lep-Rb介导信号通路参与脑损伤后神经再生修复的作用及机制

The long form receptor Lep-Rb is considered to be essential and responsible for the principle function of leptin. Upon binding with ligand, Lep-Rb is able to homo- oligomerize and active Janus kinase (JAK2), which in turn phosphorylates itself, as well as downstream signaling pathways, including JAK2-STAT3, PI3K/Akt, and MAPK/ERK. .It has been improved that leptin’s signaling effect in neuron or neuron-like cells is cell context dependent. ERK1/2 activation seems to be the main signaling in survival, proliferation and neuronal differentiation of neuronal precursor from hypothalamus while JAK2-STAT3 is involved in neuronal differentiation but not proliferation. However, for neural stem/progenitor cells (NSCs/NPCs) from embryonic rat cerebral cortex, PI3k/Akt is the main proliferation signaling while JAK2-STAT3 is for astrocytic differentiation. The role and the underling mechanisms of different signaling pathways in neural regeneration after brain injury still remain unclear. We hypothesize that Lep-Rb and its downstream signaling pathways are involved in neural regeneration via affecting the proliferation, differentiation and migration of NSCs/NPCs. The activation of different signal pathways is various and depends on the different stages of NSCs/NPCs maturation. JAK2-STAT3 is the most important one and plays complex roles in neural regeneration via enhancing astrocyte regeneration..Here, in order to improve our hypothesis, following works are going to be taken. The proliferation, differentiation and migration of NSCs/NPCs, both in vitro and in vivo, are going to be investigated after leptin treatment in ischemic/anoxic insults. Activation of various signaling pathways will be identified along the maturation of NSCs/NPCs, followed by the observation of NSCs/NPCs biological behavior alteration after site-directed Mutagenesis of Tyr985, Try1077, Tyr1138 of Lep-Rb, and/or agonist/inhibitor of MEK/ERK, PI3K/Akt, JAK2-STAT3 signaling pathways is used. Then the target proteins of JAK2-STAT3, such as SOCS3 and GFAP, the number of new-born astrocytes/neurons and the level of cytokines, like inflammatory cytokines and neurotrophic factors, in the extracellular space are going to be detected. Our results will add new data for better understanding of the role of Lep-Rb and its downstream signaling pathways in neural regeneration and may potentially be utilized to develop new strategy for brain repair.

瘦素受体Lep-Rb介导信号通路参与脑保护作用及机制不同，其对神经再生修复的影响及机制仍未明。我们推测：不同信号通路差异性激活，影响神经干/前体细胞增殖分化，参与脑损伤后神经再生修复，其中JAK2-STAT3通路发挥重要作用。本项目拟在大鼠脑缺氧模型，以行为学及形态学方法观察Lep-Rb介导信号通路激活后神经再生及功能修复；在神经干/前体细胞缺氧模型，以流式细胞分选、蛋白定量及免疫染色技术观察不同信号通路激活与细胞增殖分化相关性；利用定点突变技术构建Lep-Rb突变体及以激动剂、拮抗剂干扰信号通路活性，分别在细胞、组织及动物整体水平观察细胞增殖分化改变，分析其参与脑损伤神经再生作用及细胞与分子机制；检测JAK2-STAT3通路靶基因表达及星形胶质细胞再生，微透析检测脑损伤局部细胞因子水平，分析该通路激活对神经再生修复的意义，补充Lep-Rb参与神经再生理论，为开发脑损伤治疗新策略提供依据。

项目关注瘦素受体lep-Rb介导不同信号通路的差异性激活及其对NSCs/NPCs增殖、分化、迁移的影响和作用机制，对于完善Lep-Rb参与脑损伤后神经再生理论、开发leptin联合NSCs/NPCs治疗脑损伤新策略有重要意义。本项目通过体外细胞模型、发育期内源性高leptin水平持续暴露以及脑损伤动物模型，证实：1）体外条件下，不同浓度leptin可促进NSCs/NPCs存活、增殖及向神经元方向分化，其中低浓度与中、高浓度leptin处理组之间存在显著差异，但对NSCs/NPCs向星形胶质细胞方向分化无明显影响；上述作用过程中，MAPK/ERK、PI3K/Akt及JAK2-STAT3信号通路存在差异性激活，其中MAPK/ERK信号通路激活不受leptin浓度影响，而PI3K/Akt及JAK2-STAT3磷酸化程度随leptin浓度增高而增加；MAPK/ERK及PI3K/Akt主要与NSCs/NPCs存活、增殖相关，而JAK2-STAT3信号通路主要与NSCs/NPCs分化有关，但对具体分化方向的影响可能仍存在时间差异性。2）发育期内源性高leptin水平持续暴露对NSCs/NPCs存活、增殖、发育及向神经元方向分化有抑制作用，而对其向星形胶质细胞分化有促进作用；MAPK/ERK、PI3K/Akt信号通路参与对NSCs/NPCs存活及增殖的影响，而JAK2-STAT3信号通路参与促进NSCs/NPCs向星形胶质细胞分化。3）脑损伤后损伤灶周围成熟细胞表达Lep-Rb水平增加，提示对leptin的敏感性增高，而神经发生区域NSCs/NPCs表达Lep-Rb水平无改变，提示对leptin相对不敏感性。综合上述结果，提示：脑损伤后给予leptin的治疗策略，需综合考虑时间与空间因素；其神经保护作用及促进神经再生作用的机制，可能既依赖于下游信号通路差异性激活，从而对不同类型细胞产生直接作用，也可能与Lep-Rb介导JAK2-STAT3信号通路激活，从而促进星形胶质细胞再生、进而促进脑损伤后神经功能修复有关。有关细胞间相互作用以及最佳的leptin作用时间窗，仍需更为深入的研究。