【作者】 殷梧；

【导师】 胡兵；

【作者基本信息】 中国科学技术大学 ， 神经生物学， 2014， 博士

【摘要】 复杂的神经系统都是由神经元和胶质细胞这两种主要的细胞类型构成的,而髓鞘化则是一种胶质细胞粘附到神经元轴突上并引发质膜极化形成多层髓鞘的包裹过程。中枢神经系统(central nervous system, CNS)由少突胶质细胞负责髓鞘化,而外周神经系统(peripheral nervous system, PNS)的髓鞘化则是由施旺细胞完成。常见的中枢神经系统脱髓鞘疾病,例如多发性硬化症(multiple sclerosis, MS),是一种由中枢神经系统遭到免疫系统攻击而引发的髓鞘损伤和轴突缺失的神经退行性疾病。为了探索中枢神经系统脱髓鞘的原因并寻找有效的治疗方法,需要进一步研究发育过程中的髓鞘化和脱髓鞘化。LINGO-1蛋白是一种由富亮氨酸重复序列(Leucine-rich repeat, LRR)、免疫球蛋白结构域(immunoglobulin domain, Ig)和轴突生长抑制蛋白(neurite outgrowth inhibitory protein, Nogo)受体相互作用蛋白1构成的中枢神经系统跨膜蛋白,是少突胶质细胞分化的负调控因子。在多种动物模型中,靶向抑制LINGO-1可以促进神经元存活、轴突再生、少突胶质细胞分化和再髓鞘化。尽管在啮齿类动物模型上的研究加深了对LINGO-1的了解,但是在斑马鱼(Danio rerio)神经发育和髓鞘化过程中,其作用还不清楚。本文旨在研究lingo1基因表达的时空模式及Lingo1b蛋白在斑马鱼发育中的作用。首先我们使用生物信息学方法分析了斑马鱼lingo1基因的分子结构。相对哺乳动物,斑马鱼同源lingo1有两个基因拷贝：lingola和lingo1b。斑马鱼Lingo1b蛋白具有类似哺乳类的蛋白结构并可能在中枢神经系统中发挥作用,在脊椎动物中也是高度保守的。利用体外合成lingo1基因的地高辛(digoxin, DIG)标记的RNA探针,我们通过整体原位杂交(whole-mount in situ hybridization, WISH)技术分析了lingo1基因表达的时间和空间模式。通过实时荧光定量PCR (real-time quantitative-polymerase chain reaction, Q-PCR)分析和蛋白免疫印迹(western blot, WB)分析进一步发现斑马鱼lingo1b基因的mRNA表达始于受精后1天(day post-fertilization, dpf),而蛋白表达始于2dpf。在斑马鱼lingo1b的反义吗啡林(morpholino oligonucleotide, MO)基因敲减实验中,我们首先体外合成了含有MO靶向序列的绿色荧光蛋白(green fluorescent protein, GFP)报告mRNA序列以及Lingo1b蛋白抗体以分析基因敲减的特异性和效率。对4dfp斑马鱼lingo1b的Mo1基因敲减后的表型进行分析,发现对Lingo1b蛋白的表达抑制会导致例如黑色素减少、眼睛萎缩和脊椎(spinal cord, SC)弯曲等发育异常,暗示了其对神经系统发育的影响。通过透射电镜(transmit electron microscopy, TEM)成像和髓鞘的G-ratio分析,我们发现Mo1敲减后毛特纳细胞轴突(Mauthner axons, MAs)和其它神经元轴突的髓鞘变厚并出现了过早髓鞘化,进一分析也暗示lingo1b的敲减能够促进少突胶质细胞的分化与成熟。对EM图像的进一步分析,我们发现伴随着髓鞘的增厚,毛特纳细胞的轴突周长变小。通过针对MAs的整体免疫组化(whole-mount immunohistochemisty, WIHC)标记和共聚焦成像分析则进一步显示了lingo1b敲减后MAs的平均直径下降了,暗示了敲减后引起的过早髓鞘化可能会影响初级运动神经元(primary motor neurons, pMNs)的发育。我们采用自发运动和眼动反应(optokinetic response, OKR)行为学分析来进一步研究过早髓鞘化和运动神经元异常发育所导致的结果。图像分析软件(Image-Pro Plus, IPP)定量数据显示lingo1b敲减后,单位时间内自发运动的累积距离(accumulation distance, Ace Dist)和眼动反应的频率均发生了下降。利用生物信息学分析,我们从斑马鱼基因组DNA中预测并克隆了lingo1b基因的启动子序列,并整合到含有转座酶(TOl2)或巨核酸酶(I-Scel)位点以及荧光报告序列的表达载体上。通过激光共聚焦成像对显微注射后的斑马鱼胚胎的荧光信号模式进行了分析,初步构建了Lingolb特异性标记的转基因斑马鱼品系Tg(lingo1b:EGFP)。我们的研究结果显示,斑马鱼lingo1基因是保守的并在中枢神经系统中特异表达。吗啡林基因敲减的结果则显示斑马鱼Lingo1b蛋白在神经系统发育过程中,对于少突胶质细胞分化成熟以及髓鞘化进程是一个重要的负调控因子。而形态学和行为学的结果则提示了由lingo1b基因敲减引起的过早髓鞘化可能会影响运动神经元的发育。这些结果为深入了解神经系统损伤修复机制以及以后大规模筛选潜在的治疗脱髓鞘疾病的药物分子提供了一个新的平台。更多还原

【Abstract】 The complex nervous systems consist of two major cell types, neurons and glia cells. Myelination is a wrapping process that glial processes attach to the axons and trigger the polarization of the plasma membrane to form multilayer myelin sheath tightly. The myelin is produced by two types of specialized glial cells, oligodendrocytes in the central nervous system (CNS) and Schwann cells in the peripheral nervous system (PNS). Demyelinating diseases include multiple sclerosis, which is a neurodegenerative disease characterized by immune attacks on the CNS, resulting in myelin sheath damage and axonal loss. To explore the causes of CNS demyelination and to find effective treatment approaches, it is necessary to study the demyelination and myelination during the development closely.Leucine-rich repeat (LRR) and immunoglobulin (Ig) domain-containing neurite outgrowth inhibitory protein (Nogo) receptor-interacting protein-1(LINGO-1) is a CNS specific transmembrane protein and has been identified as a negative regulator of oligodendrocytes differentiation. Targeted LINGO-1inhibition promotes neuron survival, axon regeneration, oligodendrocyte differentiation, and remyelination in diverse animal models. Although studies in rodent models have extended our understanding of LINGO-1, its roles in neural development and myelination in zebrafish (Danio rerio) are not yet clear. Here we aim to study the temporal and spatial expression pattern of lingo1and the roles of Lingo1protein during zebrafish development.We first studied the molecular structure of lingo1in zebrafish by bioinformatics tools. Zebrafish have two copies of the lingol gene(lingo1a and lingo1b) that is present as a single copy in mammals. The Lingolb protein has a similar protein structure to the mammals and may play a role in the nervous system. A phylogenetic tree was constructed and showed that zebrafish Lingol was highly conserved within vertebrates. After the specific digoxin-labeling RNA probe was synthesized, we analyzed the temporal and spatial expression pattern of lingol by whole-mount in situ hybridization (WISH). The expression of lingo1b started1(mRNA) and2(protein) days post-fertilization (dpf) in the CNS by real-time quantitative-polymerase chain reaction (Q-PCR) and western blot (WB) analysis, In addition, by virtue of morpholino oligonucleotide (MO) knockdown technology, the MO targeted green fluorescent protein (GFP) mRNA report sequence and antibodies of Lingolb protein were synthesized in vitro for the knockdown specificity and efficiency analysis. The morphology analysis of4dpf lingo1b knockdown zebrafish showed some nervous system developmental abnormalities, including less dark pigment, small eyes, and a curly spinal cord (SC). The transmit electron microscopy (TEM) image and G-ratio calculation of lingo1b knockdown showed a thick myelin and an early-onset myelination of the Mauthner axons (MAs) and other axons, further analysis also implied that knockdown of Lingolb protein may promote the oligodendrocytes differentiation and maturation. We further measured the total and MA circumferences with TEM in4dpf larvae. The significantly decreased MA circumferences were accompanied with the increased myelin sheath thickness. To further confirm the decreasion in MA diameter, we counted the average diameter of MAs using whole-mount immunohistochemisty (WIHC) labeling confocal image. These results implied that the early-onset myelination caused by lingo1b knockdown may inhibit the development of primary motor neurons (pMNs). The outcome of early-onset CNS myelination and abnormal motor neuron development has been shown by the spontaneous movement analysis and larvae optokinetic response (OKR) tests. The accumulation distance (Acc Dist) value of spontaneous movement and the frequencies of eye movements were both decreased after lingolb knockdown that analyzed with Image-Pro Plus (IPP)6software. The lingo1b gene promoter sequences were cloned from zebrafish genomic DNA after bioinformatics predication and inserted into recombinant plasmid with transposase (Tol2) or meganuclease (I-Scel) sites and fluorescent protein report sequence. The fluorescent signal patterns of injected embryos were analyzed with confocal image, and the primary Lingolb specific gene marker of transgenic zebrafish lines Tg (lingo lb:EGFP) were also primarily constructed.Our results, to date, indicated that the lingo1is highly conserved and specific expressed in zebrafish CNS. Furthermore, the MOs knockdown effects of lingo1b suggested that Lingolb may play a role as a negative regulator of myelination and oligodendrocyte differentiation during zebrafish development. The morphology and ethology results also implied that the early-onset myelination caused by lingo1b knockdown may inhibit the development of primary motor neurons. These may provide a new platform for further understanding mechanisms underlying nervous system damage and repair as well as benefit to the large-scale potential compounds screening for the demyelinating disease in the future.更多还原