磷酸二酯酶7B介导海马齿状回突触重塑参与成瘾记忆提取的信号通路及microRNA-132的调控作用

Phosphodiesterase7B (PDE7B) is a member of PDE7 family. It is mainly distributed in the brain, and specifically hydrolyzes cAMP to inhibit the activity of PKA, eventually result in the phosphorylation of CREB. Many researches have proved that inhibition of PDE7 could not only promote adult neurogenesis of the dentate gyrus (DG) in the hippocampus, which means enhanced structure plasticity, but also strengthen the hippocampus-dependent memory of rodents. However, nearly nothing has known about the role of PDE7B in drug-addicted memory. We found in preliminary study that the 3’UTR region of PDE7B contains a sequence that can be specifically bound by microRNA-132 (miR-132) via bioinformatics, overexpression of miR-132 decreased the mRNA level of PDE7B, which suggests that miR-132 may play a role in addiction memory by regulating the expression of PDE7B. The aims of our study are 1) to detect the density/morphology of dendritic spine and LTP induction in the DG, as well as the expression of PDE7B, p-CREB and miR-132 in morphine CPP rats; to investigate whether overexpression or knock down of PDE7B could reverse the change in synaptic plasticity and affect the morphine CPP expression; 2) to investigate the miR-132 target sites in PDE7B in cultured N2a cell; 3) to observe whether knock down of miR-132 could change the density/morphology of dendritic spines and LTP induction in the DG, and whether the expression of PDE7B and morphine CPP will be changed. Our purpose is to clarify whether PDE7B mediated morphine CPP-induced synaptic plasticity in the DG and whether it is regulated by miR-132.

已知磷酸二酯酶7（PDE7）参与成体海马齿状回神经元新生和学习记忆，然而PDE7在成瘾记忆中的作用尚无报道。鉴于PDE7B主要表达在大脑，且预实验发现，过表达microRNA-132（miR-132）可以降低PDE7B的mRNA水平，提示miR-132可以调控PDE7B的表达。本课题计划：1）检测吗啡CPP表达后海马齿状回神经元的树突棘密度/形态、LTP诱导以及PDE7B的mRNA和蛋白水平；过表达/敲低PDE7B是否可以逆转上述改变；2）在N2a细胞中确定miR-132结合PDE7B mRNA的特异位点；3）CPP测试前敲低齿状回miR-132，观察其对PDE7B mRNA和蛋白水平，以及突触可塑性和吗啡CPP表达的影响。旨在探索PDE7B是否通过调控齿状回神经元的树突棘密度/形态和突触传递，来影响吗啡CPP的表达，该过程是否受miR-132的调控，试图找到可以消除成瘾记忆的潜在靶点。

成瘾记忆与生理记忆的形成和提取的机制非常相似，二者都以神经元结构和功能的可塑性改变为基础。研究成瘾记忆提取后突触重塑的机制，对于寻找能够消除成瘾记忆的靶分子极为重要。已知磷酸二酯酶7（PDE7）参与成体海马齿状回（dentate gyrus, DG）神经元新生和学习记忆，然而PDE7在成瘾记忆中的作用尚不清楚。此外，我们的前期研究提示PDE7A和7B的表达可能受microRNA-132（miR-132）所调控。而有研究证实，miR-132与神经元的结构和功能可塑性以及学习记忆密切相关。由此我们提出了以下科学假说：miR-132通过抑制PDE7A和7B的表达，调节cAMP/PKA/CREB信号通路，进而影响海马齿状回神经元的结构可塑性，并介导吗啡成瘾记忆的提取。我们采用吗啡条件性位置偏爱模型来模拟成瘾记忆形成和提取过程，结果发现：（1）吗啡CPP测试后，海马齿状回中miR-132的水平上调，PDE7A和PDE7B的表达减少，同时CREB的磷酸化水平上调，并伴有DG神经元树突树复杂度增加和树突棘密度增加；（2）吗啡CPP测试前，抑制DG中PDE7的功能，可以促进吗啡CPP的表达；（3）吗啡CPP测试前，特异性敲低DG中的miR-132，能恢复吗啡CPP大鼠DG中原本降低的PDE7A和PDE7B，还可翻转DG神经元结构的变化并抑制吗啡CPP的表达；（4）吗啡CPP测试前，同时抑制DG中的miR-132和PDE7的功能，不仅能恢复吗啡CPP的表达，还能部分逆转DG神经元结构的变化。上述结果提示吗啡条件化记忆的形成和提取过程中，会有miR-132水平的升高，它能通过抑制PDE7A和PDE7B等靶基因的表达，诱发海马DG神经元发生突触重塑，最终形成顽固的成瘾记忆。该结果为揭示成瘾记忆提取的突触机制，寻找消除成瘾记忆的分子靶点提供了新的线索。