Rheb1/mTORC1 调控的印记基因DLK1在出生后脑组织髓鞘形成中的作用

Oligodendrogenesis and myelination of axons are essential events for the formation of normal brain structure. We have shown that genetic deletion of Rheb1 impairs mTORC1 activity and causes deficit in oligodendrogenesis and myelination in the brain and spinal cord (CNS). As a result of defective Rheb1 signaling, the genomic imprinted gene—DLK1 is upregulated. This proposal examines the hypothesis that misexpression of DLK1 in oligodendrocyte precursor cells (OPCs) impairs OPC differentiation and myelination in the CNS. To test our hypothesis, we generated Cre-dependent transgenic mouse (Rosa26-DLK1) that can express DLK1 Tg in neural stem cells (Nestin-Cre driver) or OPCs (Olig1-Cre driver). We will first complete the analysis Rosa26-DLK1;Nestin-Cre mouse and confirm the defects in oligodendrogenesis and myelination in the CNS. Then we will analyze the oligodendrocyte-intrinsic inhibitory effect of DLK1 Tg (Oligo1-Cre) on oligodendrogenesis and myelination. To understand how DLK1 inhibits OPC differentiation, we will assay if DLK1 affects the expressions of cell cycle regulators and transcription factors in OPCs, and activates Wnt pathway. To better understand the pathways affected by DLK1 Tg in OPCs, we will use TRAP-RNAseq to identify mRNAs that are altered in OPCs expressing DLK1 Tg. These studies will link mTor signaling to misexpression of the genomic imprinted gene and provide novel insight into the mechanism by which mTOR regulates the formation of brain structure. The validation of DLK1 as a novel inhibitor of oligodendrogenesis opens up a new avenue for promoting adult oligodendrogenesis for myelin repair.

少突胶质细胞（OLs）和髓鞘发育是脑组织形成中的重要事件。我们前期研究表明遗传敲除Rheb1使mTORC1活性降低并导致中枢神经系统OLs和髓鞘发育异常，且Rheb1信号异常引起印记基因DLK1表达升高。我们假设：少突胶质细胞前体细胞（OPCs）中DLK1的异常表达会引起成熟OLs和髓鞘形成障碍。为了验证假设，我们构建了在神经干细胞和OPCs中过表达DLK1的转基因小鼠。通过对两种转基因小鼠的检测来明确OLs自主性表达的DLK1对成熟OLs和髓鞘形成的抑制作用。通过检测DLK1是否激活OPCs中的 Wnt信号及转录调控因子，并用TRAP-RNAseq技术筛选OPCs中过表达DLK1发生变化的mRNA揭示DLK1调控OLs分化的机制。以上研究有助于我们揭示mTOR调控脑组织结构形成的新机制。证实DLK1是一种新的OLs分化的抑制因子将为脱髓鞘疾病的治疗开辟一条新路。

大脑的髓鞘生成障碍与神经发育失调和精神紊乱疾病相关，然而造成髓鞘生成障碍的机制仍不清楚。异常的神经元信号可以影响疾病中的髓鞘发育甚至是髓鞘再生。通过对神经元特异性敲除Rheb1小鼠的研究，我们阐明了一种导致髓鞘发育缺陷的神经元调控机制。我们发现，神经元中Rheb1的缺失严重影响少突胶质细胞分化和髓鞘形成。Rheb1的这种作用与神经元Dlk1表达升高有关；而神经元特异性Rheb1和Dlk1双敲除可缓解Rheb1敲除引起的髓鞘缺陷。在体外实验中，重组DLK1蛋白抑制培养的少突胶质前体细胞分化和髓鞘蛋白表达。DLK1蛋白可以与锚定在少突胶质细胞膜上的DLK1复合物受体相互作用，启动细胞内信号级联反应，抑制少突胶质细胞分化。这些结果表明，Rheb1信号通路介导了髓鞘发育过程中神经元与少突胶质细胞之间的相互作用，而Rheb1缺失引起的DLK1表达异常导致了髓鞘发育缺陷。本研究为研究疾病中神经元/神经胶质相互作用与髓鞘发育缺陷开辟了新的途径。