NBS1缺失突变导致小颅畸形的分子机制研究

发布时间：2018-05-20 04:47

本文选题：DNA损伤反应(DDR) + Nbs1　；参考：《北京协和医学院》2012年博士论文

【摘要】：Nijmegen breakage syndrome (NBS)是一种人类染色体不稳定性综合征,具有头小畸形(microcephaly)的突出临床表现,是由NBS1基因的次形态突变所引起。NBS1(小鼠称Nbn)基因的编码产物Nbsl是DNA损伤反应(DNA damage response,DDR)的一个关键蛋白分子。Nbsl与另外两个蛋白Mre11和Rad50共同组成Mrell-Rad50-Nbsl (MRN)复合体,该复合体在DNA损伤反应、DNA双链断裂修复、细胞周期检验点激活和维持基因组稳定性方面发挥着重要的作用。增殖活跃和代谢旺盛的细胞常常容易因为复制叉阻滞和代谢产生的活性氧导致大量内源性的DNA损伤。为了维持基因组的稳定性和完整性,细胞必须激活一系列复杂而有序的DNA损伤修复机制来修复损伤的DNA。Nbs1或MRN复合体在早期阶段就能感应存在于基因组上的损伤并通过激活ATM-Chk2-和ATR-Chk1-p53信号途径来调控细胞周期检验点以阻滞细胞周期的进程,为修复受损的DNA赢得足够的时间。p53的活化能有效地激活程序性细胞死亡来及时地清除掉不能被修复的受损细胞,避免细胞发生恶性转化而形成肿瘤。为了探讨人类NBS脑发育缺陷的病理机制,我们首先利用Cre-Loxp的技术在小鼠的中枢神经系统特异性敲除Nbn基因(称NbnCNS-del小鼠)后,获得了类似于NBS病人具有的microcephaly的表型。NhnCNS-del小鼠表现为生长迟缓,在早期就出现小脑发育不全、共济失调、白内障、视神经结构的破坏和变性、白质发育障碍和胶质细胞的功能紊乱。尽管如此,这些研究仍然没有从根本上揭示microcephaly发生的具体分子病理机制。因此,本研究旨在应用NbnCNS-del小鼠模型深入探讨Nbs1在大脑发育中的作用和导致microcephaly的分子机理。我们的研究发现：NbnCNS-del小鼠大脑皮质的大小比正常小鼠要小,胼胝体也相对地变薄,模拟了人microcephaly的表现。NbnCNS-del小鼠大脑皮质的Ⅱ/Ⅲ层(外颗粒层／外锥体细胞层)中的细胞数目明显地减少,并且Ⅳ和V层(内颗粒层／内锥体细胞层)中的颗粒细胞和大锥体细胞数也显著地减少,因而导致了细胞密度的降低和大脑皮质板层结构的变薄。γH2AX的免疫组化染色结果表明在胚脑新皮质快速发育的过程中Nbs1的缺失导致了大量内源性DNA损伤的聚集。Western blotting的结果显示Nbs1的缺失影响了ATR-Chk1途径的活化,Chkl-S345的磷酸化受到明显的抑制。神经前体细胞的免疫荧光实验证实了Nbs1的缺失影响了DDR分子如Chk1、BRCAl、Mrell和Mcph1在DNA损伤位点上的募集和foci的形成。Nbs1还具有维持Mcph1蛋白稳定性的作用。Nbs1缺失的大脑皮质神经元细胞仍然保留了ATM-Chk2途径对DNA损伤反应的能力,因而ATM-Chk2途径的活化激活了由p53介导的Hzf蛋白持续性且特异性地高表达在Nbsl突变的大脑皮质区。Hzf与p53结合后,特异性地调控p53对下游靶基因的反式激活作用,促进了p53下游靶分子p21的表达,同时抑制了Bax、Noxa和Puma的转录激活,导致了发育期大脑皮质神经元细胞的增殖减弱和凋亡抑制,最终NbnCNS-del小鼠形成了microcephaly。Trp53的失活突变能够基本上恢复Nbs1缺陷小鼠的microcephaly表型。综上所述,我们的研究揭示了发育期不同部位的神经元在应答内源性DNA损伤时激活的凋亡反应具有组织空间的特异性,p53的双重作用导致了Nbs1缺失的大脑皮质和小脑对凋亡的差异性反应。本研究证明了Nbs1在神经发生中起到的重要作用。
[Abstract]:Nijmegen breakage syndrome (NBS) is a human chromosome instability syndrome with a protruding clinical manifestation of the head small deformity (microcephaly). It is a key protein molecule of the.NBS1 (mouse called Nbn) gene, caused by the secondary mutation of the NBS1 gene, which is a key protein molecule of the DNA damage reaction (DNA damage). The other two proteins, Mre11 and Rad50, together constitute the Mrell-Rad50-Nbsl (MRN) complex, which plays an important role in the DNA damage response, the repair of the double strand breaks of DNA, the activation of cell cycle checkpoints and the maintenance of genomic stability. In order to maintain genomic stability and integrity, to maintain genomic stability and integrity, cells must activate a series of complex and orderly DNA damage repair mechanisms to repair damaged DNA.Nbs1 or MRN complexes that can induce damage to the genome at the early stage and activate ATM-Chk2- and ATR-Chk1-p53 letters in order to maintain genomic stability and integrity. The cell cycle test point is regulated to block the process of cell cycle, and the activation energy of.P53 to repair damaged DNA can effectively activate programmed cell death to remove the damaged cells that can not be repaired in time, and avoid the malignant transformation of cells to form tumors.
In order to explore the pathological mechanism of human NBS brain development defects, we first use Cre-Loxp technique in the specific knockout of the Nbn gene in the central nervous system of mice (called NbnCNS-del mice), and the phenotype of.NhnCNS-del mice with microcephaly similar to those of NBS patients is growth retardation, and cerebellar dysplasia appears in the early stage. Ataxia, cataract, optic nerve structure damage and degeneration, white matter development disorder and glial cell dysfunction are still not fundamentally revealed in these studies. Therefore, the purpose of this study is to explore the application of NbnCNS-del mouse model to the development of Nbs1 in the development of the brain. Use and cause the molecular mechanism of microcephaly.
Our study found that the size of the cerebral cortex in NbnCNS-del mice was smaller than that in normal mice, and the corpus callosum was also relatively thinner, which simulated the human microcephaly expression in the.NbnCNS-del mice of the brain cortex of the second / third layer (outer granular layer / outer pyramidal cell layer), and the number of cells in the IV and V layers (inner granular layer / inner cone thin). The number of granular cells and large pyramidal cells in the cell layer also decreased significantly, resulting in the decrease of cell density and the thinning of the lamellar structure of the cerebral cortex. The results of immunohistochemical staining of gamma H2AX showed that the deletion of Nbs1 resulted in the aggregation of a large number of endogenous DNA injuries aggregated.Western blotting during the rapid development of the neocortex of the embryonic brain. The results show that the deletion of Nbs1 affects the activation of the ATR-Chk1 pathway and the phosphorylation of Chkl-S345 is obviously inhibited. The immunofluorescence test of the neural precursor cells confirms that the deletion of Nbs1 affects the DDR molecules such as Chk1, BRCAl, Mrell and Mcph1 are raised on DNA damage sites and foci form.Nbs1 also has the effect of maintaining the stability of the protein. The.Nbs1 deficient cerebral cortical neurons still retain the ability of the ATM-Chk2 pathway to respond to DNA damage, so the activation of the ATM-Chk2 pathway activates the p53 mediated Hzf protein persistently and specifically highly expressed in the Nbsl mutation of the cerebral cortex.Hzf and p53, specifically regulating the trans activation of the p53 to the downstream target gene. It promotes the expression of p21 in the downstream target molecule of p53, and inhibits the transcription activation of Bax, Noxa and Puma, which leads to the decrease of proliferation and inhibition of apoptosis in the cerebral cortical neurons of the developmental stage. Finally, the NbnCNS-del mice have formed the microcephaly.Trp53 inactivation mutation to basically restore the microcephaly phenotype of Nbs1 deficient mice.
To sum up, our study revealed that the apoptotic response activated by neurons in different parts of the developmental phase in response to endogenous DNA damage has the specificity of tissue space. The dual action of p53 leads to the difference in the apoptosis of the cerebral cortex and cerebellum missing from Nbs1. This study proved that Nbs1 plays an important role in neurogenesis. Use.
【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2012
【分类号】：R363

【相似文献】