the transcriptional activation of CREB (cAMP-response element binding protein), then CREB up-regulates the expression level of Neuritin via binding to the promoter region of Neuritin. Our in-vivo experiment found that purinergic receptor P2Y1 and Neuritin up-regulated in the regenerating RLNs. Furthermore, the positive effect of P2Y1 promoting axonal regeneration was almost abolished by the neutralized antibody against Neuritin in the cultured primary motoneurons. As a member of G-protein coupled protein family, P2Y1 receptor exerts an extensive and important effect in the physiopathological process through modulating the signaling molecules of PKA, ERK/MAPK, p38/MAPK, PI-3K/AKT and so on. These signaling pathways mentioned above have proven to be closely correlated to the transcriptional activation of CREB. Whether P2Y1 receptor regulates the Neuritin expression via those signaling pathways is unknown yet. Together, we hypothesized that P2Y1 receptor may modulate the transcriptional activation of CREB to regulate the Neuritin via PKA or ERK/MAPK or p38/MAPK or PI-3K/AKT signaling pathway. For this purpose, we will establish a mice model of RLN regeneration and the cultured primary motoneurons. In vivo and in vitro experiments, using some specific drugs and gene interference, are designed to demonstrate the spatio-temporal expression and localization of P2Y1, CREB and Neuritin in the process of RLN regeneration, and elucidate the signaling transduction mechanisms of P2Y1 regulating CREB /Neuritin. This study is expected to explore a potentially therapeutic target to promote axonal regeneration of RLN. The satisfactory rehabilitation of laryngeal function may be obtained after laryngeal reinnervation by this novel therapeutic strategy.

喉返神经轴突再生程度不足是声带麻痹患者喉功能恢复不佳的重要原因。新近发现的神经突起因子Neuritin是外在因素共同作用的关键下游靶点，我们的前期研究及文献报道其在神经轴突再生中起着重要作用，但关于其上游调控机制尚不太清楚。申请人证实喉返神经再生过程中嘌呤能受体P2Y1和Neuritin表达上调，而抑制Neuritin可显著阻断P2Y1促轴突生长效应。P2Y1受体可激活ERK、p38、PI-3K等信号通路，后者与CREB转录激活密切相关。由此推测：P2Y1受体可能通过ERK、p38或PI-3K调控CREB/Neuritin通路，发挥促进神经轴突再生的作用。本课题拟通过在体和离体实验，采用组成型激活/失活技术，从分子、细胞、组织和在体水平，阐明P2Y1调控CREB/Neuritin的信号转导机制及对轴突再生的作用，旨在为促进喉返神经再生和喉功能恢复，寻找新的治疗靶点，提供理论及实验依据。

神经轴突再生程度不足是喉面神经功能恢复不佳的重要原因，而嘌呤能受体P2Y1在神经轴突再生过程中发挥重要作用。本项目成功构建神经再生室及P2Y1受体药物干预的稳定动物模型。并采用诱发肌电图检测并记录肌肉动作电位的最大幅度、潜伏期；通过Masson染色观察单位面积内的肌纤维数目和平均直径；采用神经干甲苯胺蓝染色观察再生神经有髓神经纤维的数目；通过透射电镜观察再生神经的超微结构。证实激活P2Y1受体能明显促进神经轴突再生，从而促进肌肉功能恢复。并利用上述动物模型在术后1、2、3、4、6周5个不同的时间点切取神经再生室及远端分支，采用双重免疫荧光标记及Peggy Sue微量蛋白定量分析、Real Time-PCR等分子生物学研究方法，观测P2Y1受体在轴突再生过程中表达的时空改变，并检测Neuritin表达的时空改变。证实神经损伤早期P2Y1受体被激活，可以通过调控Neuritin促进轴突的延伸，从而促进神经的再生；而阻断P2Y1受体可以抑制Neuritin的表达，并引起再生的有髓神经纤维数量减少，阻碍神经的再生。另外采用双重免疫荧光检测不同处理因素下PI-3K、ERK1/2信号分子表达的时空改变；并用Peggy Sue微量蛋白定量分析上述信号分子表达的时间改变，检测其活性改变对神经再生的作用。结果证明ERK/MAPK、PI3K/Akt信号通路在神经损伤后被激活，且ERK1/2、 PI3K的表达具有各自的时间效应，不同的信号通道在不同时间被激活，这可能与神经再生的调节有显著的关系。此外，激活P2Y1受体上述2个信号分子表达显著增加，阻断P2Y1受体，其表达显著减少，也说明神经损伤后P2Y1受体对这两个信号通路的调控作用。离体实验部分，成功构建了原代运动神经元培养模型，通过P2Y1激动剂和阻断剂干预下，发现P2Y1受体可调控Neuritin 促进运动神经元轴突生长的作用。本项目旨在为促进喉面神经再生，寻找新的治疗靶点，提供理论及实验依据。