【作者】 王宁；

【导师】 石宇；

【作者基本信息】 重庆医科大学 ， 临床检验诊断学， 2025， 硕士

【摘要】 背景Wnt信号通路是调控胚胎发育、干细胞命运决定及组织稳态的核心信号网络之一。自1982年Nusslein-Volhard和Wieschaus首次在果蝇中发现Wnt基因以来,该通路在进化保守性、分子机制及疾病关联性方面已积累海量研究。经典Wnt/β-catenin通路需要于Wnt配体、细胞表面受体Frizzled(Fzd)和共受体低密度脂蛋白受体相关蛋白(LRP5/6)的结合。其中,低密度脂蛋白受体相关蛋白6(LRP6)作为经典Wnt信号通路的共受体,通过介导配体结合与信号转导,调控β-catenin依赖的转录激活。研究表明,LRP6缺失导致小鼠颅骨缝早闭(craniosynostosis)和长骨缩短,与人类Robinao综合征表型相似;肝源性PEDF通过竞争性结合LRP5/6,将会抑制Wnt信号过度激活,维持隐窝-绒毛轴的分区化。本课题组前期通过文库建构与质谱分析发现ephrin B2与诱导LRP6剪切密切相关。在此基础上,我们通过动物模型建构对ephrin B2诱导Wnt共受体LRP6剪切的分子机制与其表型进行研究。方法1.爪蛙模型中敲低ephrin B2诱导LRP6剪切过程中的不同蛋白观察其作用的分子机制:利用Morpholino显微注射敲低ephrin B2、LRP6不同片段后明确其相互作用的区域;敲低不同的金属蛋白酶,发现参与剪切的是ADAM10;构建不同截短和突变形式的LRP6来不断精确LRP6的具体剪切位置。2.在细胞中明确LRP6受ephrin B2和ADAM10参与剪切具体氨基酸位置(APQA)后,构建LRP6 APQA-PPPS突变小鼠,使其变为低等动物不被剪切的氨基酸序列,观察小鼠表型的研究:设计LRP6APQA-PPPS突变小鼠进行一系列行为学实验,发现突变小鼠出现了海马区主导的学习记忆能力下降表型;离体LTP电生理实验进一步验证了LRP6基因突变小鼠海马体的学习记忆功能受损情况;应用免疫荧光技术观察对照组和突变小鼠海马体区域少突胶质细胞的增殖情况。结果1.爪蛙模型中明确ADAM是ephrin B2诱导Wnt共受体LRP6剪切的剪切酶。爪蛙模型中敲低ephrin B2和LRP6,明确是ephrin B2的胞内PDZ结构与LRP6的的胞内区域结合诱导LRP6剪切;敲低不同金属蛋白酶,发现ADAM10是ephrin B2诱导Wnt共受体LRP6剪切的脱落酶;因ADAM偏好的P-X-X-↓-X剪切模式预测LRP6胞外域近跨膜区是剪切酶作用位点,并进一步缩小范围,确定为第1368-1369区域。2.对比陆生生物与两栖及水生生物LRP6剪切序列不同,只有陆生脊椎动物APQA序列能够被ephrin B2和ADAM10诱导剪切,我们建构了LRP6 APQA-PPPS突变小鼠模型,进行行为学实验。通过一系列行为学实验明确LRP6 APQA-PPPS突变小鼠基础运动能力及疼痛感知无变化而学习记忆能力减退;体外电生理观察发现LRP6基因突变小鼠影响学习记忆能力的海马体区域突触功能受损;影响突触功能的星形胶质细胞在海马体区域减少。结论我们在爪蛙胚胎和人细胞系中明确ADAM是ephrin B2诱导的LRP6剪切的剪切酶,LRP6的剪切位点精确在序列第1368、1369位。通过对比不同生物这一区域序列,发现陆生哺乳生物在第1367-1369高度保守为APQS,而两栖生物、水生生物为PPPS且不同种属间有所差异。我们感兴趣LRP6在高等陆生脊椎动物中这一剪切是否赋予其新的功能,因此我们构建了LRP6 APQA-PPPS突变导致LRP6不能被剪切的小鼠。PPPS突变小鼠胚胎期发育未发现明显肉眼可见的异常,我们进行了一系列行为学实验发现LRP6突变小鼠出现了学习记忆能力下降的表型。进一步的实验表明,学习记忆能力下降是由于影响突触功能的星形胶质细胞在海马体区域减少导致的突触功能受损。更多还原

【Abstract】 BackgroundThe Wnt signaling pathway is one of the central signaling networks regulating embryonic development,stem cell fate determination and tissue homeostasis.Since the first discovery of Wnt genes in Drosophila by Nüsslein-Volhard and Wieschaus in 1982,the pathway has accumulated a vast amount of research in terms of evolutionary conservatism,molecular mechanisms,and disease relevance.Activation of the classical Wnt/β-catenin pathway relies on the binding of Wnt ligands to the cell surface receptor Frizzled(Fzd)and the co-receptor low-density lipoprotein receptor-related proteins 5/6(LRP5/6).Among them,low-density lipoprotein receptor-related protein 6(LRP6),a co-receptor of the classical Wnt signaling pathway,regulatesβ-catenin-dependent transcriptional activation by mediating ligand binding and signaling.LRP6 deficiency leads to premature closure of the cranial suture(craniosynostosis)and shortening of the long bones in mice,which is similar to the phenotype of Robinow syndrome in humans.Hepatic-derived PEDF will inhibit the over-activation of Wnt signaling and maintain the partitioning of the cryptochondral axis by competitively binding to LRP5/6.Our group found that ephrin B2 is closely related to the induction of LRP6 cleavage through library construction and mass spectrometry analysis.On this basis,we investigated the molecular mechanism of ephrin B2 inducing the cleavage of Wnt co-receptor LRP6 and its phenotype by constructing an animal model.Methods1.Different proteins during ephrin B2-induced LRP6 cleavage were knocked down in the clawed frog model to observe the molecular mechanism of their action:Morpholino microinjection was utilized to knock down the different fragments of ephrin B2 and LRP6 to clarify their interaction regions;different metalloproteinases were knocked down,and it was found that it was ADAM10 that was involved in the cleavage;and the specific LRP6 fragment was constantly refined using Morpholino technology;and the specific LRP6cleavage was found to be the same as that of LRP6.Morpholino technology was utilized to constantly pinpoint the specific cleavage position of LRP6.2.Constructing LRP6 APQA-PPPS mutant mice to observe the phenotype of mice:designing LRP6 APQA-PPPS mutant mice to conduct a series of behavioral experiments,and found that the mutant mice showed a hippocampus-dominated phenotype of reduced learning and memory ability;in vitro LTP electrophysiological experiments further verified the impaired learning and memory function in the hippocampus of the LRP6 mutant mice;applying immunofluorescence technology to observe the learning and memory function of control and control groups;and applying the immuno-fluorescence technology to observe the learning and memory function of control group and control group.Immunofluorescence technique was applied to observe the proliferation of oligodendrocytes in the hippocampus of control and mutant mice.Results1.In the clawed frog model,it was clearly shown that ADAM was the shedding enzyme for ephrin B2-induced cleavage of the Wnt co-receptor LRP6.Knockdown of ephrin B2 and LRP6 in the clawed frog model clarified that the intracellular PDZ structure of ephrin B2 binds to the intracellular region of LRP6 to induce LRP6 cleavage;knockdown of different metalloproteinases revealed that ADAM10 is the shedding enzyme of ephrin B2 to induce Wnt co-receptor LRP6 cleavage;and due to the preferred P-X-X-↓-X cleavage mode of ADAM It was predicted that the near transmembrane region of the extracellular domain of LRP6 was the site of cleaving enzyme action,and further narrowed down the range and identified as region 1368-1369.2.Comparing the sequence differences between terrestrial organisms and amphibious and aquatic organisms in the same region,we constructed the LRP6 APQA-PPPS mutant mouse model for behavioral experiments.Through a series of behavioral experiments,we clarified that LRP6 APQA-PPPS mutant mice showed no change in basic motor ability and pain perception,but reduced learning and memory ability;in vitro electrophysiological observation revealed that LRP6 mutant mice had impaired synaptic function in the hippocampus region affecting learning and memory ability;and astrocytes affecting synaptic function were reduced in the hippocampus region.ConclusionWe clarified in the clawed frog model that ADAM is the shedding enzyme for ephrin B2-induced cleavage of LRP6,and the shedding site of LRP6 is precisely at sequence positions 1368 and 1369.By comparing this region of the sequence in different organisms,we found that terrestrial mammals are highly conserved as APQS at positions 1367-1369,whereas amphibians and aquatic organisms are PPPS and vary among species.We wondered whether the difference in this shear site of LRP6 is related to aquatic to terrestrial evolution,so we constructed LRP6 APQA-PPPS mutant mice.To observe the mutant mice,we conducted a series of behavioral experiments and found that LRP6 mutant mice showed a phenotype of reduced learning memory ability.Further experiments showed that the decline in learning memory ability was due to a star affecting synaptic function.更多还原