基于成体神经干细胞的分化命运研究吗啡成瘾记忆的调控机制

The role of adult neurogenesis in drug addiction is a focus in the current field of neuroscience, and may contribute to most potential therapeutic strategies. The lineages of differentiated adult neural stem cells in the dentate gyrus determines the quantities and proportions of new-born neurons and glial cells, and is therefore a crucial factor in adult neurogenesis. According to our previous in vitro studies, morphine could activate ERK via PKCε-dependent pathway, and in turn modulates miR-181a in a post-transcriptional way. MiR-181a then inhibits the expression of its target, Prox1, thus inhibiting neuronal differentiation. Although the regulatory effects of addictive drugs on neural stem cells are widely reported, the relationship between neural stem cell differentiation and drug memory remains unclear. In our current project, we will use lentivirus that modulates Prox1 overexpression in the adult neural stem cells of both wild type and PKCε knockout mice, in order to confirm the regulation of adult hippocampal neural stem cell differentiation by PKCε/Prox1. Furthermore, we will study the effect of this pathway on the acquisition, extinction and re-consolidation of morphine-induced drug memory by using the model of conditioned place preference. Moreover, we will precisely control the time points of Prox1 overexpression, in order to elucidate the effects of Prox1 at different stages of morphine addiction. Overall, this project is due to contribute to the therapeutic strategies of drug addiction with novel ideals.

成体神经发生在药物成瘾中的作用是当前的研究热点之一，有望发展为极具潜力的治疗策略。齿状回成体神经干细胞的分化命运决定了新生神经元和胶质细胞的数量和比例，是成体神经发生的关键因素。我们的前期的体外研究表明，吗啡可通过PKCε激活ERK，并经转录后加工途径调控miR-181a，进一步抑制其靶基因Prox1的表达，最终抑制成体神经干细胞向神经元分化。虽然成瘾药物对神经干细胞的调控作用已多有报道，但有关成体神经干细胞分化命运和药物成瘾记忆关系的研究仍不足。本项目拟用慢病毒载体调节野生型和PKCε基因敲除小鼠神经干细胞的Prox1的表达量，以证明PKCε/Prox1对海马成体神经干细胞分化的调控，并进一步在条件位置偏爱模型上研究该通路对吗啡成瘾记忆获得、消退和重现的影响。在此基础上，通过精确调控Prox1的表达时间，深入探讨其在成瘾不同阶段的调控作用。本项目将为阿片类药物成瘾的治疗策略提供新的思路。

本项目用慢病毒载体调节野生型和PKCε基因敲除小鼠神经干细胞的Prox1的表达量，以证明PKCε/Prox1对海马成体神经干细胞分化的调控，并进一步在条件位置偏爱模型上研究该通路对吗啡成瘾记忆获得、消退和重现的影响。首先，用5-溴脱氧尿嘧啶核苷（BrdU）示踪法，以及通过慢病毒载体在小鼠海马齿状回神经干细胞上过表达Prox1，证明了PKCε/Prox1信号通路对成年小鼠齿状回神经发生的调控作用：PKCε基因敲除可促进成体神经干细胞向神经方向分化，且在基因敲除小鼠上，吗啡促进干细胞向星形胶质细胞分化的作用被阻断，而Prox1的过表达则可促进海马神经干细胞分化为神经元，进而促进成体神经发生，并可逆转吗啡介导的神经干细胞向星形胶质细胞的分化。我们进而通过条件位置偏爱（CPP）模型进一步研究了PKCε/Prox1信号通路对小鼠吗啡成瘾记忆的调控作用，发现PKCε基因敲除可延长吗啡成瘾记忆的消退期，增强吗啡诱导的成瘾记忆重现，而Prox1的过表达同样可减缓小鼠吗啡成瘾记忆的消退过程，并可增强成瘾记忆重现的强度。用X射线定点辐照法抑制小鼠的成体海马神经发生功能后，发现吗啡成瘾记忆的维持时间明显缩短，证明成体神经发生对成瘾记忆的维持有重要作用，以上结果证明了PKCε/Prox1信号通路可通过调控成体神经发生干预吗啡的成瘾记忆。在此基础上用Tet-On调控表达系统在成瘾小鼠CPP训练的前和后两个阶段诱导Prox1的过表达，发现Prox1在CPP训练前的过表达能延长成瘾记忆的持续时间，而在训练后的过表达则缩短成瘾记忆的维持时间，证明了Prox1对吗啡成瘾记忆的调控作用取决于其表达的时间。综合上述结果，本项目揭示了阿片类药物成瘾记忆形成、消退与复吸的调控机制，对于解决成瘾药物精神依赖的问题有重要的意义。